Journal of Research in Biology An International Scientific Research Journal

Original Research

Smoke toxicity effect of Artemisia Parviflora against

dengue vector Aedes Aegypti
Journal of Research in Biology

Authors: ABSTRACT:
Uma Devi R and Lakshmi D.
Background and Objectives: This paper reports the smoke toxicity effect of
Asteraceae plant species against the dengue vector, adult Aedes aegypti. These plants
are used traditionally as source of medicine.
Institution:
Department of Life
Methods: The mosquito coils were prepared by using leaves, root, and the stem of
Science, Karpagam
University,Coimbatore, Artemisia Parviflora by mixing with coconut shell and charcoal powder as burning
Tamil Nadu, India. material. Test mosquito coil were compared with commercially available coils. The
percentage of unfed mosquitoes and % of population reduction was calculated.
Karpagam College of
Education, Karpagam Results: Smoke emerged from the coil made up of leaves showed maximum
University, Coimbatore, protection 51% and population reduction was 83.8%.Contol II showed highest toxic
Tamil Nadu, India. effect and more population. The smoke from root and stem coils showed moderate
amount of protection.

Conclusion: The result suggested that the smoke toxic effect of Artemisia Parviflora
Corresponding author: affects the central nervous system and hence affects the neuroendocrine system to
Lakshmi D. inhibit the hatchability of eggs and reduces the egg laying capacity as well the egg
hatchability of the mosquitoes.

Email: Keywords:
umadevi.suba@gmail.com. Aedes aegypti, Artemisia Parviflora, smoke toxicity, knock down effect.

Web Address: Article Citation:

http://jresearchbiology.com/ Uma Devi R and Lakshmi D.
documents/RA0235.pdf. Smoke toxicity effect of Artemisia Parviflora against dengue vector Aedes Aegypti.
Journal of Research in Biology (2012) 2(4): 273-280

Dates:
Received: 23 Apr 2012 Accepted: 07 May 2012 Published: 15 May 2012

This article is governed by the Creative Commons Attribution License (http://creativecommons.org/

licenses/by/2.0), which gives permission for unrestricted use, non-commercial, distribution and
reproduction in all medium, provided the original work is properly cited.

273-280 | JRB | 2012 | Vol 2 | No 4

Journal of Research in Biology
An International
Scientific Research Journal www.jresearchbiology.com

INTRODUCTION
Blood-feeding arthropods are responsible for the
transmission of a large number of diseases world-wide.
Insect-transmitted disease remains a major cause of
illness and death worldwide (Pavela,2009).Mosquitoes
are the most important single group of insects in terms of
public health importance, which transmit a number of
diseases, such as malaria, filaria, dengue, japanese
encephalitis, etc. causing million of deaths every year
(Rahuman et al., 2008). There are approximately 3,500
species of mosquitoes grouped into 41 genera ((NCID,
2004). Aedes aegypti, a vector of Dengue, Dengue
hemorrhagic fever and chikungunya which is a widely
distributed tropical and subtropical disease, is now
endemic in more than 100 countries and threatens the
health of approximately 2.5 billion people. Worldwide,
around 80 million people are infected annually at an
attack rate of 4% (Monath, 1994). In recent years, Aedes
aegypti (Diptera:Culicidae) spread the virus chikungunya
which affected the southwest Indian ocean islands in
2005, spread out to india, and resulted in an ongoing
outbreak that has involved >1.5 million patients (Taubitz
et al.,2007).
Today, the environmental safety of an insecticide
is considered to be of paramount importance. Insecticide
applications are although highly efficacious against the
target species, vector control is facing a threat due to the
development of resistance to chemical insecticides
resulting in rebounding Victoria capacity (Liu et al.,
2006). Furthermore, they are responsible for substantial
hazards to a variety of non-target organisms and
environment in the form of biomagnifications (Gold et
al., 2001). In recent years, many researchers have been
looking for new botanical insecticides. Plants contain
many chemicals, which are important in their defense
against insects. They fall into several categories
including repellents, feeding deterrents, toxins and
growth regulators. There are thousands of chemical
compounds that act in one or more of these ways.
274
Devi and Lakshmi, 2012
Sukumar et al., (1991) reviewed the bioactivity of 344
plant species against mosquitoes. They showed that some
photochemical acts as general toxicants to all life stages
of mosquitoes, whereas others interfere with growth and
reproduction or act on the olfactory receptors eliciting
responses of attractancy or repellency. Uma Devi et al.,
2010 reported the larvicidal activity of ethanolic leaf
extract of Artemisia parviflora tested against A.stephens.
Smoke is the most widely used means of
repelling mosquitoes utilized in the rural tropics.
Waste plant materials are frequently burned in Sri Lanka
as a mosquito repellent, even though indoor residual
spraying has been carried out by the government for
many years (Silva, 1991). In rural Guinea Bissau, 86%
of residents used an unimpregnated bed net in
conjunction with mosquito coils or plant-based smoke
(Palsson and Jaenson, 1999a). In the Solomon Islands a
recent survey revealed that fire with coconut husks and
papaya leaves was more prevalent among the other
personal protections from mosquitoes, being used by
52% of residents (Dulhunty et al., 2000). In the Gambia
L. cheraliera leaves are traditionally used as mosquito
repellents. L. javanica is commonly found in Southern
Africa and is frequently used as a repellent (Lukwa,
1994). Hot leaf infusion of the leaves of these plants are
traditionally used as remedies for a variety of ailments,
including malaria. The leaves have a strong lemon smell
(Van Wyk et al., 1997) to which belief in its healing
abilities can probably be attributed. L. cheraliera is also
burned in the Gambia as a mosquito repellent smoke
(Aikins et al., 1994). A thorough study carried out in
Zimbabwe by Lukwa et al. (1999) revealed that 29% of
the population used plants to protect themselves from
mosquitoes, mainly by burning the leaves of L. javanica.
From this literature survey, we could be
certained that there were no information was available on
the smoke toxicity activities with respect to the
experimental plant species given here. Therefore present
study aimed to evaluate the smoke toxicity effect of
Journal of Research in Biology (2012) 2(4): 273-280
Devi and Lakshmi, 2012
Artemisia parviflora plant parts against the adult Aedes
aegypti. Artemisia parviflora come under the family
Asteraceae. The family Asteraceae comprises of many
aromatic and medicinal plants. Artemisia parviflora
(Asteraceae) is an important medicinal plant found in
Western Ghats, northern Himalayas, Coimbatore hills,
Nilgiris and hills of Travancore above 3000 feet.
Artemisia parviflora is an important medicinal plant
belonging to family Asteraceae, commonly used for skin
diseases, cuts and wounds (Kimothi and Shah, 1989). It
is also considered for the treatment of high blood
pressure, diabetes, and anthelmintic (Ahmad et al.,
2006). These activities are because of complex mix of
phytochemicals present in A.parviflora. It also possesses
anti-viral properties (Ambasta, 1986; Anonymous,
1985).
MATERIALS AND METHODS
Colonization of Aedes aegypti
Collection of eggs
The eggs of A.aegypti were collected from
National Institute for Communicable Diseases (NICD),
Mettupalayam, Coimbatore,Tamil Nadu, India without
expose to any insecticide and in and around
Coimbatore,India at different breeding habitats with the
help of a O type brush. The eggs were then brought to
the laboratory and transferred to 18 x 13 x 4 cm size
enamel trays containing 500 ml water and kept for larval
hatching. They were hatched and reared have been still
maintained from many generations in the laboratory. The
eggs and larvae obtained from this stock were used for
different experiments.
Maintenance of larvae
The freshly hatched larvae were fed with dog
biscuits and yeast at 3:1 ratio. The feeding was continued
till the larvae transformed into the pupae stage. The
larvae reared in plastic cups. Water was changed
alternate days. The breeding medium was regularly
checked and dead larvae were removed at sight. The
Journal of Research in Biology (2012) 2(4): 273-280
normal cultures as well as breeding cups used for any
experimental purpose during the present study were kept
closed with muslin cloth for preventing contamination
through foreign mosquitoes.
Maintenance of pupae and adult
The pupae were collected from culture trays and
were transferred to glass beakers containing 500 ml of
water with the help of a sucker. The pupae containing
glass beaker were kept in 90 x 90 x 90 cm size mosquito
cage for adult emergence. The cage was made up of
wooden frames and covered with polythene sheets on
four sides (two laterals, one back and other one upper)
and the front part was covered with a muslin cloth. The
bottom of the cage was fitted with strong cardboard. The
freshly emerged adults were maintained 27.2C, 75 -
85% RH, under 14L: 10D photoperiod cycles. The adults
were fed with 10% sugar solution for a period of three
days before they were provided an animal for blood
feeding.
Blood feeding of adult A.aegypti and egg laying
The females were fed by hand every alternate
days at 6.00 a.m. Feeding mosquitoes on human arm for
experimental purposes was suggested by Judson (1967)
and Briegel (1985). Both females and males were
provided with 10% glucose solution as described by
Villani et al., (1983) on cotton wicks. The cotton was
always kept moist with the solution and changed every
day. Theoder and Parsons (1945) noticed that glucose as
well as ordinary sugar appeared equally attractive to the
mosquitoes. An egg trap (cup) lined with filter paper
containing pure water was always placed at a corner of
the cage. This arranged made collection of
eggs easier.
Smoke toxicity test
Artemisia parviflora (leaves stem and roots) used
for smoke toxicity assay. The mosquito coils were
prepared by following the method of Saini et al., (1986)
with minor modifications by using 2.5 gram of plant
ingredients, 4 grams of coconut shell and charcoal
275

powder as burning material. These ingredients were
thoroughly mixed with distilled water to form a
semisolid paste. A Mosquito coil (0.6 cm thickness) was
prepared manually and shade dried. The control coils
will be prepared by without the plant ingredient. The
experiments were conducted in glass chamber measuring
140 X 120 X 60 cm. A window measuring 60 X 30 cm
was situated at mid bottom of one side of the chamber.
One hundred 3-4 days old blood starved adult
female mosquitoes were released into the chamber and
were provided with 10% sucrose solution. A belly
shaven pigeon was kept tied inside the cage in
immobilized condition. The experimental chamber was
tightly closed. The experiment was repeated five times
on separate days including control groups using
mosquitoes of same age. The data were pooled and
average values were subsequently used for calculations.
Control was maintained in two sets. One set was run with
coil lacking the active ingredient of plant powder
(control I) another one was a commercial coil (control 2),
which was used for positive control to compare the
effectiveness of plant coils. After the experiment was
over, the fed, unfed (active and dead) mosquitoes were
counted. The protection given by the smoke from plant
samples against the biting of A.aegypti was calculated in
terms of percentage of unfed mosquitoes due to
treatment
Number of unfied mosquitoes in treatment -
Number of unfed mosquito in control 1
= 100
Number of mosquitoes treated
The live blood fed mosquitoes were reared in a
mosquito cage, measuring 30 x 30 x 15 cm. The top and
bottom of the cage were fit with glass and all other sides
were covered with muslin cloth. Water soaked raisins
and a 5% sucrose solution soaked in cotton balls were
provided as a food source. Water containing powdered
yeast and dog biscuits were also kept inside the cage in a
glass bowl for oviposition. The eggs from the cage were
276
Devi and Lakshmi, 2012
collected daily till all the mosquitoes died. A total 50-
100 eggs were allowed to hatch in plastic trays
measuring 30 x 25 x 6 cm, containing about 2.5 liters of
unchlorinated tap water. Hatched larvaes were fed with
a mixture of dog biscuits and yeast powder in the ratio of
2:1 and water in the tray was changed daily. Survival and
dead instars were counted and reduction in the
population from the smoke treated
mosquitoes were calculated using the formula.
Number of larvae hatched in control 1 -
Number of larvae hatched in treated
Population reduction (%) 100
Number of larvae hatched in control 1
Statistical analysis
Data were analyzed using analysis of variance
(ANOVA) and means separated by Duncans multiple
range tests (DMRT).
RESULTS AND DISCUSSION
Table 1 provides the results of smoke toxicity
effect of A. parviflora leaf on A.aegypti. The control was
maintained without plant ingredients. It acts as negative
control. The commercially available (Mortein) mosquito
coil was used as positive control. One hundred, 4-3 days
starved A.aegypti were used. After the treatment of the
plant, the fed and unfed mosquitoes were counted. There
were 19 fed and 81unfed mosquitoes counted after the
treatment of leaf smoke exposure,21fed and 79 unfed
from roots and 24 fed and 76 unfed mosquitoes were
counted after the treatment of stem smoke exposure of A.
parviflora. The comparisons of these plant parts the
smoke from leaves showed very high efficacy. The
effect of plant leaves showed good smoke toxicity effect
on A.aegypti. This may be due to the presence of active
chemical compounds in the leaves. Plant derived smoke
contains an array of chemicals with different mode of
action, which kill mosquitoes. The smoke from the blank
coil also showed considerable toxic effect and control II
showed higher toxic effect against A.aegypti.
Table 2 shows the smoke toxicity effect of
Artemisia parviflora from ((leaves, stem and root)
Journal of Research in Biology (2012) 2(4): 273-280
Devi and Lakshmi, 2012

Table 1: Smoke toxicity effect of Artemisia parviflora parts against biting activity of Aedes aegypti.
Artemisia parviflora No. of mosquitoes Unfed mosquitoes % unfed over
Fed mosquitoes Total
parts used tested Alive Dead control I
Leaf 100 19c 61a 20a 81ab 51a
bc b c ab
Root 100 21 49 30 79 49ab
a c b b
Stem 100 24 40 36 76 46b
a d e c
Control I* 100 70 30 0 30 0c
cd c a a
Control II* 100 18 40 42 82 -
Within a column means followed by the same letter(s) are not significantly different at 5% level by
DMRT.Control I* -Negative control - blank without plant material Control II*-Positive control - mortein coil

different parts ensured population of the Aedes aegypti. product in low communities in India. The common active
The numbers of eggs laid by the alive, fed females were ingredients in coil are various pyrethroids and different
shown. Number of eggs laid and the hatchability were toxic chemicals and frequently contain octa
greatly reduced or affected by the exposure of smoke. chlorodiprophyl ether include undefined geno toxicagent
Only 5 of 25 mosquitoes oviposited a total of 1357 eggs (Pauluhn and Mohr, 2000). Plant derived smoke contains
of which only 950 eggs had hatched. The percentage of an array of chemicals which has been used since early
reduction was 83.8% in the plant exposed mosquitoes in time to deter mosquitoes and it is cheap target specific
the leaf smoke, 82.6% from root and the 53.7% from and highly toxic to adult mosquitoes at low dose. Smoke
stem. The leaves showed a significant effect on the toxicity from Moringa oleifera (Prabhu et al., 2011)
fecundity and hatchability. The smoke exposure affects Mesua ferra (Someshwar et al., 2011) Spathodea
the central nervous system and hence affects the campanulata (Aarthi and K Murugan.,2010),
neuroendocrine system to inhibit the hatchability of eggs C.occidentalis Abirami Dhandapani and Murugan
and reduces the egg laying capacity as well as the egg Kadarkarai (2011) Albizzia amara and Ocimum
hatchability of the mosquitoes. basilicum (Murugan et al., 2007) considerably affect the
The control of adult mosquitoes has been mosquito survival and pronounced high repellent
considered too, either by adulticiding or preventing potential. In the present smoke toxicity test also the plant
method such as repellency or mosquito coil burning. showed a good smoke repellency activity. The smoke
There are four major types of insecticidal products used exposed females also laid only limited number of eggs,
by general people in their residence like aerosols, in that also only a limited hatched out. This reduces the
mosquito coil, liquid vaporizers and vaporizing mats, out mosquito population also. The results of this study
of which mosquito coils are preferred as anti-mosquito indicated that A.parviflora leaves enhances in the smoke

Table 2. Smoke toxicity effect of Artemisia parviflora parts ensured population of Aedes aegypti.

Artemisia No. of No of egg rafts Total No Total No. of % of reduction in

parviflora mosquitoes laid by fed of eggs Larvae hatched population over
parts used tested mosquitoes from the eggs rafts control I
Leaf 25 5bc 1357a 950a 83.8a
Root 25 8b 955c 440c 53.7b
Stem 25 7b 1357a 950a 82.6 ab
Control I* 25 12ab 1048b 675b 28.9c
Control II* 25 4c 380e 165d -

Within a column means followed by the same letter(s) are not significantly different at 5% level by DMRT.
Control I* -Negative control - blank without plant material Control II* Positive control - mortein coil

Journal of Research in Biology (2012) 2(4): 273-280 277


toxicity test and it may be an effective alternative to
conventional synthetic insecticides for the control of
A.aegypti smoke emitted from the A.parviflora also
showed a good knock down effect. The smoke exposure
affects the central nervous system and hence affects the
neuroendocrine system to inhibit the hatchability of eggs
and reduces the egg laying capacity as well as the egg
hatchability of the mosquitoes. According to Thangam
and Kathiresan (1992) stated that smoke from burning
various dry materials has been used since early times to
determine insects, especially mosquitoes. Pandian et al.,
(1995) studied powdered preparations of the leaves of
Adhatoda vasica, Azadirachta indica and Ocimum
sanctum, which on burning with charcoal produced
smoke that repelled Armigeres subalbatus and Culex
quinquefasciatus and prevented their biting activity for 6
8 h. However, compared with the previous study, our
study showed greater smoke toxicity. Traditional
repellents not only provide protection against mosquito
bites but also curtail disease transmission. Hence, these
plant parts can be preferably employed for the
development of mosquito coil in future.
CONCLUSION
The finding of the present investigation revealed
that A.parviflora has good toxic and repellent effect
against Aedes aegypti. Smoke from medicinal plants are
tremendous to the human beings, also they are
biodegradable and easily available.
ACKNOWLEDGEMENT
The authors are thankful to Karpagam
University, Coimbatore, Tamil Nadu, India and National
Institute of Communicable Disease Mettupalayam, for
providing facilities for completing this research work.
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 Journal of Research in Biology An International Scientific Research Journal

Original Research

Bioremediation of oil contaminated soil using biosurfactant

produced by Pseudomonas aeruginosa
Journal of Research in Biology

Authors: ABSTRACT:
Pradeep NV1, Anupama1, Biosurfactants are surfactants that are biologically produced from bacteria,
Anitha G2, Renukamma2, yeasts and fungi. Examples include Pseudomonas aeruginosa which produce
Sudhakar Avvaru2. biosurfactants from various substrates including sugars, oil and wastes. Biosurfactants
Afreen SS2. have the property of reducing surface and interfacial tension. These are biodegradable
and nontoxic. In the present study saw dust and rice husk were used as substrates for
biosurfactant production. Pseudomonas aeruginosa was isolated from contaminated
soil sample collected from diesel contaminated site for the production
Institution:
1. Assistant Professor, of Biosurfactant. The fermentative production of biosurfactant from
Department of Pseudomonas aeruginosa was carried out by solid state fermentation (SSF) using two
Biotechnology, Ballari low cost substrates (rice husk and saw dust). These substrates were inoculated with
st rd th th th
Institute of Technology and P. aeruginosa and surface tension was measured on 1 , 3 , 5 , 7 and 9 day. The
th
Management, Bellary, India. highest reduction of surface tension (22.9 mN/m) was obtained using saw dust on 7
day. Produced biosurfactant have been tested for remediation of oil contaminated
2. B.E Scholar, Department soil. The contaminated soil was prepared in the laboratory by mixing oil, sand and
of Biotechnology, Ballari seed (diesel contaminated soil) in the ratio of 10:2:1. Contaminated soil was further
Institute of Technology and transferred to containers and was allowed for acclimatization. Three containers were
Management, Bellary, India. used in this study 1) Containing contaminated soil without biosurfactant (C1). 2)
Container containing contaminated soil with 4 g of biosurfactant per kg of soil (C2). 3)
Containing contaminated soil with 8 g biosurfactant per kg of soil (C3). The oil
remaining in soil was determined by solvent extraction method using n-hexane. Oil
removal was high in the C2 container which contained 4 g of biosurfactant per kg of
soil.
Corresponding author:
Keywords:
Pradeep NV.
Biosurfactant, Solid state fermentation, Bioremediation,
Pseudomonas aeruginosa, Surface tension.

Email: Article Citation:

nagamallivpradeep@gmail.com
Pradeep NV, Anupama, Anitha G, Renukamma, Sudhakar Avvaru, Afreen SS.
Bioremediation of oil contaminated soil using biosurfactant produced by
seudomonas aeruginosa.
Journal of research in Biology (2012) 2(4): 281-286

Web Address: Dates:

http://jresearchbiology.com/
documents/RA0236.pdf.
Received: 23 Apr 2012 Accepted: 14 May 2012 Published: 23 May 2012

This article is governed by the Creative Commons Attribution License (http://creativecommons.org/

licenses/by/2.0), which gives permission for unrestricted use, non-commercial, distribution and
reproduction in all medium, provided the original work is properly cited.

281-286 | JRB | 2012 | Vol 2 | No 4

Journal of Research in Biology
An International
Scientific Research Journal www.jresearchbiology.com

INTRODUCTION
Biosurfactants are diverse group of surface-
active chemical compounds that are produced by a wide
variety of microorganisms. Some biosurfactants are
known to have therapeutic applications as antibiotics,
antifungal or antiviral compounds. Biosurfactants can
also be used in the bioremediation of soil (Youssef et al.,
2004).
Biosurfactants can be synthesized by many
different microorganisms and are grouped into six major
classes based on the producing microorganism. These
classes are glycolipids, phospholipids, polysaccharide
lipid complexes, lipoproteinslipopetides, hydroxylated
and cross-linked fatty acids, and the complete cell
surface (Urum & Pekdemir., 2004). The two major
classes of biosurfactants include lipopeptides and
glycolipids, lipopeptides being synthesized by many
bacilli and other species, and the latter being synthesized
by Pseudomonas species (Youssef et al., 2004). These
molecules have attracted considerable scientific attention
due to lower toxicity, higher biodegradability, activity at
extreme temperatures, pH and salinity and possibility of
their production through fermentation using low cost
agro-based substrates. Carbon substrate is an important
limiting factor affecting the production of microbial
surfactants. The type of carbon substrate used for
production has been reported to influence both the
quality and quantity of biosurfactants (Das et al., 2009).
The most widely distributed environmental
pollution can be attributed to oil contamination, caused
by tanker accidents, storage tank ruptures, pipeline leaks
and transport accidents (Margesin, 2000). This
contamination causes significant environmental impacts
and presents substantial hazards to human health (Lu et
al., 2009). Bioremediation involves the acceleration of
natural biodegradation processes in contaminated
environments (Calvo et al., 2009). Biosurfactants have
been recommended and classified for environmental
applications because they are cost-effective and readily
282
Pradeep et al., 2012
biodegradable with low or no environmental toxicity and
are more effective than the synthetic surfactants (Urum et
al., 2003). Bioremediation of petroleum hydrocarbon
contaminated soils has been recognized as an efficient,
economic, versatile, and environmentally sound
treatment (Liu et al., 2008).
Pseudomonas aeruginosa is a typical strain for
rhamnolipid production and can utilize vegetable oil or
glycerol as the sole carbon source (Zhang et al., 2005). A
group of biosurfactants that has been studied extensively
is the rhamnolipids from Pseudomonas aeruginosa
(Mulligan, 2000).
The success of bioremediation is dependent upon
the microbial ability to degrade these complex mixtures
and their rate limiting kinetics. Higher rates of
degradation of hydrocarbons are often achieved with a
bacterial enrichment consortium isolated from the
environment that needs bio restoration. Bacterial
consortia display a wide array of metabolic mechanisms
in the breakdown of diesel oil components, including
production of surface-active agents and emulsifiers
(Bento et al., 2005). The combined behaviour of two
immiscible liquids such as oil and water that result in the
formation of the emulsions is important in the
application of surfactants in oil-contaminated soil
treatment (Urum & Pekdemir., 2004).
In the present study, diesel contaminated soil was
collected and Pseudomonas aeruginosa was isolated for
the production of biosurfactant. The produced
biosurfactant was used for the bioremediation of
contaminated soil.
MATERIALS AND METHODS
Microorganism, media composition
Diesel contaminated soil was used for the
isolation of Biosurfactant producing microorganism.
Serial dilution followed by streaking technique was
carried out for the isolation of the organism. Cetrimide
agar was used for culture maintenance and preparation of
the inocula.
Journal of Research in Biology (2012) 2(4): 281-286
Pradeep et al., 2012
Gram staining and Biochemical characterization of
Pseudomonas aeruginosa
The isolated strain was characterized
conventional methods like gram staining
biochemical tests such as gelatin hydrolysis, citrate
utilization, nitrate reduction, indole, starch, MRVP and
catalase test.
Inoculum and Substrates
Petri plates were inoculated with 1ml of
inoculum and incubated at controlled temperature for the
production of biosurfactant. Rice husk and saw dust were
collected from rice mill and carpenter shop respectively
and were used as substrates for the production of
biosurfactant. Substrates were sterilized in autoclavable
petri plates prior to use. Minimum water content was
added to maintain the moisture content required for the
growth of Pseudomonas aeruginosa.
Biosurfactant production from saw dust and rice
husk
Five petri plates numbered 1 to 5 were prepared
for each substrate and inoculated with P. aeruginosa.
Plates with samples were analysed on every alternative
st rd th th th
days viz., 1 , 3 , 5 , 7 and 9 day of fermentation.
Fermented substrates were transferred to conical flask
and 100 ml of water was added to it. Flasks were kept in
rotary shaker for 2 hrs. Substrates were filtered using
filter cloth, filtrate was centrifuged at 4000 rpm for 15
min and supernatant (crude biosurfactant) was collected.
Measurement of surface tension
Stalagmometer was used to measure surface
tension of samples. Surface tension (S.T) was calculated
using the formula (Turkovskaya et al., 2001 and
Veenanadig et al., 2000).
(density of sample) *(drops of water)
* (S.T. of water)
S.T. of sample =
(Density of water) *(drop of sample)
Where surface tension of water =72 mN/m
Density of water = 1g/ml
Journal of Research in Biology (2012) 2(4): 281-286
Preparation of contaminated soil
The soil (sand) was collected and was sieved
by using sieve shaker machine. Soil particle size of 4.75 m
and was used for this study. Three containers were used for
contamination of soil. A fixed mass of 4 kg sand was
measured and placed in each container. Further, soil was
contaminated with oil (used two wheeler oil) and seed
(diesel contaminated soil) in the ratio of 10:2:1 at room
temperature and it was allowed to contaminate for 10-12
days.
Addition of Biosurfactant
After contamination of soil, 4 g and 8 g of
biosurfactant per kg of soil were added to containers C2
& C3 respectively and C1 was kept as a blank (no
biosurfactant was added).
Determination of Total Petroleum Hydrocarbon
(TPH) using n-hexane extraction
The soil samples present in the containers were
analyzed using TPH method by n-hexane extraction to
determine the oil content remaining in the soil samples.
Four grams of soil sample was taken and mixed with 20
ml of distilled water and stirred for 5 mins. The sample
was acidified using HCl and 10 ml of n-hexane was
added and mixed for 10 mins. The sample was
transferred to separating funnel and was allowed to stand
for 10 mins. The topmost layer contained n-hexane and
oil. Bottom layer containing water was drained; the top
layer was transferred to evaporating dish and was placed
in hot air oven for one hour to evaporate the n-hexane.
The TPH was measured using the formulae:
TPH in the contaminated soil% = (W2-W1)*100/
Wt of soil in g
Where, W1 = weight of the empty dish.
W2 = weight of the dish with separated oil after dried
in oven at 103C for 2 hours.
RESULTS AND DISCUSSION
Gram staining and Biochemical characterization
The results of Gram staining and biochemical
283
 Pradeep et al., 2012

tests are reported in Table 1. The growth of

microorganism on specific media, results of gram
staining and biochemical tests indicates that the organism
is Pseudomonas aeruginosa.
Production of Biosurfactant
In order to economize the biosurfactant
production, the low cost carbon sources, saw dust and
rice husk were used. Biosurfactant was produced using Figure 1: Surface tension of biosurfactant produced
Pseudomonas aeruginosa with rice husk and saw dust as from P. aeruginosa.
substrates. Microbial molecules which exhibit high 2297 using orange peel as the carbon source reduced the
surface activity and emulsifying activity are classified as surface tension of culture broth and the final surface
biosurfactants. These molecules reduce surface and tension reached from a value of 57 mN/m to a level up to
interfacial tensions in both aqueous solutions and 31.3 mN/m.
hydrocarbon mixtures making them potential agents for Surface tension decreased rapidly from 72 to 30
bioremediation (Bento et al., 2005). Biosurfactant mN/m with increase in the Rhamnolipid concentration up
activity can be measured by changes in surface and to 40 mg/L (Whang et al., 2008).
interfacial tensions and emulsification/emulsion Dubey and Juwarkar (2001) used glucose,
stabilization. In this work, a reduction in surface tension distillery and whey wastes for the production of
to 22.9 mN/m was achieved with monoculture isolates, a biosurfactant using P. aeruginosa and obtained a
th
defined consortium and saw dust as substrate on 7 day reduction in surface tension of 27 mN/m by using the
of SSF. above mentioned substrates.
Solid state fermentation Bioremediation of Oil Contaminated Soil
Solid state fermentation was carried out using Oil contaminated soil was bioremediated in the
two substrates viz., rice husk and saw dust. The presence of biosurfactant. Oil content in the
reduction in surface tension is as shown in Figure 1. contaminated soil was tested using TPH (Total
There were significant reduction in the surface Petroleum hydrocarbon) method using n-hexane
tension using saw dust as it can be used as a source for extraction. Percentage of TPH in the contaminated soil
producing biosurfactant due to the significant reduction was measured and is as shown in Figure 2. C1 contained
in surface tension. no biosurfactant, C2 and C3 contained 4 g and 8 g of
George and Jayachandran (2008) reported the biosurfactant per kg of contaminated soil respectively.
production of biosurfactant by P. aeruginosa MTCC TPH in the soil was measured every day; TPH on the
Table 1: Results of Gram staining and first day was 6% in all the containers. The oil percentage
biochemical tests.
in C1, C2 and C3 containers slowly reduced and reached
TEST RESULT
Grams staining Gram Negative a steady state on 8th day. It was observed that the oil
Catalase test Positive percentages were 2.75, 1.3 and 2 in C1, C2 and C3
Citrate utilization Positive
respectively. The results indicated that 4 g of
Indole production Negative
Starch hydrolysis Negative biosurfactant per kg of contaminated soil was optimum.
Methyl red Negative The TPH analysis was stopped when there was no
Voges Proskauer Negative
Gelatin hydrolysis Positive considerable difference in two consecutive readings.
284 Journal of Research in Biology (2012) 2(4): 281-286
Pradeep et al., 2012

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Rhamnolipid Biosurfactants Produced through
CONCLUSIONS Submerged Fermentation Using Orange Fruit Peelings as
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Pseudomonas aeruginosa was isolated from diesel
Ligia Rodrigues, Ana Moldes, Jose Teixeira, Rosario
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Biochemical Engineering Journal. 28:109-116.
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286 Journal of Research in Biology (2012) 2(4): 281-286

 Journal of Research in Biology
Studying physical and emotional aspects of self-care among patients with
cancer referred to the chemotherapy ward of Shiraz Namazi Hospital.
Journal of Research in Biology
Authors:
Fatemeh Vizeshfar,
Mahboobe Magharei.
Institution:
Faculty members of Shiraz
University of Medical
Sciences, Hazrat-e-Fatemeh
Nursing and Midwifery
College, Shiraz-Iran.
Corresponding author:
Fatemeh Vizeshfar.
Email:
VizeshfarF@sums.ac.ir.
Phone No:
0098711-6474250
Fax:
00987116474252
Web Address:
http://jresearchbiology.com/
documents/RA0186.pdf.
Journal of Research in Biology
An International
Scientific Research Journal
An International Scientific Research Journal
Original Research
ABSTRACT:
Introduction: Being diagnosed with cancer is a painful experience for affected persons
as well as their relatives. Both disease and it's complication create a variety of
symptoms that influence the way of self-care and facing patients with this matter in
their prognosis.
Objective: This research has been carried out with the aim to study the way of self-
care and knowledge of patients under Chemotherapy regarding the physical and
emotional aspects of self-care.
Materials and Methods: This is a descriptive-analytical study and research population
was consisting of patients under Chemotherapy referred to the Namazi Hospital
among which, 134 persons were selected randomly.
Results: Results indicated that, 101 cases (75.4%) were women with mean age of 46.8
years. Most of samples (64 persons, 47.8%) were illiterate. 113cases (84.3%) were
married. Majority of samples (64 cases, 47.8%) were diagnosed with cancer and 72.4%
of them used multi-drug treatment. 52.2% of samples had little knowledge about
chemotherapy complication and 53.0% of them had no specific source of obtaining
information. There was a statistically significant relation between physical care score
and level of education P<0.0004).
Discussion: According to the results of this research, educating cancer patients about
self-care is very important and nurses should have more emphasis on teaching
programs in order to promote quality of life (QOL) and knowledge of chemotherapy
patients.
Article Citation:
Fatemeh Vizeshfar, Mahboobe Magharei.
Studying physical and emotional aspects of self-care among patients with cancer
referred to the Chemotherapy ward of Shiraz Namazi Hospital.
Journal of Research in Biology (2012) 2(4): 287-295
Dates:
Received: 10 Jan 2012 Accepted: 18 Jan 2012 Published: 23 May 2012
This article is governed by the Creative Commons Attribution License (http://creativecommons.org/
licenses/by/2.0), which gives permission for unrestricted use, non-commercial, distribution and
reproduction in all medium, provided the original work is properly cited.
287-295 | JRB | 2012 | Vol 2 | No 4
www.jresearchbiology.com

INTRODUCTION AND OBJECTIVE:
Cancer is a potential risk factor for mortality and
creates important difficulties in different personal,
familial and social aspects of patient's life Heidary,
(2009). Patients with cancer experience various
behavioral alterations including depression, weakness,
sleep disturbance and cognitive dysfunction. These
behavioral Co-morbidities become apparent throughout
the process of diagnosis and treatment of cancer and can
remain as such during individual's life time Guadagnolo,
(2009). Cancer is the third main cause of death in Iran.
The standardized prevalence rate is 98-110 per 100000
among both females as well as males. The male to
female standard ratio is 1:12. The most prevalent type of
cancer among women and men is Breast cancer and
Stomach cancer respectively. The mortality rate due to
cancer is estimated to be 41.1-65 per 100000 for both
sexes Ingenta contennect cancer incidence and mortality
in Iron (2009).
According to the data issued by Ministry of
Health, cancer is the most common cause of death in Iran
after Cardio-Vascular diseases and accidents MH, Somi
et al., (2009). In the US alone, it is estimated that 2008 a
total of 565,650 patients will have died from cancer,
whereas 1,437,180 will have been diagnosed. Thus,
despite undeniable advancements in early diagnostics
and progress in reducing morbidity through therapeutic
efforts (Deisboeck, 2009).chemotherapeutics are the
most effective treatment for metastatic tumors
Gottesman et al., (2002)
Patients with cancer have many problems in
maintaining the style and quality of their life and are
confronting with psychological complication like fearing
and physical complication like pain and weakness.
Therapeutic side-effects and treatment failure cause
many difficulties and consequently death of the patients.
Most of patients can adapt themselves with disease based
on social, cognitive and emotional resources depending
upon their situation before illness, social and economical
288
Vizeshfar and Magharei, 2012
security and familial relation Html patient empowerment
through supportive care (2009). Promoting self-care is an
attitude that should be created and supported from early
part of life. Health behaviors such as Breast Self
Examination (BSE) can help empowering women in
order to take responsibility and control over their health
promotion Karayurt et al., (2008). Cancer diagnosis
often creates a crisis because, person is facing with
various complication in relation with disease and it's
treatment such as death, the circumstance of continuity
with disease and uncertain future. Cancer and it's
treatment causes morbidities and undermines the quality
of life among survivors Mantazeri et al., (2009).
Many studies have been carried out in relation
with the effects of physician-patient communication on
self-care behaviors. Self care program with the aim to
promote knowledge and skill of patient for managing the
facts of facing with disease and self-care in normal living
environment includes important skills in relation with
disease, treatment and prevention of complication.
Recognition of disease symptoms, medication
use, management of physical and emotional stresses, self
-monitory activities, exercise, diet, smoking
abandonment, non consumption of alcohol and social
and familial support are some of the examples of self-
care. These programs will prepare and empower patients
to care their health. Patients should undertake the basic
role and main responsibility of caring their own health
Arar et al., (2006).
MATERIALS AND METHODS:
This sectional and descriptive-analytical study
has been carried out with the aim to survey physical and
emotional aspects of self-care among patients with
cancer under chemotherapy in Shiraz Namazi Hospital
during 2007-2008. Research population was consisting
of patients with different types of cancer who were under
chemotherapy among which 134 patients were selected
randomly. Data were collected using a questionnaire
Journal of Research in Biology (2012) 2(4): 287-295
Vizeshfar and Magharei, 2012

consisting of two parts. First part included some
questions regarding demographic information of samples
and second part contained questions about the
importance of periodic examinations to determine the
complication of regime therapy drugs doing such
examinations, and also knowledge of patients about
prevention of side effects, emotional problems resulted
from chemotherapy and management methods of such
side effects. According to the patient responses to the
questions of second part of the questionnaire, its grading
and score varied between zero to 20. Collected data were
analyzed using SPSS software, descriptive (mean,
frequency) ANOVA and t-test were found out.
RESULTS:
According to the results of this research, patients
were aged between 16 to 79 with a mean of 46.8 years.
Majority of the samples (101) were women (75.4%) and
only 33(24.6%) of them were men. Concerning
education, majority of the samples (64 cases, 47.8%)
were illiterate, 34 cases (25.4%) had high school
education, 25 cases (18.7%) studied up to the level of
primary and guidance and just 11 cases (7.0%) had
degree of diploma or more one hundred thirteen (84.3%)
of samples were married, 16 cases (11.9%) unmarried
and 5 cases (3.7%) were widow widower or divorced.
Eighty seven (64.9%) of samples were housewives, 23
cases (17.2%) had free job, 17(12.7%) were clerks, four
cases (3.0%) were unemployed and three cases (2.2%)
were students. Breast cancer with sixty four cases
(47.8%) were the most cancer diagnosed followed by
Colon and Rectum cancer with twenty eight cases
(20.9%), Lung cancer with 20 cases (14.9%), Lymphoma
and Leukemia with seven cases (5.2%), Uterine and
Ovary cancer with six cases (4.5%), Bone cancer with
five cases (3.7%) and Brain cancer with four cases
(3.0%). Tables 1 to 3 show the frequency distribution of
samples according to demographic characteristic and
diagnosis type.
The number of referrals for chemotherapy varied
from first with twenty persons (15.0%) to 22nd reference
with one case (0.8%). The maximum referring times
belonged to fourth time with 23 cases (17.3%) of
referrals. Ninethy seven persons (72.4%) positioned
under multi-drug regime-therapy and 37 cases (27.6%)
were under uni-drug regime-therapy.
In response to the question that whether they
have done periodic examinations (blood, urine, stool,
etc.) for the diagnosis of chemotherapy side effects, one
hundred ten cases (82.1%) gave positive, 21 cases
(15.7%) gave negative and three cases (2.2%) had no
answer for this question. But, among those who had

Table 1: Frequency distribution of subjects' responses to the questions on physical aspects of self-care
Frequency (%) percent
Questions correct wrong correct wrong
answer answer answer answer
Do you consider the instructions or tips related to nutrition,
105 29 78.9 21.6
in order to prevent chemotherapy complications?
How do you prevent the constipation? 44 90 32.8 67.2
What is your procedure to care for your mouth and teeth? 31 103 20.4 79.6
Which one of procedures do you follow to prevent urinary
29 106 20.9 79.1
complications?
What are the local symptoms to be reported in case of
33 91 32.1 67.9
intravenous injection?
Is it necessary to avoid pregnancy during the treatment? 46 89 33.6 66.4
What are the symptoms of medication sensitivity? 28 106 20.9 79.1
What are the proceedings necessary to keep the skin healthy
13 121 9.4 90.3
during chemotherapy?
What do you do for preventing alopecia? 24 110 17.9 82.1

Journal of Research in Biology (2012) 2(4): 287-295 289

 Vizeshfar and Magharei, 2012

Table 2: Frequency distribution of subjects' Table3: Frequent distribution of subject kind of cancer
responses to the questions on mental manifestations Kind of cancer No. of Cases Percentage
experienced during chemotherapy
Breast cancer 64 47.8
frequency (%) percent Colon and rectomic 28 20.9
questions
Yes No Yes No Brain.c 4 3
Do you feel constant
53 81 39.6 60.4 Lymphoma&
stiffness of your body? 7 5.2
leukemia
Do you experience
99 35 73.9 26.1 Bane. C 5 3.7
disturbing mind?
Are you often irritable? 88 46 65.7 34.3 Uterus and ovaryc 6 4.5
Are you able to control Lung . c 20 14.9
39 95 29.1 70.9 Total 134 100
your disturbance?

given positive answer, 87 persons (64.9%) did these
examinations as to obey physician prescription and were
not aware of importance and reason of doing such
examinations.
In response to the question about "Which
symptoms do you consider to be important in your
physical condition that, inform your physician"?, seventy
cases (52.2%) mentioned one or several physical
symptoms such as fever, ecthyma, sore throat, colour
change of stool and other symptoms related to
chemotherapy drugs. One hundred twelve samples
(83.6%) considered fatigue to be the most important side
effect of chemotherapy and 75 cases (56.0%) mentioned
resting as an effective factor to avoid such symptom.
In response to the question on what points should
be observed to reduce side effects of chemotherapy
related to the nutrition, one hundred five persons (78.4%)
had selected correct answers such as avoidance from
eating so spicy foods, reducing food volume and
increasing times of eating, avoiding raw meat and
vegetable and using anti-emesis drugs.
Finally in response to the question on "What to
do to prevent constipation"? ninethy cases (67.2%) had
no correct information or had incomplete information.
One hundred three persons (79.6%) had no
knowledge about necessary cares of mouth and teeth and
106 cases (79.1%) were unaware of preventing side
effects of chemotherapy. Eighty nine cases (66.4%) did
not know that, they should prevent pregnancy during
chemotherapy and 132 (98.5%) of them did not know
290
about the duration of pregnancy prevention. Ninety one
persons (67.9%) had no knowledge that, the intravenous
injection site should be controlled and what symptoms in
this relation should be reported. One hundred six persons
(79.1%) did not recognize the symptoms of drug
sensitivity and 121 cases (90.3%) did not know what to
do for caring their skin during chemotherapy.
About the Alopecia complication, Twenty
persons (14.9%) mentioned their reaction, as to wear a
hat or using a wig, 35 cases (26.1%) got anxious, 55
cases (41.0%) avoided looking at the mirror and finally
24 persons (17.9%) exposed themselves to others sight.
One hundred ten persons (82.1%) did not know what to
do for caring themselves about such complication.
Majority of samples (71 cases, 53.0%) did not
have specific source to obtain information regarding self-
care and 50 of them (37.3%) had mentioned physician as
their information source. The total scores of self-care
about physical symptoms varied from minimum of 3 to
maximum of 16 with the mean of 8.8 cases.
Concerning the psychological aspects of self-
care, the following results were obtained; in response to
the question whether they feel that, their body is
continuously hard, 50 cases (37.3%) gave positive
answer, 81 cases (60.4%) gave negative answer and three
of them (2.2%) have not given any answer. Ninety nine
samples (73.9%) experienced disturbing thoughts, 88
cases (65.7%) felt that, they are irritable and only 39
persons (29.1%) were able to control their anxiety.
Journal of Research in Biology (2012) 2(4): 287-295
Vizeshfar and Magharei, 2012
In response to the question that, what do you do
when you are anxious and worried, 109 persons (81.3%)
were not aware of appropriate reactions such as making
themselves busy, expressing their feelings and other
suitable stress controlling methods.
Concerning their relationship with others, 109
cases (81.3%) mentioned seclusion impatience for
making relation with others and unnecessary to have
relation with others. 71 samples (53.0%) were often
angry, 39 cases (29.1%) did not accept that their life is
pleasurable, 58 persons (43.3%) had difficulty in
decision making and 35.8% of them had feeling of
disappointment and worthlessness.
The scores of knowledge about emotional
aspects of self-care in chemotherapy ranged between 6 to
20 with mean of 15 that are higher than scores of
knowledge about physical aspects of self-care in
chemotherapy.
There was significant statistical relation between
obtained scores from physical aspects and education
level (P< 0.004) meaning that, the knowledge about
physical aspects of self-care in chemotherapy increased
with increase in educational level.
Furthermore, there was significant statistical
relation between obtained scores from physical aspects
of self-care in chemotherapy and the number of referrals
for chemotherapy (P<0.001) and the knowledge of
patients had increased with increase in the number of
referrals.
DISCUSSION AND CONCLUSION:
This research was carried out on subjects aged
between 16-79 with mean of 46.8 years and majority of
them were women. In the study done by Montazeri et-al.
(2008) and Sheibani et al., (2009), the mean age of
samples were 43.4 and 55 years respectively (Sheibani
2009). But the age of participants in the study of Sadler
et al., (2007) were above 40 years.
Journal of Research in Biology (2012) 2(4): 287-295
It seems that, the age of cancer incidence in our
country is lower than that of other countries and has been
reduced to the beginning of middle age. This change
could be due to the changes in life style.
The mean age of samples in the research done by
Okibia was 29.13 years Okobia et al., (2006) and in the
research of Montazeri, it was 54.1 years (Mantazeri,
2009).
The majority of samples in this research (47.8%)
were illiterate and married (84.3%). Merchant et al..,
(2007) in their study observed that, most participants
were single and educated Madlin et al., (2008).
Rezaianzadeh (2009), in his study observed that, 18.0%
of samples were illiterate and 82.0% of them were
married Rezaianzadeh et al., (2009). The difference in
the education level and marital status is attributed to
socio-economic differences among various societies.
Breast cancer ranking 47.8% was the most
prevalent among research samples followed by Colon
and Lung cancer. Somidet et al., (2009) in their study
showed that, the 5 most common cancers in women are
Stomach, Breast, Colorectal, Anus and Bladder cancers
(Somim et al., 2009). In Iran, Lung cancer is one of the
five leading tumors and its incidence rate is increasing
among men and women Hosseini et al., (2009). Breast
cancer is the main and prevailing diagnosis among
cancers in developing countries and is estimated to be
10.0% in developed countries like USA and Europe. The
incidence of Breast cancer in Iran is reported to be 6.7%
Rezaianzadeh et al., (2009). Approximately, 50000 of
new cases of cancer are adding every year to the patients
population in Iran.
Digestive system (Stomach, Esophagus, and
Colon) is the most involved organ ranking 38.0% among
all cancers in women Mohebbi et al., (2008). Screening
possibilities, doing diagnosis tests, medical
examinations, level of knowledge in the society, socio-
economical differences and cultural factors in different
societies are effecting these amounts.
291

In the present research, there was a statistical
significant relation between referral times and
knowledge of patients in relation with physical aspects of
self-care (P<0.001). Times of referring will be resulted in
obtaining information from physician, nurse, books and
other patients and media. Sadler (2007) came into
conclusion that, knowledge about Breast cancer is
associated with the guidelines for screening to women
having health problems and adventured for their Breast
cancer therefore, early diagnosis of this type of cancer is
very important Sadler et al., (2007).
The results of studies in Nigeria indicated that,
the knowledge of women in relation with Breast cancer
is low and only few numbers of participants responded
correctly to the questions regarding the symptoms of
Breast cancer (Cokobia) Okobia et al., (2006).
Motlhews et al., (2006) observed that, 20.0% of
samples positioned at a poor health situation and had lots
of problems in relation with treatments Matthews et al.,
(2006). Other studies showed that, there are knowledge
limitation regarding aspects of therapeutical-biologic of
cancer and also low use of screening services among
study population Mcmullin et al., (2008). Guadagnolo et
al., (2009) observed that, there is little knowledge in
relation with screening tests among Americans
Guadagnolo et al., (2009).
In present research, the majority of samples
(72.4%) followed multi-drug regime therapy. Regime
therapy depends upon the type of diagnosis and
physician opinion that was so variable, but the important
point was the high drug side effects among patients.
Gotlordo (2008), Ladish (2009) and Modlio (2008) in
their studies observed that, majority of patients followed
multi-drug regime-therapy and suffered from side effects
of drugs Gottardo et al., (2008) Lidosh and Stratakis
(2009) Claudio (2007).
The results of present study in connection with
the physical aspects of self-care were an evidence of low
knowledge in relation with doing periodic examinations.
292
Vizeshfar and Magharei, 2012
44.9% had lack of knowledge and chemotherapy side
effects were reported in 47.8% of samples. The score of
knowledge about physical aspects of self-care was
associated with Chemotherapy complication. Digestion,
teeth, mouth, skin, alopecia and urinary complication
were prevalent among patients. There was statistical
significant difference between the score of physical
aspect of self-care and education level (P<0.004),
number of referral times for Chemotherapy (P<0.001)
and marriage status (P<0.001).
The results of study carried out by Rezaian
(2009) indicated that, 18.0% of samples had low
education, 9.0% were single and the rate of 5-years
survival was associated with disease stage and diagnosis
type Rezaianzadeh et al., (2009). Parter et al., (2009) in
their research observed that, patients who placed at a low
level in relation with self-care, reported the highest
physical complication resulted from chemotherapy Parter
et al., (2008). Arar et al., (2006) in their study found that,
76.0% of patients encountered with physical distress and
disease symptoms Arar et al., (2006). Merchant et al.,
(2007) in their research carried on 1100 patients found
that, most of participants were unmarried and
participated women have low knowledge about screening
tests Merchant et al., (2007).
Cancer diagnosis in patients causes disorder in
their useful function, Co morbidity and cognitive
disorders Somim et al., (2009). In the present research,
the score of knowledge about emotional aspects of self-
care were between 6 to 20 with the mean of 15 and no
significant statistical relation was observed between the
study variables and these scores. Montazeri (2008)
observed that, the Quality of life (QOL) in patients with
Breast cancer is an important matter that should be
considered carefully because, it creates important
information in relation with therapeutical decisions and a
part of QOL is related with emotional aspects and
feelings of patients about disease, treatments and its
complication Montazeri (2008). According to Yang
Journal of Research in Biology (2012) 2(4): 287-295
Vizeshfar and Magharei, 2012
(2008), cancer diagnosis raises the level of stress and
confronting method of this stress is effective on QOL
Html patient empowerment through supportive care
(2009). Patients suffering from cancer often have some
difficulties in their life style that resulted in emotional
complication. According to Rosenbaun, emotional
complication deeply effects the patients somatic
Rosenbaum et al., (2009).
Claudio et al., (2007) by self-evaluation from
patients with cancer concluded that, screening
examination have been so useful from the view point of
patients. Anxiety, fear, lack of knowledge, distress and
lack of self-confidence are among barriers which cause
patients not to receive necessary treatment and drug side
effects can not be prevented Claudio et al., (2007).
Awadalla et al., (2007) observed that, effective methods
to create motive and promotion of QOL of patients with
cancer have led to an increase in their longevity. Married
patients with higher level of education and better
occupation have higher QOL therefore, by emotional and
social support; the quality of life of patients could be
increased.
FINAL CONCLUSION:
Self-care is an important factor in presenting
health cares by health team and this team should teach
different aspects of self-care (physical-emotional
aspects) to patients in order to promote their health and
increase their QOL. Paying attention to the self-care will
cause the screening tests of early diagnosis of cancers
and effective treatments resulting in the increment of
longevity and QOL of patients with cancer. Most of
researches have emphases on the need for cancer
screening programs, public education and knowledge
regarding cancer diagnosis and health education to the
health providers Sadjadi et al., (2009); Goa et al., (2002).
The most important knowledge deficit problem
about early diagnosis of cancer is the confronting
circumstance with disease and its therapeutical side
Journal of Research in Biology (2012) 2(4): 287-295
effects Karayurt et al., (2008); Mantazeri et al., (2009);
Okobia et al., (2006); Merchant et al., (2007); Mcmullin
et al., (2008).
ACKNOWLEDGEMENT:
This project has been carried out with financial
support of researching assistant of Shiraz University of
Medical Sciences. Thereby, the project authors sincerely
acknowledge university researching assistant and
research center of clinical sciences of Namazi Hospital
(Mr. Zare and Mrs. Golami) for their cooperation.
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 Journal of Research in Biology
Efficient callogenesis and Shoot organogenesis from nodal explants of
Rosa hybrida L. (Lovely girl)
Journal of Research in Biology
Authors:
Nighat Seema1,
Kiran Yasmin Khan1,
Mohammad Zakaria2,
Hina Fazal3.
Institution:
1. Department of Plant
Sciences, Quaid-i-Azam
University, Islamabad
Pakistan.
2. Post Graduate College,
Mardan. Pakistan.
3. Pakistan Council of
Scientific and Industrial
Research (PCSIR)
Peshawar complex, Pakistan.
Corresponding author:
Nighat Seema.
Email:
seemazakaria@gmail.com.
Web Address:
http://jresearchbiology.com/
documents/RA0219.pdf.
Journal of Research in biology
An International Scientific
Research Journal
An International Scientific Research Journal
Original Research
ABSTRACT:
The callogenic and shoot organogenic potential of ornamental plant, Rosa
hybrida were investigated. Callus induction and shoot regeneration were induced
from nodal explants of garden plants incubated on Murashige and Skoog (MS)-
medium supplemented with different concentrations of Plant Growth Regulators
(PGRs). The best callus induction (80-85%) was observed on explants incubated on MS
-medium supplemented with 2.0 mg l-1 6-benzyladenine (BA) and 1.5 mg l-1 BA along
with 0.5 mg l-1 -Naphthaleneacetic acid (NAA) or 0.5 mg l-1 2,4 dichlorophenoxyacetic
acid (2,4 D) after four weeks of culture. When MS-medium supplemented with 1.5 mg
l-1 BA alone induced 90% shoot organogenesis after 30-days following culture. 2.0 mg l-
1
BA in combination with 1.0 mg l-1 Kin (Kinetin) also induced 90 % shooting. Moreover,
when shoots were transferred to an elongation medium, the longest shoots (3.9 cm)
were observed with similar composition of PGRs. The regenerated shoots were
inoculated on rooting medium supplemented with different concentrations of Indole
Butyric Acid (IBA), NAA and Indole Acetic acid (IAA) but no rooting was observed.
Keywords:
Rosa hybrida; Callogenesis, shoot organogenesis, 6-Benzyladenine.
Article Citation:
Nighat Seema, Kiran Yasmin Khan, Mohammad Zakaria, Hina Fazal.
Efficient callogenesis and Shoot organogenesis from nodal explants of
Rosa hybrida L. (Lovely girl).
Journal of Research in Biology (2012) 2(4): 296-302
Dates:
Received: 16 Mar 2012 Accepted: 03 Apr 2012 Published: 23 May 2012
This article is governed by the Creative Commons Attribution License (http://creativecommons.org/
licenses/by/2.0), which gives permission for unrestricted use, non-commercial, distribution and
reproduction in all medium, provided the original work is properly cited.
296-302 | JRB | 2012 | Vol 2 | No 4
www.jresearchbiology.com

INTRODUCTION
Rosa hybrida L. (Lovely girl) belongs to the
family Rosaceae and is one of the popular ornamental
plants worldwide. Rosa hybrida (R. hybrida) is also
called queen of flowers due to its beauty and fragrance
(Shabbir et al., 2009). More than 20, 000 cultivars were
produced from eight wild species in the genus Rosa. The
cut flowers of R. hybrida are used in social events,
religious rituals, in medicines and also used for the
production of important secondary metabolites Vitamins
C and essential oils (Kim et al., 2003; Shabbir et al.,
2009).
According to the literature cited, R. hybrida is
easily propagated through cutting, grafting and layering
asexually but do not give true-to-type plants. Other
limiting factors in the conventional propagation of R.
hybrida are slow multiplication rate and susceptibility to
bacterial and fungal diseases. Furthermore, germplasm
conservation in seed bank is not pragmatic due to
heterozygous nature induced through cuttings (Nair and
Gupta, 2006; Khosravi et al., 2007; Razavizadeh and
Ehsanpour, 2008). In order to facilitate multiplication
rate and to get disease free plant, an efficient protocol is
needed for in vitro regeneration of R. hybrida. In vitro
regeneration is a potential source for mass propagation of
ornamental and medicinally important plants (Ahmad et
al., 2010; Abbasi et al., 2010). Razavizadeh and
Ehsanpour (2008) reported that in vitro regeneration
enhance multiplication rate, disease free plants, non
seasonal production and germplasm conservation in R.
hybrida.
The overall objective of current research was to
develop an efficient regeneration protocol for R. hybrida
from nodal explants to produce genetically pure, healthy
and vigorous plants in relatively short span of time.
MATERIALS AND METHODS
Nodal explants were collected from 60-days-old
garden plant of R. hybrida. Before use, the explants were
297
Seema et al., 2012
given a quick rinse in 70% ethanol for 2 min, then
immersion in 0.1% mercuric hypochloride for 1 min,
followed by three rinses with sterile-distilled water. The
aseptic explants were cut and cultured on MS basal
medium (Murashige and Skoog, 1962) supplemented
with/without BAP, 2, 4-D, NAA, Kinetin or IBA,
separately or in combination. The phases of callus
induction, shooting, shoot elongation and shoot
multiplication were sub-cultured/carried out in similar
medium. Data on response of explants, number of shoots
per explants were collected after day-15 and 40,
respectively, and regenerated shoots were excised and
sub-cultured on similar medium after day-40. All
cultures were incubated in controlled environmental
conditions with a 16-h photoperiod under cool
-2 -1
fluorescent white light (~50molm s ). The design of
all experiments was a complete randomized block, and
each experiment consisted of 2-3 explants per flask and
eight replicate culture flasks per plant growth regulator
treatment.
RESULTS AND DISCUSSION
Callogenesis is considered as a significant
feature of indirect organogenesis and for research on
biologically active molecules in ornamental and
medicinal species (Abbasi et al., 2010). Investigations of
in vitro regeneration were accomplished with callus
induction, maintenance of calli, shoot organogenesis and
shoot multiplication. The effects of various PGRs such as
BA, 2, 4-D and NAA alone or BA in combination with 1
mg l-1 2, 4-D or 1 mg l-1 NAA on indirect organogenesis
were evaluated (Figure 1A-1I). Nodal explants of R.
hybrida used in present study responded to all PGRs
used (Figure 3). Best callus induction was recorded on
MS medium supplemented with 2.0 mg l-1 BA (85%) and
1.5 mg l-1 BA with 0.5 mg l-1 2, 4-D (80%; Figure 1A-
1I). Callus induction recorded for 2, 4-D (25%) and
NAA (30%) in combination with BA was significantly
lower than other PGRs, and no callus was observed on
Journal of Research in Biology (2012) 2(4): 296-302
Seema et al., 2012

A B C

D E F

G H I
Fig. 1. Efficient Callogenesis in R. hybrida L. (A-I) Callus formation after day-15 of culture on
MS medium incorporated with different concentrations of BA in combination with 2,4-D and
NAA. Best callogenic response was recorded on 2.0 mg L-1 of BA along with 0.5 mg L-1 of NAA.

MS0 medium. However, addition of 2, 4-D (0.5 to 1.0
mg l-1 ) or NAA (0.5 to 1.0 mg l-1) to the medium
-1
containing BA (0.5 to 2.0 mg l ) enhanced callus
-1
induction (70-85%) to comparative levels of 3.0 mg l
BA and 0.5 mg l-1 BA with 0.5 mg l-1 2, 4-D or 0.1 NAA
-1
mg l (Figure 3). Abbasi et al., (2010) reported in recent
study that addition of NAA in medium containing BA/
GA3 enhanced callus induction during in vitro
regeneration of Silybum marianum. From the current
experiment it was observed that the medium containing
Cytokinins in combination with auxins enhanced callus
induction. A similar report was also observed by Ahmad
et al., (2010) that different concentration of BA promote
callogenesis in important medicinal plant Piper nigrum
L. There was distinct difference in appearance of callus
medium supplemented with different phytohormones.
Auxin and Cytokinins have chief effects on callus
induction and regeneration, varying their concentration
in the medium, cause differences in amount, rate and
growth pattern of explants.
Data on shoot regeneration were recorded after 4
-5 weeks of subculture (Figure 4). The highest shooting
(90%) was recorded for leaf explants cultured on
medium containing 1.5 mg l-1 BA alone and 2.0 mg l-1
BA along with 1.0 mg l-1 of Kinetin. Moderate
concentrations of BA have shown highest shooting

Journal of Research in Biology (2012) 2(4): 296-302 298

 Seema et al., 2012

A B C

D E F

G H I

Fig. 2. Direct and indirect shoot organogenesis in R. hybrida L. (A-D) Callus subculture on medium containing
different concentrations of BA alone or either in combination with Kin and SA. Best response of indirect
shoot organogenesis was recorded after subculture on medium containing 2.0 mg L -1 of BA. (E-I) Direct shoot
organogenesis from nodal explants was recorded on 2.0 mg L -1 of BA in combination with 1.0 mg L-1 of NAA.

response, however higher concentration has shown
inhibitory action. In the current experiment it was also
observed that the medium containing BA and Kinetin
along with SA (Adenine Sulphate) inhibit shooting. For
-1
the combination containing 0.5 mg l of BA and Kinetin
along with 0.5 mg l-1I SA induced maximum 30%
shooting in nodal explants. Also the addition of 2, 4-D
and SA in medium already containing BA significantly
inhibited % shoot induction. These findings were similar
to the observations of Kanchanapoom et al., (2010).
Nonetheless, BA was more effective than other PGRs
299
used in current report. However, Razavizadeh and
Ehsanpour (2008) find 70% shooting was induced in the
medium containing combination of TDZ and BA.
Shabbir et al., (2009) concluded from his work that
combination of BA and Kin is effective in in vitro shoot
formation from apical meristem of R. hybrida. Best
shooting of 85% has been produced when the medium
was supplemented with 3 mg l-1 of BA. The findings of
Tang et al., (2010) was also in agreement with our data
that combination of BA and NAA induced 97%
regeneration in Lilium leucanthum.
Journal of Research in Biology (2012) 2(4): 296-302
Seema et al., 2012

0 10 20 30 40 50 60 70 80 90 Kinetin produced three shoots/ explant. Furthermore 8.4

3.0 BA
3.0+BA0.5+ NAA
0.5 NAA bc
2.0 BA+ 0.5+NAA
2.0 BA 0.5 NAA a shoots/ culture were obtained on medium containing 2.0
1.0 BA +1.0 NAA
1.0 BA + 1.0 NAA ab

Plant Growth Regulators (mg/l)

1.0 BA + 0.5 NAA
1.0 BA + 0.5 NAA b mg l-1 BA and 0.5 mg l-1 Kinetin in rose plant (Shabbir et
0.5 BA
0.5+BA0.2+ NAA
0.2 NAA c
0.5 BA
0.5+BA0.1+ NAA
0.1 NAA cd al., 2009). The observation of Saglam (2010) are similar
1.0 BA
1.0+BA1.0+ 1.0
2,4-D
2,4-D a
0.5 BA
0.5+BA1.0+ 1.0
2,4-D
2,4-D a to the current study that combination of BA and NAA
3.0 BA
3.0+BA0.5+ 0.5
2,4-D
2,4-D cd
2.5 BA
2.5+BA0.5+ 0.5
2,4-D
2,4-D ab produced 6.79 cm shoot in Onobrychis sativa LAM.
2.0 BA
2.0+BA0.5+ 0.5
2,4-D
2,4-D ab
1.5 BA
1.5+BA0.5+ 0.5
2,4-D
2,4-D a Combination of TDZ (0.05 mg l-1), Kinetin (0.2 mg l-1)
1.0 BA
1.0+BA0.5+ 0.5
2,4-D
2,4-D ab
0.5 BA
0.5+BA0.5+ 0.5
2,4-D
2,4-D
0 10 20 30 40 50
bc
60 70 80 90
and NAA (0.1 mg l-1) induced six shoot/explant (Ozel
% Callus induction
and Arsalan, 2006). Addition of BA in shoot
Fig. 3. Effects of various concentrations of BA with 2,4
-D and NAA. Best % callus induction response in R. regeneration medium significantly enhanced number of
hybrida L was observed on MS medium containing 2.0 shoots/explant (Murashige and Skoog, 1962) in different
mgl-1 of BA in combination with 0.5 mgl-1 of NAA.
Data were collected after 4 weeks of culture. Values Rosa spp. (Hameed et al., 2006). Previously, the
are means of 3 replicates. Each columns with common presence of 3.0 mg l-1 BA alone produced 2.6 shoots per
letters are not significantly different at P<0.05.
explant (Nak-Udam et al., 2009). Surprisingly, shoot
Highest number (7.6) of shoots/explant was induction was recorded for all of PGRs tested in present
-1 -1
recorded for 2.0 mg l of BA along with 1.0 mg l study (Figure 2). The highest mean shoot length (3.9 cm)
Kinetin and lowest number (1.0) of shoots/explant was was observed in the presence of 2.0 mg l-1 BA in
recorded for 0.5 mg l-1 BA and 0.5 mg l-1 Kinetin and combination with 1.0 mg l-1 Kinetin (Figure 6).
combination of 0.5 mg l-1 of BA and Kin along with 2.0 Significantly similar mean shoot length of 3.7 cm was
-1
mg l of SA (Figure 5). It was observed that addition of recorded for 2.0 mg l-1 BA alone or 2.0 mg l-1 BA in
SA in medium incorporated with either BA or Kin combination with 0.5 mg l-1 Kinetin. However lower
significantly inhibited number of shoots/explant. Similar mean shoot length was observed with the addition of 2.0
findings were previously reported for other plant species mg l-1 SA in medium containing 0.5 mg l-1 of BA and
(Ahmad et al., 2010). Kanchanapoom et al., (2010) Kinetin.
reported that the addition of 13.3 mM BA and 9.3 mM
0 1 2 3 4 5 6 7 8
0 10 20 30 40 50 60 70 80 90 0.5 BA0.5
+ Kin + 2.0 SA
BA+Kin+2.0 SA d
0.5 BA+ Kin+2.0 SASA d 1.0 BA1.0+BA+0.1
0.1 NAA
0.5 BA+Kin+2.0 c
NAA
0.5 0.5
BA+ Kin+1.5 SA
BA+Kin+1.5 SA de 2.0 BA2.0+BA+0.5
0.5 NAA NAA c
0.5BA + Kin+ 1.0SA
0.5 BA+Kin+1.0 SA e 2.0 BA+ 1.0 KinKin
2.0 BA+1.0 bc
Plant Growth Regulators (mg/l)

0.5BA + Kin+ 0.5SA

Plant Growth Regulators (mg/l)
0.5 BA+Kin+0.5 SA de 1.0 BA+ 1.0 KinKin
1.0 BA+1.0 d
2.0 BA+ 1.0 KinKin
2.0 BA+1.0 a 0.5 BA+ 1.0 KinKin
0.5 BA+1.0 d
1.0 BA+ 1.0 Kin
1.0 BA+1.0 Kin c 2.0 BA+ 0.5 KinKin
2.0 BA+0.5 c
0.5 BA+ 1.0 KinKin
0.5 BA+1.0 cd 1.0 BA+0.5
1.0 BA+ 0.5 KinKin d
2.0 BA+ 0.5 KinKin
2.0 BA+0.5 ab 0.5 BA+0.5
0.5 BA+ 0.5 KinKin cd
1.0 BA+ 0.5 KinKin
1.0 BA+0.5 c 2.0 2.0
KinKin cd
0.5 BA+ 0.5 KinKin
0.5 BA+0.5 b 1.5 1.5
KinKin cd
2.0 Kin 1.0 1.0
KinKin d
2.0 Kin ab 0.5 Kin d
1.5 Kin
1.5 Kin b 0.5 Kin
1.0 Kin 3.0 BA
1.0 Kin cd 3.0 BA
2.5 BA c
0.5 Kin
0.5 Kin 2.5 BA
cd 2.0 BA a
1.5 BA
1.5 BA a 2.0 BA
1.5 BA c
1.0 BA
1.0 BA 1.5 BA
1.0 BA cd
c
0.5 BA
0.5 BA 1.0 BA
0.5 BA d
d
0 10 20 30 40 50 60 70 80 90 0.5 BA 0 1 2 3 4 5 6 7 8

% Shooting No. of shoots/explant

Fig. 4. Effects of various concentrations of BA, Kin, Fig. 5. Effects of various concentrations of BA, Kin,
SA, and BA with 1 mg l-1 Kin and BA with 2.0 mgl-1 SA BA with 1 mg l-1 Kin and BA with 0.5 mgl-1of Kin and
on percent shooting in Rosa hybrida L. Data were with 2.0 mgl-1 of SA on number of shoots per explant
collected after 5 weeks of sub-culture to MS media in Rosa hybrida L. Data were collected after 4 weeks
with similar composition of plant growth regulators. of sub-culture to MS media with similar composition
Values are means of 3 replicates. Columns with of plant growth regulators. Values are means of 3
common letters are not significantly different at replicates. Columns with common letters are not
P<0.05. significantly different at P<0.05.

Journal of Research in Biology (2012) 2(4): 296-302 300

 Seema et al., 2012

0.5 BA0.5
+ BA+Kin+2.0
Kin + 2.0 SASA
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 Ahmad N, Fazal H, Abbasi BH, Rashid M,
c
1.0 BA
1.0 +BA+0.1
0.1 NAANAA ab
2.0BA
2.0 +BA+0.5
0.5 NAANAA ab Mahmood T, Fatima N. 2010. Efficient regeneration
2.0 BA+ 1.0 Kin
2.0 BA+1.0 Kin a
1.0BA
1.0 + 1.0 Kin
BA+1.0 Kin c
and antioxidant potential in regenerated-tissues of Piper

Plant Growth Regulators (mg/l)

0.5BA
0.5 + 1.0 Kin
BA+1.0 Kin cd
2.0 +
2.0BA BA+0.5
0.5 KinKin a
1.0 BA+0.5
1.0BA + 0.5 Kin Kin b nigrum L. Plant Cell Tiss. Org. Cult. 102:129-134.
0.5 +
0.5BA BA+0.5
0.5 KinKin b
2.02.0 Kin
Kin bc
1.5 Kin c
1.5 Kin
1.01.0 Kin
Kin cd Hameed N, Shabbir A, Ali A, Bajwa R. 2006. In vitro
0.50.5 Kin
Kin c
3.0 BABA
3.0
2.5 BA
b
a
micropropagation of disease free rose (Rosa
2.5 BA
2.0 BA a
2.0 BA
1.5 BA
1.51.0
BABA
b indicaL.).Mycopath. 4:35-38.
bc
1.00.5
BABA c
0.5 BA 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0
Mean shoot length (cm) Kanchanapoom K, Sakpeth P, Kanchanapoom K.
Fig. 6. Effects of various concentrations of BA, Kin,
2010. In vitro flowering of shoots regenerated from
BA in combination with Kin and SA on mean shoot
length in Rosa hybrida L. Data were collected after 5 cultured nodal explants of Rosa hybrida cv. Heirloom.
weeks of sub-culture to MS media with similar Science Asia. 36:161-164.
composition of plant growth regulators. Values are
means of 3 replicates. Columns with common letters
are not significantly different at P<0.05. Khosravi P, Kermani MJ, Nematzadeh1 GA,
Bihamta MR. 2007. A Protocol for Mass Production of
Regenerated shoots collected from shoot
Rosa hybrida cv. Iceberg Through In Vitro Propagation.
organogenesis medium were transferred to MS medium
Iranian J Biotech., 5:100-104.
supplemented with different concentrations of IBA,
NAA and IAA for root organogenesis but unfortunately Kim CK, Oh JY, Jee SO, Chung JD. 2003. In vitro
no rooting was observed. However, Oo et al., (2008) micropropagation of Rosa hybrida L. J. Plant
-1
reported that 1 mg l of NAA induced 4-5 roots per Biotechnology. 5:115-119.
culture. 1/4 MS-medium without plant growth regulators
Murashige T, Skoog F. 1962. A revised medium for
induced 75% rooting in Rosa hybrid (9,5). While Ozel
rapid growth and biassays with tobacco culture. Physiol.
and Arsalan (2006) reported that strength MS-medium
Plant. 15:473-497.
without PGRs induced 50% rooting.
Nair RR, Gupta SD. 2006. High-frequency plant
CONCLUSION regeneration through cyclic secondary somatic
From the present investigation it was clearly embryogenesis in black pepper (Piper nigrum L.). Plant
demonstrated that the BAP alone or in combination with Cell Rep. 24:699-707.
Kin induced shoot induction and multiplication and BA
Nak-Udam N, Kanchanapoom K, Kanchanapoom K.
along with NAA and 2, 4-D enhance efficient callus
2009. Micropropagation from cultured nodal explants of
formation.
rose (Rosa hybrida L. cv. Perfume Delight)
Songklanakarin J. Sci. Technol., 31:583-586.
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Chaudhary MF, Khan MA. 2010. Shoot regeneration of rose (Hybrid Rosa spp.) by In Vitro culture technique.
and free-radical scavenging activity in Silybum GMSARN. 12-14.
marianum L. Plant Cell Tiss. Org. Cult. 101:371-376.
Ozel CA, Arsalan O. 2006. Efficient micropropagation
of english shrub rose Heritage under in vitro

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 Journal of Research in Biology An International Scientific Research Journal

Original Research

Diversity and habitat preferences of butterflies in Gorumara National Park,

West Bengal, India
Journal of Research in Biology

Authors: ABSTRACT:
Das RP, Saha GK, De JK and
Sanyal AK.
Institution:
1. Entomology and Wildlife
Biology Research Laboratory,
Department of Zoology,
University of Calcutta, 35
Ballygunge Circular Road,
Kolkata-700019, West Bengal,
India.
2. Entomology and Wildlife
Biology Research Laboratory,
Department of Zoology,
University of Calcutta, 35
Ballygunge Circular Road,
Kolkata-700019, West Bengal,
India.
3. Zoological Survey of India,
M-Block, New Alipore, Kolkata
-700053, West Bengal, India.
Gorumara National Park is located towards the foothills of Eastern Himalayas
in northern part in the state of West Bengal. A systematic survey on diversity,
abundance and habitat preferences of butterflies was carried out from October 2009
to January 2011 to explore the butterfly richness of the protected area. An amazing
170 species of butterflies belonging to 109 genera, 21 sub-families, and five families
were recorded from different habitats. Nymphalidae and Lycaenidae are the dominant
families with maximum species count of 54 and 50 respectively; they are followed by
Hesperiidae (33), Pieridae (18) and Papilionidae (15). Among the listed butterflies,
69 species are habitat specific; whereas 101 species are habitat generalists. Species
count reaches the peak during pre-monsoon (April-May) period. Post-monsoon
(October-November) and monsoon (June-September) periods have moderate species
diversity and winter (December to March) period has least diversity. The abundance of
butterflies is related to better availability and access to the larval host plants and
nectar plants. In general forests and grasslands offer a better combination of habitat
to the butterflies.

4. 66, Dum Dum Road, Ahana

Keywords:
Apartment, Flat-1C, Kolkata- Indian butterflies, diversity, habitat preference, Gorumara National Park,
700074, West Bengal, India. West Bengal, Eastern Himalayas.

Corresponding author: Article Citation:

Saha GK.
Das RP, Saha GK De JK and Sanyal AK.
Email: Diversity and habitat preferences of butterflies in Gorumara National Park, West
1. rudraprasaddas@hotmail.com Bengal, India.
2. gkszoo@gmail.com Journal of Research in Biology (2012) 2(4): 303-314
3. jkdezsi@yahoo.com
4. asokzsi@yahoo.co.in Dates:
Phone No: Received: 29 Dec 2011 Accepted: 11 Jan 2012 Published: 24 May 2012
1. +91-98305-17019
2. +91-94331-82500
3. +91-94324-99611
4. +91-94325-99095
This article is governed by the Creative Commons Attribution License (http://creativecommons.org/
Web Address: licenses/by/2.0), which gives permission for unrestricted use, non-commercial, distribution and
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303-314 | JRB | 2012 | Vol 2 | No 4

Journal of Research in Biology
An International
Scientific Research Journal www.jresearchbiology.com
 Das et al., 2012

INTRODUCTION Sal Savannah (Champion & Seth, 2005). 10% of the total
West Bengal is the only state in India consisting
area is grassland which is mainly confined to the river
of high peaks of the Himalayas in the northern extremes,
plains. Also, sizeable area is covered with bamboo
coastal regions down south, and regions such as plateau
brakes. Within the 326 identified plant species, the
and Gangetic delta intervening in between. The total
commonest tree species are Shorea robusta C.F. Gaertn,
recorded forest covered area of the state is 13.38%
1805, Dalbergia sisoo Roxb., Albizia spp.,
(11,879 sq. km km) which is well below the national
Acacia catechu (L.) Willd., Oliv., 1806, Bombax ceiba
average of 23%. The state has a chain of five national
L., 1753, Terminalia myriocarpa van Heurck & Mll.
parks, fifteen wildlife sanctuaries, two tiger reserves, and
Arg., Lagerstroemia parviflora Roxb. etc. The National
one biosphere reserve. Adequate rainfall, nutrient rich
Park is also a home for 48 mammalian species, 193
soil profile, and rich ecological diversity of the state -
species of birds, 29 species of reptiles including seven
support and favour a great diversity of flora and fauna.
species of turtles, 40 species of fishes and other macro
Gorumara National Park is a medium-sized
and micro-fauna (Anonymous, 2007). However, there is
protected area covering only 79.45 sq. km. This park is
a considerable study gap so far as the butterfly fauna is
situated in the Jalpaiguri district of northern West Bengal
concerned. The main objective of the present study was
between 264712.5N to 264325.6 and 88
to conduct a systematic survey on diversity, abundance
524.2E to 88477.3E. The area comes under the bio
and habitat preferences of butterfly communities in
-geographic zone of 7B-Lower Gangetic Plains (Rodgers
Gorumara National Park.
& Panwar, 1988). Three main rivers - Murti, Indong and
Garati flow through the area and become intermingled
MATERIALS AND METHODS
before joining River Jaldhaka which forms the park
The field surveys were carried out in Gorumara
boundary in the eastern side. Also, there are few seasonal
National Park from October 2009 to January 2011
streams in the National Park, which remain dry almost
in-connection with the assigned project entitled
throughout the year except monsoon. The overall climate
Feasibility study regarding re-introduction of Pygmy
of the area is humid (maximum relative humidity
Hog (Porcula salvania Hodgson, 1847) at Gorumara
75-100%), with maximum recorded temperature of 37C
National Park, Jalpaiguri, West Bengal. Three camps
(in summer) and a minimum of 4C (in winter).
namely Bamni (North Range), Garati and Medla (both in
However, a considerable drop of night temperature
South Range) were primarily selected as base camps.
occurs during winter and sometimes winter nights are
These camps are located in the transition zones between
severe. South-west Monsoon is the main source of
high forests and grasslands. This area falls under tropical
rainfall and maximum precipitation occurs between June
monsoon climate with four distinct season, summer or
to September. Average annual rainfall is about 350 cm.
pre-monsoon (April-May), monsoon (June-September), a
The terrain of Gorumara National Park is differentiated
short autumn or post-monsoon (October-November) and
into distinct high plateau and plain area, both runs
winter (December-March). A total of six surveys (pre-
parallel from north to south. The soil profile of the area
monsoon-2, monsoon-1, post-monsoon-2, winter-1) each
is of alluvial and bhabar formations. The recorded four
having 15 days of duration were conducted. Special
major forest types are - Northern Dry Deciduous Seral
emphasize were given on pre-monsoon and post-
Sal Khair Sisoo Association, Eastern Bhabar and Terai
monsoon periods, as because, these are the known
Sal, Sub-Himalayan Secondary Wet-mixed forests and
seasons when abundance of butterflies remain high
304 Journal of Research in Biology (2012) 2(4): 303-314
Das et al., 2012
(Wynter-Blyth, 1957; Kunte, 1997). The forest trails in
different representative habitats were used as fixed
transects and total six such transects, each having 2km of
length were used for this purpose. The entire survey was
conducted on foot for seven hours per day; consisting of
two sessions 0700 h to 1100 h and 1300 h to 1600 h
(Borkar & Komarpant, 2004). Butterflies were sampled
following Pollard Walk technique (Pollard & Yates,
1993). Identification of butterflies was done in the field.
More emphasize were given on direct sighting and or
photographic evidences. Some rare and small butterflies
which are difficult to identify were caught and closely
observed after placing them in clear glass bottle. Then
they were released to the same habitat from where they
were caught. However, enough precautions were taken,
so that, by no means the entire procedure can cause any
damage to the target specimens. Photographs of both
upper and underside of the respective specimen were
also taken for further references (when possible). No live
or dead specimens were collected from the field. The
relative abundance of each species was estimated based
on sighting records in a single day for the entire period of
sampling (Rajasekhar, 1995). Then, butterflies were
broadly categorized into four groups: rare (<1%),
uncommon (1-5%), common (6-30%) and abundant
(>30 %) depending on their relative abundance.
Figure 1: Seasonal variation of butterfly families in
Gorumara National Park, West Bengal, India
(October 2009 to January 2011).
Journal of Research in Biology (2012) 2(4): 303-314
The butterflies were identified using standard
literatures: Watson (1891); Evans (1932); Talbot (1939
& 1947); Wynter-Blyth (1957); Corbet & Pendlebury
(1992); DAbrera (1982, 1986 & 1998); Haribal (1992);
Larsen (2004); Colin Smith (2006) and Kehimkar
(2008). The classification scheme followed here is based
on Ackery (1984).
RESULTS & DISCUSSION
During the entire survey from October 2009 to
January 2011, a total of 170 species of butterflies
belonging to 5 families, 21 sub-families and 109 genera
were recorded from Gorumara National Park (Table 1).
Nymphalidae was the dominant family, with highest
species count (54), followed by Lycaenidae (50),
Hesperiidae (33), Pieridae (18) and Papilionidae (15).
It is apparent from the analysis of the recorded
data that, the availability of butterflies is distinctly
influenced by the respective seasons (Figure 1).
Maximum 122 species of butterflies were recorded in pre
-monsoon (April-May), followed by 89 species in post-
monsoon (October-November) and 75 species monsoon
(June-September). However, a mere 41 species were
recorded in winter (December to March). 18 species
(Papilionidae - 1, Pieridae - 6, Lycaenidae - 4, and
Nymphalidae - 7) namely, Common Rose -
Atrophaneura aristolochiae (Fabricius, 1775), Common
Figure 2: Habitat-wise distribution of butterfly
families in Gorumara National Park, West Bengal
India (October 2009 to January 2011).
305

Grass Yellow - Eurema hecabe (Linnaeus, 1758),
Common Emigrant - Catopsilia pomona (Fabricius,
1775), Mottled Emigrant - Catopsilia pyranthe
(Linnaeus, 1758), Yellow Orange-tip - Ixias pyrene
(Linnaeus, 1764), Red-spot Jezebel - Delias descombesi
(Boisduval, 1836), Psyche - Leptosia nina (Fabricius,
1793), Western Centaur Oakblue - Arhopala
pseudocentaurus (Doubleday, 1847), Common Tit -
Hypolycaena erylus (Godart, [1824]), Purple Sapphire -
Heliophorus epicles (Godart, [1824]), Common Pierrot -
Castalius rosimon (Fabricius, 1775), Common Five-ring
- Ypthima baldus (Fabricius, 1775), Common Sergeant -
Athyma perius (Linnaeus, 1758), Common Sailer -
Neptis hylas (Linnaeus, 1758), Plain Sailer - Neptis
cartica Moore, 1872, Chocolate Pansy - Junonia iphita
(Cramer, [1779]), Grey Pansy - Junonia atlites
(Linnaeus, 1763), and Lemon Pansy - Junonia lemonias
(Linnaeus, 1758) were recorded in all four seasons
during which the surveys were conducted (Table 1).
Overall species diversity in-connection with seasonal
variation is more pronounced in papilionids and
nymphalids; they are followed by lycaenids, pierids and
hesperiids (Figure 1).
Further, the population dynamics of individual
species vary in relation to habitat (Figure 2). More than
90% (154 species) are found in forested areas, whereas,
67% (114 species) and 8% (14 species) of the total
butterfly fauna were encountered from grassland and
bamboo habitats respectively (Table 1). Among the listed
butterflies, 69 species are habitat specific and 101
species are habitat generalists. Of the total habitat
specific butterflies, 53 are forest dwellers, 15 are
grassland species and a single species is restricted to
bamboo patches only. However, 11 species of the habitat
generalists butterflies (Lycaenidae -1, Nymphalidae -8,
and Hesperiidae -2) are found in all three habitats -
forest, grassland and bamboo. These are Common Tit -
Hypolycaena erylus (Godart, [1824]), Common Evening
Brown - Melanitis leda (Linnaeus, 1758), Common
306
Das et al., 2012
Palmfly - Elymnias hypermnestra (Linnaeus, 1763),
Common Bushbrown - Mycalesis perseus (Fabricius,
1775), Dark-brand Bushbrown - Mycalesis mineus
(Linnaeus, 1758), Common Five-ring - Ypthima baldus
(Fa br i ci us, 1775), Comm on Four -r ing -
Ypthima huebneri Kirby, 1871, Lemon Pansy - Junonia
lemonias (Linnaeus, 1758), Great Eggfly -
Hypolimnas bolina (Linnaeus, 1758), Common Red-eye
- Matapa aria (Moore, [1866]), and Chestnut Bob -
Iambrix salsala (Moore, [1866]) (Table 1). Analysis of
the data also revealed that altogether forests and
grasslands offer a better combination of habitat to the
butterflies; as many as 88 species are common to these
habitats. This may be due to better availability and
access to the larval host plants and nectar plants in these
areas. However, both the combination of forest-bamboo
and grassland-bamboo habitats possess very poor, almost
nil (2 and 0 respectively) diversity (Figure 2).
Common Lantana - Lantana camara L., 1753,
Common Floss Flower - Chromolaena odorata (L.) R.M.
King & H. Rob., 1970, and Climbing Hempweed -
Mikania micrantha H.B.K., 1820 although considered as
invasive alien plant species, are the major source of
nectar for butterflies in both forest and grassland habitats
in the National Park (Kehimkar, 2000). Butterflies were
also seen nectaring regularly on two native Indian plants,
Hill Clerodendrum - Clerodendrum viscosum Vent. and
Indian Turnsole - Heliotropium indicum L., 1753. During
pre-monsoon season some butterflies (especially
papilionids, sometimes representatives of other families
also) were gathered in large numbers at selected sites like
sandy river-banks, damp soil patches etc. for mud-
puddling. This behaviour mostly performed by the male
butterflies, to get some important nutrients (such as
sodium, calcium, phosphate etc.) which are required for
spermatophore formation (Krushnamegh, 2002;
Smetacek, 2002). However, butterflies (nymphalids and
lycaenids) were also found sitting on over-ripe fruits
(rich in alcohol), bird-droppings, fresh elephant dung,
Journal of Research in Biology (2012) 2(4): 303-313
Das et al., 2012

dead and decaying animals, faeces of carnivores etc. Society, Kuala Lumpur. 595.

DAbrera B. 1982. Butterflies of the Oriental Region,

CONCLUSION
Part I: Papilionidae, Pieridae & Danaidae. Hill House,
Gorumara National Park is not a big area, but, its
Victoria, Australia. 288.
geographic position, good floral diversity, different
habitats, adequate source of water and unique climate DAbrera B. 1985. Butterflies of the Oriental Region,
boast a rich diversity of butterfly communities. The Part II: Nymphalidae, Satyridae & Amathusiidae. Hill
present study will be helpful for further long-term House, Victoria, Australia. 296.
monitoring of the butterfly fauna and for planning
DAbrera B. 1986. Butterflies of the Oriental Region,
conservation initiatives in Gorumara National Park.
Part III: Lycaenidae & Riodinidae. Hill House, Victoria,
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ACKNOWLEDGEMENTS
Authors are thankful to Forest Directorate Evans WH. 1932. The Identification of Indian
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Smetacek P. 2002. The Study of Butterflies: 5 -

Congregations, Courtship and Migration. Resonance 7:6-
14.

Smith C. 2006. Illustrated Checklist of Nepals

Butterflies. Walden Book House, Kathmandu, Nepal.
129.

Talbot G. 1939. The Fauna of British India, including

Ceylon and Burma: Butterflies, Vol. I. Taylor and
Francis, London. 600.

Talbot G. 1947. The Fauna of British India, including

Ceylon and Burma: Butterflies, Vol. II. Taylor and
Francis, London. 506.

Watson EY. 1891. Hesperiidae Indicae: being a reprint

of Descriptions of the Hesperiidae of India, Burma and
Ceylon. Vest and Company, Mount Road. Madras. 161.

Wynter-Blyth MA. 1957. Butterflies of the Indian

Region. Bombay Natural History Society, Mumbai. 523.

Table Continued
308 Journal of Research in Biology (2012) 2(4): 303-313
 Table 1: Systematic list of butterflies recorded from Gorumara National Park, West Bengal, India (from October 2009 to January 2011) [A - abundant,
C - common, M - monsoon, PoM - post-monsoon, PrM - pre-monsoon, R - rare, U - uncommon, W - winter]
Habitat Relative
Species Common Name Distribution (season-wise)
Forest Grassland Bamboo Abundance
A. Super-family: PAPILIONOIDEA
Das et al., 2012

I. Family: PAPILIONIDAE
a. Sub-family: PAPILIONINAE
1 Graphium sarpedon (Linnaeus, 1758) Common Bluebottle + + - PrM, M, PoM C
2 Graphium doson (C. & R. Felder, 1864) Common Jay + + - PrM, M, C
3 Graphium agamemnon (Linnaeus, 1758) Tailed Jay + + - PrM, M, C
4 Graphium antiphates (Cramer, [1775]) Five-bar Swordtail + - - PrM, M, C
5 Papilio clytia Linnaeus, 1758 Common Mime + + - PrM, M, C
6 Papilio polytes Linnaeus, 1758 Common Mormon + + - PrM, M, PoM A
7 Papilio helenus Linnaeus, 1758 Red Helen + - - PrM, PoM C
8 Papilio nephelus Boisduval, [1836] Yellow Helen + - - PrM, M, C
9 Papilio memnon Linnaeus, 1758 Great Mormon + - - PrM, M, C
10 Papilio protenor Cramer, [1775] Spangle + - - PrM, M, C

Journal of Research in Biology (2012) 2(4) : 303-314

11 Papilio demoleus Linnaeus, 1758 Lime Butterfly + + - PrM, PoM C
12 Papilio paris Linnaeus, 1758 Paris Peacock + - - PrM, R
13 Atrophaneura varuna (White, 1842) Common Batwing + - - W R
Atrophaneura aristolochiae (Fabricius,
14 Common Rose + + - PrM,M,PoM,W A
1775)
15 Triodes helena (Linnaeus, 1758) Common Birdwing + - - M U
II. Family: PIERIDAE
a. Sub-family: COLIADINAE
16 Eurema andersoni (Moore, 1886) One-spot Grass Yellow + + - M U
17 Eurema blanda (Boisduval, 1836) Three-spot Grass Yellow + + - PrM, M, PoM C
18 Eurema hecabe (Linnaeus, 1758) Common Grass Yellow + + - PrM,M,PoM,W A
19 Eurema laeta (Boisduval, [1836]) Spotless Grass Yellow + + - R
20 Gandaca harina (Horsfield, [1829]) Tree Yellow + + - PrM U
21 Catopsilia pomona (Fabricius, 1775) Common Emigrant + + - PrM,M,PoM,W A
22 Catopsilia pyranthe (Linnaeus, 1758) Mottled Emigrant + + - PrM,M,PoM,W A
b. Sub-family: PIERINAE
23 Ixias pyrene (Linnaeus, 1764) Yellow Orange-tip + + - PrM,M,PoM,W A
24 Hebomoia glaucippe (Linnaeus, 1758) Great Orange-tip + - - PrM C
25 Appias libythea (Fabricius, 1775) Striped Albatross + + - PrM, PoM, W C
26 Appias lyncida (Cramer, [1777]) Chocolate Albatross + + - PrM, M, PoM C
27 Pieris canidia (Sparrman, 1768) Indian Cabbage White + + - PrM, PoM C
28 Cepora nerissa (Fabricius, 1775) Common Gull + + - PrM, PoM, W C

309
 29 Cepora nadina (Lucas, 1852) Lesser Gull + + - PoM U

310
30 Delias pasithoe (Linnaeus, 1767) Red-base Jezebel + + - PoM, W C
31 Delias descombesi (Boisduval, 1836) Red-spot Jezebel + + - PrM,M,PoM,W C
32 Delias hyparete (Linnaeus, 1758) Painted Jezebel + + - PrM U
33 Leptosia nina (Fabricius, 1793) Psyche + - - PrM,M,PoM,W C
III. Family: LYCAENIDAE
a. Sub-family: PORITIINAE
34 Poritia hewitsoni Moore, [1866] Common Gem + - - W U
b. Sub-family: MILETINAE
35 Spalgis epius (Westwood, [1851]) Apefly + - - PrM U
c. Sub-family: CURETINAE
36 Curetis acuta Moore, 1877 Angled Sunbeam + + - PrM, M, PoM C
d. Sub-family: THECLINAE
Arhopala pseudocentaurus (Doubleday, Western Centaur Oak-
37 + - - PrM,M,PoM,W C
1847) blue
38 Arhopala amantes (Hewitson, 1862) Large Oakblue + - - PrM U
39 Arhopala atrax (Hewitson, 1862) Indian Oakblue + - - W U
40 Arhopala fulla (Hewitson, 1862) Spotless Oakblue + - - PrM U
41 Surendra quercetorum (Moore, [1858]) Common Acacia Blue + - + M, PoM U
42 Loxura atymnus (Stoll, [1780]) Yamfly + - - PrM U
43 Cheritra freja (Fabricius, 1793) Common Imperial + - - PoM U
44 Remelana jangala (Horsfield, [1829]) Chocolate Royal + + - M U
45 Horaga onyx (Moore, [1858]) Common Onyx + - - PoM R
46 Hypolycaena erylus (Godart, [1824]) Common Tit + + + PrM,M,PoM,W A
47 Zeltus amasa (Hewitson, [1865]) Fluffy Tit + - - PrM, M U
48 Chliaria othona (Hewitson, 1865) Orchid Tit + - - PrM, PoM U
49 Sinthusa nasaka (Horsfield, 1829) Narrow Spark + - - PrM R
50 Rapala iarbus (Fabricius, 1787) Indian Red Flash + + - PoM, W C
51 Rapala pheretima (Hewitson) Copper Flash + + - PoM, W C
52 Rapala manea (Hewitson, [1863]) Slate Flash + + - PrM, W C
53 Rapala varuna (Horsfield, [1829]) Indigo Flash + + - PoM U
54 Rapala nissa (Kollar, [1844]) Common Flash + - - W R
55 Catapaecilma elegans Druce, 1873 Common Tinsel + - - PrM, PoM U
56 Spindasis vulcanus (Fabricius, 1775) Common Silverline + + - PoM U
e. Sub-family: LYCAENINAE
Das et al., 2012

Journal of Research in Biology (2012) 2(4): 303-314

57 Heliophorus epicles (Godart, [1824]) Purple Sapphire + + - PrM,M,PoM,W A
 f. Sub-family: POLYOMMATINAE
58 Anthene emolus (Godart, [1824]) Common Ciliate Blue + + - PrM, M, PoM C
59 Anthene lycaenina (R. Felder, 1868) Pointed Ciliate Blue + + - M, PoM C
60 Caleta elna (Hewitson, [1876]) Elbowed Pierrot + + - PrM, M C
61 Castalius rosimon (Fabricius, 1775) Common Pierrot + + - PrM,M,PoM,W A
62 Tarucus nara (Kollar, 1844) Rounded Pierrot + + - PoM U
Das et al., 2012

63 Petrelaea dana (de Nicville, [1884]) Dingy Lineblue + - - PrM, M U

64 Nacaduba kurava (Moore, [1858]) Transparent 6-Lineblue + - - M U
Prosotas aluta coelestis (Wood-Mason & de
65 Banded Lineblue + - - PrM, M C
Nicville, [1887])
66 Prosotas nora (C. Felder, 1860) Common Lineblue + + - PrM, M C
67 Prosotas dubiosa indica (Evans, [1925]) Tailless Lineblue + + - PrM, M C
68 Jamides bochus (Stoll, [1782]) Dark Cerulean + - - PrM, PoM C
69 Jamides celeno (Cramer) Common Cerulean + - - PoM R
70 Jamides alecto (C. Felder, 1860) Metallic Cerulean + + - PrM, PoM C
71 Catochrysops strabo (Fabricius, 1793) Forget-me-not + + - PrM, PoM C
72 Catochrysops panormus (C. Felder, 1860) Silver Forget-me-not + - - PrM R
73 Leptotes plinius (Fabricius, 1793) Zebra Blue - PoM U

Journal of Research in Biology (2012) 2(4) : 303-011

+ +
74 Lampides boeticus (Linnaeus, 1767) Pea Blue + + - PrM, PoM C
75 Zizeeria karsandra (Moore, 1865) Dark Grass Blue + + - PrM, M, C
76 Pseudozizeeria maha (Kollar, [1844]) Pale Grass Blue + + - PrM, PoM C
77 Zizula hylax (Fabricius, 1775) Tiny Grass Blue + + - PoM, U
78 Zizinia otis (Fabricius, 1787) Lesser Grass Blue + + - PrM, PoM C
79 Megisba Malaya (Horsfield, [1828]) Malayan + - - PrM, U
80 Acytolepis puspa (Horsfield, [1828]) Common Hedge Blue + - - PrM, PoM C
81 Chilades lajus (Stoll, [1780]) Lime Blue + + - W U
g. Sub-family: RIODININAE
82 Abisara echerius (Stoll, [1790]) Plum Judy + - - PoM R
83 Zemeros flegyas (Cramer, [1780]) Punchinello + + - PrM, M, W A
IV. Family: NYMPHALIDAE
a. Sub-family: DANAINAE
84 Tirumala limniace (Cramer, [1775]) Blue Tiger + + - PrM, PoM C
85 Tirumala septentrionis (Butler, 1874) Dark Blue Tiger + + - M, PoM U
86 Danaus genutia (Cramer, [1779]) Striped Tiger + + - PrM, PoM C
87 Danaus chrysippus (Linnaeus, 1758) Plain Tiger + + - PrM, M C
88 Parantica aglea (Stoll, [1782]) Glassy Tiger + + - PrM, M, PoM C
89 Parantica sita (Kollar, [1844]) Chestnut Tiger + + - PrM, PoM U
90 Euploea radamanthus (Fabricius, 1793) Magpie Crow + + - PrM, M, C
91 Euploea mulciber (Cramer, [1777]) Striped Blue Crow + + - PrM, M, PoM C

311
92 Euploea klugii Moore, [1858] Brown King Crow + + - M R
 Long-branded Blue
93 Euploea algea (Godart, 1819) - PrM R

312
+ +
Crow
94 Euploea core (Cramer, [1780]) Common Crow + + - PrM, M C
b. Sub-family: CHARAXINAE
95 Charaxes bernardus (Fabricius, 1793) Tawny Rajah + - - M, PoM C
96 Charaxes marmax Westwood, [1847] Yellow Rajah + - - PrM R
c. Sub-family: SATYRINAE
Common Evening
97 Melanitis leda (Linnaeus, 1758) + + + PrM, PoM, W C
Brown
98 Lethe europa (Fabricius, 1775) Bamboo Treebrown - - + PoM U
99 Elymnias hypermnestra (Linnaeus, 1763) Common Palmfly + + + PrM, PoM U
100 Mycalesis anaxias Hewitson, [1862] White-bar Bushbrown + + - PrM U
101 Mycalesis visala Moore, [1858] Long-brand Bushbrown - + - PoM R
102 Mycalesis perseus (Fabricius, 1775) Common Bushbrown + + + PrM, M, W C
103 Mycalesis mineus (Linnaeus, 1758) Dark-brand Bushbrown + + + PrM, M, C
104 Orsotrioena medus (Fabricius, 1775) Nigger + + - PrM U
105 Ypthima baldus (Fabricius, 1775) Common Five-ring + + + PrM,M,PoM,W A
106 Ypthima huebneri Kirby, 1871 Common Four-ring + + + PrM, M, PoM C
107 Ypthima asterope (Klug, 1832) Common Three-ring - + - PrM U
d. Sub-family: HELICONIINAE
108 Cethosia cyane (Drury, 1773) Leopard Lacewing + - - PrM, U
109 Cirrochroa aoris Doubleday, [1847] Large Yeoman + - - PrM, PoM U
110 Phalanta phalantha (Drury, [1773]) Common Leopard + + - PrM, PoM, W C
e. Sub-family: LIMENITINAE
111 Moduza procris (Cramer, [1777]) Commander + + - PoM U
112 Athyma perius (Linnaeus, 1758) Common Sergeant + + - PrM,M,PoM,W C
113 Athyma cama Moore, 1858 Orange Staff Sergeant + + - PoM U
114 Athyma ranga Moore, 1857 Blackvein Sergeant + + - PrM, PoM C
115 Athyma nefte (Cramer, [1780]) Colour Sergeant + + - PrM, M U
116 Pantoporia hordonia (Stoll, [1790]) Common Lascar + + - PrM, PoM C
117 Neptis hylas (Linnaeus, 1758) Common Sailer + + - PrM,M,PoM,W C
118 Neptis ananta Moore, 1858 Yellow Sailer + - - PrM U
119 Neptis cartica Moore, 1872 Plain Sailer + + - PrM,M,PoM,W C
120 Neptis soma Moore, 1858 Sullied Sailer + + - PrM U
121 Tanaecia lepidea (Butler, 1868) Grey Count + + - PrM, M C
122 Tanaecia julii (Lesson, 1837) Common Earl + + - PrM, M C
f. Sub-family: CYRESTINAE
123 Dichorrhagia nesimachus (Doyre, [1840]) Constable - - PrM R
Das et al., 2012

Journal of Research in Biology (2012) 2(4) : 303-314

+
 g. Sub-family: BIBLIDINAE
124 Ariadne ariadne (Linnaeus, 1763) Angled Castor - + - PoM U
125 Ariadne merione (Cramer, [1777]) Common Castor - + - PrM U
h. Sub-family: NYMPHALINAE
126 Symbrenthia hippoclus (Cramer, [1779]) Common Jester + + - PrM, PoM C
127 Vanessa indica (Herbst, 1794) Indian Red Admiral + + - W U
Das et al., 2012

128 Vanessa cardui (Linnaeus, 1758) Painted Lady - + - W U

129 Junonia orithiya (Linnaeus, 1758) Blue Pansy - + - PrM, PoM U
130 Junonia hierta (Fabricius, 1798) Yellow Pansy - + - PrM, PoM C
131 Junonia iphita (Cramer, [1779]) Chocolate Pansy + + - PrM,M,PoM,W A
132 Junonia atlites (Linnaeus, 1763) Grey Pansy + + - PrM,M,PoM,W C
133 Junonia almana (Linnaeus, 1758) Peacock Pansy + + - PrM, PoM, W C
134 Junonia lemonias (Linnaeus, 1758) Lemon Pansy + + + PrM,M,PoM,W A
135 Hypolimnas bolina (Linnaeus, 1758) Great Eggfly + + + PrM, M C
136 Kallima inachus (Boisduval, 1864) Orange Oakleaf + - + PrM, PoM U
137 Doleschallia bisaltide (Cramer, [1777]) Autumn Leaf + - - M U
B. Super-family: HESPERIOIDEA

Journal of Research in Biology (2012) 2(4) : 303-314

V. Family: HESPERIIDAE
a. Sub-family: COELIADINAE
138 Hasora badra (Moore, [1858]) Common Awl + - - PrM, M, W C
139 Badamia exclamationis (Fabricius, 1775) Brown Awl + - - PrM, M, PoM C
Choaspes benjaminii (Gurin-Mneville,
140 Indian Awlking + - - PoM U
1843)
141 Bibasis sena (Moore, [1866]) Orange-tail Awl + - - M R
142 Hasora chromus (Cramer, [1780]) Common Banded Awl + - - M U
b. Sub-family: PYRGINAE
143 Celaenorrhinus leucocera (Kollar, [1844]) Common Spotted Flat + - - PrM, PoM U
144 Spialia galba (Fabricius, 1793) Indian Skipper + + - PrM U
145 Sarangesa dasahara Moore, [1866] Common Small Flat + - - W C
146 Coladenia agni (de Nicville, [1884]) Brown Pied Flat + - - M R
147 Pseudocoladenia dan (Fabricius, 1787) Fulvous Pied Flat + - - PrM, PoM C
Common Yellow-
148 Gerosis bhagava (Moore, [1866]) + - - M R
breasted Flat
149 Tagiades gana (Moore, [1866]) Suffused Snow Flat + - - PoM R
150 Tagiades japetus (Stoll, [1781]) Common Snow Flat + - - M, PoM U
c. Sub-family: HESPERIINAE
151 Oriens goloides (Moore, 1881) Common Dartlet + + - PrM U
152 Telicota colon (Fabricius, 1775) Pale Palm Dart + + - W U
153 Telicota ancilla (Herrich-Schffer, 1869) Dark Palm Dart + + - PrM U

313
154 Cephrenes chrysozona (Pltz, 1883) Plain Palm Dart + + - PrM, M, PoM C
 155 Baoris farri (Moore, 1878) Paintbrush Swift - + - PrM, PoM U

314
156 Parnara guttatus (Bremer & Grey, [1852]) Straight Swift - + - PrM U
157 Borbo cinnara (Wallace, 1866) Rice Swift - + - PrM, M U
Pelopidas conjuncta (Herrich-Schffer,
158 Conjoined Swift - + - PrM U
1869)
159 Arnetta atkinsoni (Moore, 1878) Atkinsons Bob + + - M R
160 Matapa aria (Moore, [1866]) Common Red-eye + + + PrM, PoM, W U
161 Baracus vittatus (C. Felder, 1862) Hedge Hopper - + - PrM U
162 Iambrix salsala (Moore, [1866]) Chestnut Bob + + + PrM, M, PoM C
163 Sancus fuligo (Mabille, 1876) Coon - + - PrM R
164 Ancistroides nigrita (Latreille, [1824]) Chocolate Demon + + - PrM, W U
Notocrypta curvifascia (C. & R. Felder,
165 Restricted Demon + - - PrM R
1862)
166 Udaspes folus (Cramer, [1775]) Grass Demon + + - M U
167 Ampittia dioscorides (Fabricius, 1793) Bush Hopper + + - M, PoM C
Astictopterus jama jama C. & R. Felder,
168 Forest Hopper + + - PrM, M C
1860
169 Aeromachus pygmaeus (Fabricius) Pygmy Scrub Hopper - + - PrM, PoM U
170 Halpe porus (Mabille, [1877]) Moores Ace - + - PrM R

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Das et al., 2012
 Journal of Research in Biology
Plasma protein markers for estimating the population genetic structure of
Amazon turtle (Podocnemis expansa Schweigger, 1812)
Journal of Research in Biology
Authors:
Teixeira AS1 and
Moura AS2.
Institution:
1. Instituto Nacional de
Pesquisas da Amaznia
(INPA), Avenida Andr
Arajo, 2936, Aleixo, CEP
69060-001, Manaus, AM,
Brasil.
2. Uninorte Laureate-Centro
Universitrio do Norte -
Laureate International
Universities, Avenida
Joaquim Nabuco, 1232,
Centro, CEP 69020-030,
Manaus-AM, Brasil.
Corresponding author:
Teixeira AS.
Email:
aylton1950@yahoo.com.br
Web Address:
http://jresearchbiology.com/
documents/RA0226.pdf.
Journal of Research in Biology
An International
Scientific Research Journal
An International Scientific Research Journal
Original Research
ABSTRACT:
This study aims to test the potential of allelic frequency distributions of plasma
proteins analysed by starch gel electrophoresis for identifying Amazon turtle (Podocnemis
expansa Schweigger, 1812) stocks. Three proteins: transferrin (Tf), albumin (Alb) and general
protein (GP), and four enzymes: esterase (EST), phosphoglucomutase (PGM), malate
dehydrogenase (MDH) and superoxide dismutase (SOD) from three geographical areas in the
Amazon region, totalizing three population samples: 1) Maracarana, Rio Uatum-AM, 2)
Trombetas, Rio Trombetas-PA and 3) Monte Cristo, Rio Tapajs-PA, were tested. Out of 11
presumptive loci detected, seven were monomorphic (GP-1, GP-2, EST-1, EST-2, PGM-1, SOD-1
2
and SOD-2) and four polymorphic (Tf, Alb, MDH and PGM-2). The chi-square (X ) analysis for
testing the hypothesis of independent segregation between pairs of polymorphic loci in the
population samples, revealed no statistically significant differences. Based on Wrights
F-statistics (FIS, FIT and FST) the detected average value of FIS = 0.1347, indicates a moderate
inbreeding within the population samples, whereas the average value of FIT = 0.1912, indicates a
moderately great inbreeding in the total population sample.The detected average value of
FST = 0.0652, indicates a moderate genetic differentiation among the population samples. The
pairwise comparisons of FST point to a moderate differentiation between all comparisons made
with the Monte Cristo population sample (FST ranging from 0.061 to 0.066); but very little
differentiation was detected between Maracarana and Trombetas samples (FST = 0.0035).
A limited gene flow among the population samples was detected (Nm = 3.58). The UPGMA
dendrogram showed the lowest genetic distance between population samples of Maracarana
and Trombetas, whereas, the highest genetic distances were detected when these two samples
were compared to that of Monte Cristo. Exact test for population differentiation revealed high
levels of statistically significant differences in all pairwise comparisons made with the Monte
Cristo sample. The data point out a possible existence of distinct subpopulations stocks of
P. expansa in the sampled area, where Monte Cristo sample was always genetically different
compared to the other two population samples examined.
Keywords:
Podocnemis expansa, Amazon Basin, plasma proteins, eletrophoresis,
population genetic structure.
Article Citation:
Teixeira AS and Moura AS.
Plasma protein markers for estimating the population genetic structure of Amazon
turtle (Podocnemis expansa Schweigger, 1812).
Journal of Research in Biology (2012) 2(4): 315-337
Dates:
Received: 03 Apr 2012 Accepted: 19 Apr 2012 Published: 26 May 2012
This article is governed by the Creative Commons Attribution License (http://creativecommons.org/
licenses/by/2.0), which gives permission for unrestricted use, non-commercial, distribution and
reproduction in all medium, provided the original work is properly cited.
315-337 | JRB | 2012 | Vol 2 | No 4
www.jresearchbiology.com

INTRODUCTION
Podocnemis expansa (Schweigger, 1812),
popularly known as giant Amazon river turtle or also as
Amazon turtle in the Brazilian Amazon Basin, belongs to
the order Testudines, suborder Pleurodira, family
Podocnemididae (Bickham et al., 2007). Among the 14
species of turtles existing in the Amazon region,
P. expansa is recognized to be the largest freshwater
turtle of the genus Podocnemis in South America, being
quite exploited commercially by man (Alho, 1985, Vogt,
2008). Females of this species can measure 50 to 89 cm
long, 60 cm wide (Alfinito, 1975; Pritchard and Trebbau,
1984; IBAMA, 1989) and weigh 15 to 80 kg
(Mittermeier, 1978; Alho and Pdua, 1982; Pritchard and
Trebbau, 1984; IBAMA, 1989). The Amazon turtle is
widely distributed and it can be found in the Rios
Araguaia and Tocantins (Mittermeier et al., 1980;
Ferreira Jnior and Castro, 2003), mouth of the Rio
Amazonas, and at least up to Rios Maran and Morona,
in Peru. The geographical distribution of this species also
covers the Rios Orinoco and Essequibo, both in
Venezuela (Mittermeier et al., 1980; Pritchard and
Trebbau, 1984; Iverson, 1992), also including Rios in
Bolivia, Colombia and Guyana. In Ecuador, there has
been a decrease in the number of P. expansa at the Rio
Tiputini (Cisneros-Heredia, 2006).
According to Gilmore (1986), the capture of
Amazon turtle can be considered the most important
ethnozoological activity throughout the Amazon region,
back from the pre-Columbian times up to today.
Many adventurers and travelers who roamed
South America in the centuries that followed its
discovery highlighted several natural spectacles of the
Amazon, among which the most impressive were the
major agglomerations of the Amazon turtle in large
beaches of the Amazon and Orinoco River basins. The
spawn of Amazon turtle in large groups, with tens of
thousands of females crawling up to the beaches was at
the same time a faithful record of the abundance and
316
Teixeira and Moura, 2012
exuberance of the turtle populations of the New World
(Ferreira Junior, 2003).
After European occupation of the Amazon, the
consumption of Amazon turtle as subsistence activity
turned into an extractive production system with
characteristics of mercantilism (1700 -1900
approximately), where, the main products were butter
and oil produced from the eggs, both used for frying
food, household and public lighting (Bates, 1863; Smith,
1974). At that time, the eggs were crushed in a canoe to
break the shells. Afterwards, water was added to it
making a mixture that was left exposed to the sun for
several hours in order to extract the oil floating on the
surface. The oil was skimmed off together with shells
and boiled before being stored in clay pots (Smith, 1974;
1979; Redford and Robinson, 1991), while, the fat
extracted by this process, was mixed with the resin, and
used to caulk boats (Smith,1974; 1979). In the eighteenth
century, P. expansa used to spawn in some beaches near
the town of Itacoatiara, State of Amazonas, Brazil,
where, due to commercial interests of the Portuguese
these places started being named as Royal beach
(Pesqueiro Real das Tartarugas). The meat of the
Amazon turtle was destined for local consumption,
supplying only the regional market and troops of the
Province of Rio Negro in Barcelos, State of Amazonas,
Brazil (for details see Smith, 1979).
By 1980s, there was a change in the human
consumption of the Amazon turtle meat, where this
product, which was considered a main item and source of
protein for local people, began also to be consumed as a
delicacy in the Amazonian people's diet (Alho, 1985). In
Manaus, capital city of the State of Amazonas, Brazil,
the so-called "tartarugada", a banquet of Amazon turtle
meat prepared in different ways, is very common on
important occasions. Thus, exploitation, illegal trade and
consumption of turtles are socially important for the
higher social class, and economically important for the
lower social class who catches these animals to supply
Journal of Research in Biology (2012) 2(4): 315-337
Teixeira and Moura, 2012
the demand from the higher social class (Alho, 1985). In
Belm, capital city of the State of Par, Brazil, in
addition to the trade of fat and derivates, the
consumption of meat and eggs from the Amazon turtle,
is also practiced (Alves and Santana, 2008).
Other by-products, such as shells, fat and viscera
of the Amazon turtle are much appreciated, and used in
making medicinal ointments, soaps, cosmetics,
household items and adornments (Smith, 1974, 1979;
Alho, 1985; IBAMA, 1989; Pantoja-Lima, 2007; Alves
and Santana, 2008). The Amazon turtle meat besides
being consumed as a local delicacy has also been looked
for in restaurants (Wetterberg et al., 1976; Ferrarini,
1980; Redford and Robinson, 1991).
After several decades of intense exploitation,
Podocnemis expansa had already been considered as
endangered species by the criteria of the International
Union for Conservation of Nature and Natural Resources
(IUCN). Today, due to protection programmes
implemented by the Brazilian federal government, and
led by Chico Mendes Institute for Biodiversity
(ICMBIO) and the Center for Conservation and
Management of Reptiles and Amphibians (RAN) in the
Amazon region, this species is no longer threatened.
However, this species still shows a low risk of extinction,
therefore, it is considered a conservation dependent
species according to the IUCN criteria. The Podocnemis
species are listed in the Appendix II of the Convention
on International Trade in Endangered Species of Wild
Fauna and Flora (CITES), published as Annex II,
Normative Instruction Number 11/2005, available at the
Brazilian Institute of the Environment and Renewable
Natural Resources (IBAMA).
Protein and isozyme electrophoresis has long
been used as a powerful molecular tool for species
identification, population genetic structure, conservation
and management genetics of plants and animals
(Ferguson, 1980; Ferguson et al., 1995, Scaltsoyiannes,
1999, Zeidler, 2000, Ridgway, 2005; Verspoor et al.,
Journal of Research in Biology (2012) 2(4): 315-337
2005; Sujatha et al., 2011). Protein and isozyme
molecules known as genetic markers are encoded by
structural genes, which play the role of determining the
precise sequence of amino acids in their polypeptide
chains. Thus, whenever these molecules are
electrophoretically analysed, the bands visualized in the
gels are assumed as direct products of gene action. These
genetic markers when extracted from various biological
tissues of animal and vegetable origin (muscle, heart,
liver, whole blood, blood plasma, hemolymph, seed,
root, leaf, among others) and then analysed by
electrophoresis, reveal protein banding patterns which
can provide valuable information, primarily regarding
the correct identification of species, natural and artificial
stocks (Ferguson, 1980; Ferguson et al., 1995). Among
the most diverse biological molecules, proteins are
essential for the expression of hereditary information.
There are tens of thousands of genes coding for unique
polypeptide chains, which, either solely or in combined
forms, generate an indefinitely great number of protein
variants, a variety further embellished by allelic
diversity. So far, protein electrophoresis has provided the
main source of insight into the genetic character of
species in the wild (Verspoor et al., 2005).
Protein electrophoresis detects genetic variation
that arises from amino acid substitutions generated by
base sequence variation. These substitutions alter the
charge state or conformational character of the protein
and change its mobility when placed in a gel
electrophoresis matrix subjected to an electrical field.
However, protein electrophoresis presents some
limitations, such as not every DNA sequence variation
that leads to amino acid changes, not all amino acid
changes are detectable by this technique. Moreover,
electrophoresis screening can detect only a part of the
amino acid sequence variation, which might be present,
amino acid variation represents only part of the variation
in the DNA, protein-coding regions represent only a
small part of the overall genome, only one third of amino
317

acid substitutions and one tenth of DNA base
substitutions will be detected by this method. Despite
these limitations, protein electrophoresis analyses are
still able to provide valuable insight into the relative
amount and distribution of genetic variation which, in the
context of population genetic theory, can iteself give
significant insight into the character of a given species
(for review see Verspoor et al., 2005). Even with the
advent of DNA molecular markers, protein and isozyme
markers when separated by electrophoresis, due to the
relative simplicity, speed and relatively low cost of
analysis as compared to many others are still being
applied in studies of population genetic structure in
different taxonomic groups. For instance, electrophoretic
studies of proteins and isozymes have successfully been
used in: plants (Scaltsoyiannes, 1999; Zeidler, 2000;
Veasey et al., 2008); insects (Simon and Hebert, 1995;
Scarpassa and Tadei, 2000; Santos et al., 2003); coral
reafs (Ridgway, 2005); mollusks (Nikiforov and
Zvyagintsev, 2008); reptiles (Bock et al., 2001); fish
(Billington, 1996, Batista and Sol-Cava, 2005; Kuzmin
and Kuzmina, 2005, Verspoor et al., 2005; Zawadzki et
al., 2008); mammals (Erdoan and zbeyaz, 2004; Yiit (P. expansa). Among the various types of biological
et al., 2007; Tao et al., 2007); among many other
organisms.
Some studies on the genetic population structure
of the Amazon turtle (Podocnemis expansa) have already
been performed by means of proteins (Teixeira et al.,
1996; Moura et al., 2011), allozymes (Bock et al., 2001),
mitochondrial DNA (mtDNA) and microssatlites (Sites
et al., 1999; Pearse et al., 2006).
The first study in Amazon turtle using the allele
frequency distribution of transferrin (a blood plasma iron
binding protein), suggested a free flow of genes in seven
population samples from five geographical areas of the
Amazon (Teixeira et al., 1996). This finding has recently
been confirmed by Moura et al., (2011) who were
investigating the temporal distribution of the transferrin
a b
alleles (Tf and Tf ) in three of the population samples
318
Teixeira and Moura, 2012
originally examined by Teixeira et al., (1996).
The allozyme markers used by Bock et al. (2001)
showed population genetic structure between Amazon
turtle samples collected in Brazil and Peru. While the
DNA molecular markers (nuclear and mitochondrial) of
this species, analysed by Sites et al., (1999) in samples
collected from two river systems in the Amazon Basin
(Tapajos and Araguaia) suggested extensive within-
system gene flow (up to 275 Km) but very little gene
flow between river sytems (2400 km). Another study
with Amazon turtle based on analysis of mitochondrial
DNA and microsatellite markers, from a broad sampling
in South America (i.e. 18 different sites in Brazil,
Colombia, Peru and Venezuela) strongly indicated the
existence of genetic structure between populations
(Pearse et al., 2006).
With the exception of the works by
Teixeira et al., (1996), Bock et al., (2001) and Moura et
al., (2011) there is practically no information on the
distribution of allele frequencies at polymorphic loci of
proteins applied to the identification, conservation and
management of natural stocks of the Amazon turtle
tissues as sources of protein variants, the circulating
blood plasma has a high number of proteins
(approximately 300) (Anderson and Anderson, 2002),
which can be extracted with relative facility and used in
the analysis of genetic variability. Then, the present
study aims to test the potential of allelic frequency
distributions of plasma proteins separated by means of
starch gel electrophoresis for identifying Amazon turtle
stocks. This research may also provide a valuable
contribution to conservation and management of this
species in the region.
MATERIALS AND METHODS
Sampling sites and collection of blood samples
A capture permit for catching wild Amazon turtle
(P. expansa) was provided by the Brazilian
Journal of Research in Biology (2012) 2(4): 315-337
Teixeira and Moura, 2012

Institute of Environment and Renewable Natural sodium citrate as anticoagulant. Blood plasma specimens
Resources (IBAMA) through the Biodiversity were separated by centrifugation at 3,000 rpm for 20
Authorization and Information System (SISBIO License minutes and stored at -25C, until electrophoresis run
No. 13081-1 of 19/12/2007). In possetion of this legal (Teixeira et al., 1996). After blood collection all animals
support ninety five hatchling specimens of P. expansa were returned to their respective collection sites.
were collected from three geographical areas in the Electrophoresis procedures
Amazon region, totalizing three population samples: The electrode buffer (0.06 M lithium hydroxide
1) Maracarana (0213S; 5815W), Rio Uatum-AM and 0.30 M boric acid) and gel buffer (0.03 M tris
(30); 2) Trombetas (0120S; 5645W), Rio (hydroxymethyl) aminomethane and 0.005 M citric acid)
Trombetas-PA (34), and 3) Monte Cristo (0404S; 55 containing 1% of the electrode buffer, were prepared
38W), Rio Tapajs-PA (31)(Fig. 1). according to Ridgway et al., (1970). The buffers were
With the aid of 3 ml disposable syringes, blood adjusted to pH 8.20 with 1 M lithium hydroxide. Sigma
samples were taken from the femoral artery and placed in starch gels at a concentration of 8.35% were made up in
5 ml BD vacutainer tubes, containing 0.5 ml of 3.8% 340 ml of the gel buffer. By means of a continuous

Fig. 1. The geographic sources where the three population samples of P. expansa, were captured in the Amazon
region: 1) Maracarana, Rio Uatum; 2) Trombetas, Rio Trombetas; and 3) Monte Cristo, Rio Tapajs. Map
modified from Teixeira et al., (1996).

Journal of Research in Biology (2012) 2(4): 315-337 319


stirring process performed with a simple mechanical
stirrer, the starch mixed in buffer was cooked up to
boiling point (approximately 90C) in a 2-liter
round-bottomed flask supported by a heating mantle.
Then, the cooked gel was degassed with the help of a
vacuum pump. The soluble protein supernatants were
absorbed into 5 mm x 8 mm rectangular filter papers
(Whatman 3 MM or 17 MM), which were then inserted
on the gels. The filter paper Whatman 3 MM was used
for analysis of esterase (EST), phosphoglucomutase
(PGM), malate dehydrogenase (MDH) and superoxide
dismutase (SOD); and Whatman 17 MM for analysis of
transferrin (Tf), albumin (Alb) and general protein (GP).
In the analyses of Tf, Alb and GP, the gels were
subjected to a potential of 120 V during the first 45
minutes. The filter paper inserts were removed and the
electrophoretic run continued at 250 V for about four
hours, until the borate line had moved about 10 cm past
the insert line. For the analyses of EST, PGM, MDH and
SOD, the gels were subjected to a potential of 150 V for
one hour. The filter paper inserts were removed and the
electrophoretic run continued for about five hours, until
the borate line had moved about 10 cm past the insert
line. The alleles were classified alphabetically according
to their decreasing electrophoretic mobilities towards the
anode. In the analyses of Tf, Alb, PG, MDH and PGM,
control plasma of known genotypes were selected and
interspersed along the gels in order to genotype the
specimens more accurately.
Protein system staining
Transferrin (Tf)
The differential precipitation method with rivanol
(2-ethoxy-6,9-diaminoacridine lactate) was used for
isolation of plasma transferrin prior to electrophoresis
run as described by Jamieson and Turner (1978) with
modifications of Teixeira et al.,.(1996). Then, plasma
samples were treated with 2% rivanol solution at a ratio
of 1:1. For the revelation of transferrin molecules the
gels were stained with 1% Amido Black, diluted in a
320
Teixeira and Moura, 2012
solution prepared in 5:5:1 parts of water, methanol and
acetic acid, respectively for five minutes, according to
Jamieson and Turner (1978). Finally, the gels were
washed by successive immersions in plastic vessels
containing the aforementioned solution (water, methanol
and acetic acid) for approximately 15 hours
(Teixeira et al., 1996). The Tf locus alleles were
i d e n t i fi e d an d c l a s s i fi e d a c c or d i n g to
Teixeira et al., (1996).
Albumin (Alb)
Given that, usually the protein supernatant
obtained by the differential precipitation method with
rivanol, contains significant amounts of albumin
(Boettcher et al., 1958) this method was also tested to
isolate albumin molecules. Thus, plasma samples were
treated with 1% rivanol solution at a ratio of 1:1
(Jamieson and Turner, 1978) with modifications. For
visualization of the albumin molecules, the gels were
stained with 1% Amido Black following the procedure of
Jamieson and Turner (1978) used for staining transferrin,
except for the staining time that lasted from five to seven
minutes. The distaining procedure was the same as that
previously described for transferrin.
General protein (GP)
For the revelation of bands in the general protein
patterns, the gels were stained with the same
methodology described previously for transferrin and
albumin (i.e. 1% Amido Black).
Nomenclature and enzyme staining
The enzymes were classified according to the
Nomenclature Committee of the International Union of
Biochemistry and Molecular Biology (NC-IUBMB.
2009), where each enzyme name is written in full and
enzyme abbreviation and enzyme commission number
(EC number) in parentheses, as follow: esterase (EST-
EC 3.1.1.1), phosphoglucomutase (PGM-EC 2.7.5.1),
malate dehydrogenase (MDH-EC 1.1.1.37) and
superoxide dismutase (SOD-EC 3.15.1.1).
Journal of Research in Biology (2012) 2(4): 315-337
Teixeira and Moura, 2012
In order to visualize isozyme bands in the
zymograms, the starch gels were immersed and
incubated in staining solutions containing chemical
components (substrates, coenzymes, electron carriers,
buffer solutions, and tetrazolium salts) necessary to de-
tect enzymatic activities as described below for each
enzyme system tested.
Esterase (EST EC 3.1.1.1)
For the revelation of esterase isozyme pattern in the
gels the following staining solution was used: 3 ml of 1%
-naphthyl acetate, dissolved in acetone at 50%, 87 ml of
distilled water, 100 mg of the dye fast blue RR salt and
10 ml of 0.5 M Tris HCl pH 7.1 (Shaw and Prasad,
1970). The gel slices were placed in plastic vessels,
immersed in the above solution and later incubated at
37C in a laboratory oven for about 30 minutes or until
the development of the bands. The staining solution was
then discarded and the gels, after being washed with
distilled water, were fixed in a 10% glycerol solution
prepared in 5:5:1 parts of water, methanol and acetic
acid, respectively.
Phosphoglucomutase (PGM EC 5.4.2.2)
The staining recipe used for revealing the
phosphoglucomutase isozyme patterns in the gels was
that recommended by Alfenas et al., (1991) with
+
modifications: 14 mg of NADP , 2.8 mg PMS, 14 mg
MTT, 28 mg MgCl2, 70 mg of glucose-1-phosphate,
70 ml of 0.2 M Tris-HCl pH 8.0 and 28 ml of glucose-6-
phosphate dehydrogenase (G6PDH), which was added
last. The gels were placed in plastic vessels containing
the staining solution and then incubated in an oven at
37C for about two hours, or until the development of
the bands. The staining solution was discarded and the
gels after being washed with distilled water were fixed in
a 10% aqueous glycerol solution.
Malate dehydrogenase (MDH EC 1.1.1.37)
The malate dehydrogenase isozyme patterns
were revealed using the following staining solution: 5
+
mg of NAD , 5 mg PMS, 10 mg MTT, 25 mL of 0.5 M
Journal of Research in Biology (2012) 2(4): 315-337
malic acid pH 8.0 and 50 mL of 0.1 M Tris HCl pH 8.5
(Allendorf et al., 1977) with modifications. The gels
were immersed into this solution in plastic vessels and
then incubated in an oven at 37C for about one hour, or
until the development of the bands. The staining solution
was discarded and the gels after being washed with
distilled water were fixed in a 10% glycerol solution
prepared in 5:5:1 parts of water, methanol and acetic
acid, respectively.
Superoxide dismutase (SOD EC 1.15.1.1)
The following staining solution was used for
visualizing the superoxide dismutase pattern in the gels:
24 mg MTT plus 7 mg PMS dissolved in 70 ml of 0.05
M Tris-HCl pH 7.0 (Allendorf et al., 1977) with
modifications. The gels were immersed in the
aforementioned solution, in plastic vessels and then
incubated at 37C in the dark for approximately 2 hours,
or until the development of achromatic bands. The
staining solution was discarded and the gels were then
washed with distilled water and fixed in a 10% glycerol
solution prepared in 5:5:1 parts of distilled water,
methanol and acetic acid, respectively.
Statistical analysis
Population genetic analyses of the Amazon turtle
were performed using the following statistical
programmes: Biosys-2 (Swofford et al., 1997), TFPGA
1.3 (Miller, 1997) and POPGENE Version 1.32
(Yeh et al., 1999). The list of statistical analyses is
presented below, and the programmes used in each one
of them are shown in parentheses: 1) log-likelihood ratio
(G) and chi-square (X2) tests assuming the Hardy-
Weinberg equilibrium (POPGENE 1.32 and TFPGA
1.3); 2) estimation of allele frequencies, average
heterozygosity, proportion of polymorphic loci and mean
number of alleles per locus (POPGENE 1.32 and TFPGA
1.3); 3) estimation of genetic distances according to
Rogers (1972) as modified by Wright (1978) (TFPGA
1.3); 4) cluster analysis of genetic distances using the
Unweighted Pair-Group Method with Arithmetic
321
 Teixeira and Moura, 2012

Table 1. Allele frequencies at 11 protein and isozyme loci examined in the blood plasma
from three population samples of Podocnemis expansa.
Population samples
Locus and allele Maracarana* Trombetas* Monte Cristo* Total
N=30 N=34 N=31 N=95
Tfa 0.7333 0.8088 0.7903 0.7789
Tfb 0.2667 0.1912 0.2097 0.2211
Alba 0.9167 0.9265 0.9516 0.9316
Albb 0.0833 0.0735 0.0484 0.0684
GP-1 1.000 1.000 1.000 1.000
GP-2 1.000 1.000 1.000 1.000
EST-1 1.000 1.000 1.000 1.000
EST-2 1.000 1.000 1.000 1.000
SOD-1 1.000 1.000 1.000 1.000
SOD-2 1.000 1.000 1.000 1.000
MDHa 0.000 0.000 0.2903 0.0947
MDHb 1.000 1.000 0.7097 0.9053
PGM-1 1.000 1.000 1.000 1.000
PGM-2a 0.7037 0.7273 0.4423 0.6395
PGM-2b 0.2963 0.2727 0.5577 0.3605
*Three, one and five specimens were not genotyped at the locus PGM-2 in Maracarana, Rio Uatum;
Trombetas, Rio Trombetas and Monte Cristo, Rio Tapajs, respectively (see Table 2).

Average (UPGMA) dendrogram (TFPGA 1.3); 5) exact
tests for population differentiation (Raymond and
Rousset,1995) for making pairwise population
comparisons (TFPGA 1.3); 6) F-statistics (FIS, FIT and
FST) of Wright (1978) and estimated gene flow (Nm)
based on Nei (1978) among the population samples and
between pairs of the population samples (POPGENE
1.32); 7) linkage disequilibrium analysis between pairs
of polymorphic loci (BIOSYS 2); and 8) homogeneity
test for investigating allele frequency distributions
among population samples and between pairs of the
population samples (BIOSYS 2 and POPGENE 1.32).
Futhermore, the significance of FST values
between population samples was tested using the
chi-square contingency test of Workman and Niswander
(1970).
RESULTS
Electrophoretic analyses of three protein and four
enzyme systems from blood plasma specimens of three
Amazon turtle population samples: 1) Maracarana, Rio
Uatum-AM; 2) Trombetas, Rio Trombetas-PA; and
3) Monte Cristo, Rio Tapajs-PA revealed zones of
322
electrophoretic activities in the gels presumably encoded
by 11 loci (Table 1). Of these loci, seven proved to be
monomorphic (GP-1, GP-2, EST-1, EST-2, PGM-1,
SOD-1 and SOD-2) and four polymorphic (Tf, Alb,
MDH-1 and PGM-2). Detailed descriptions of the
electrophoregrams and zymograms follow below.
Protein systems
Transferrina (Tf)
As previously reported by Teixeira et al., (1996),
the locus Tf was found to be polymorphic revealing
electrophoretic patterns with three theoretically expected
genotypes (Tfaa, Tfab and Tfbb), presumably encoded by
codominant alleles (Tfa and Tfb). The samples showed a
good genetic balance at the intra and inter-population
levels, as revealed by chi-square (X 2) and log-likelihood
ratio (G) tests (Table 2). Homogeneity test for Tf allele
frequency distributions among the population samples
and between pairs of population samples revealed no
statistically significant differences (Table 3).
Albumina (Alb)
The electrophoregrams of albumin revealed a
polymorphism with the presence of two genotypes (Albaa
and Albab) presumably encoded by two codominant
Journal of Research in Biology (2012) 2(4): 315-337
Teixeira and Moura, 2012

Table 2. Genotype and allele frequency distributions at four polymorphic loci in the P. expansa population
samples. Chi-square (X2) and log-likelihood ratio (G) tests were applied assuming Hardy-Weinberg
equilibrium. Expected numbers of genotypes are shown in parentheses.
Population samples
Maracarana N=30 Trombetas N=34 Monte Cristo N=31 Total N=95
Tf genotypes
Tfaa 16 (16.0339) 22 (22.1642) 20 (19.2787) 58 (57.5556)
Tfab 12 (11.9322) 11 (10.6716) 9 (10.4426) 32 (32.8889)
bb
Tf 2 (2,0339) 1 (1.1642) 2 (1.2787) 5 (4.5556)
Tf allele frequencies
Tfa 0.7333 0.8088 0.7903 0.7789
Tfb 0.2667 0.1912 0.2097 0.2211
Hardy-Weinberg test
d.f. 1 1 1 1
2
X 0.001022 0.0345 0.6332 0.0708
P 0.9745 0.8527 0.4262 0.7902
G 0.001024 0.0355 0.5824 0.0697
P 0.9745 0.8504 0.4454 0.7918
Alb genotypes
Albaa 25 (25.1695) 29 (29.1493) 28 (28.0492) 82 (82.4127)
ab
Alb 5 (4.6610) 5 (4.7015) 3 (2.9016) 13 (12.1746)
Albbb 0 (0.1695) 0 (0.1493) 0 (0.092) 0 (0.4127)
Alb allele frequencies
Alba 0.9167 0.9265 0.9516 0.9316
Albb 0.0833 0.0735 0.0484 0.0684
Hardy-Weinberg test
d.f. 1 1 1 1
2
X 0.1953 0.1690 0.0526 0.4707
P 0.6586 0.6810 0.8186 0.4926
G 0.3642 0.3178 0.1017 0.8822
P 0.5462 0.5729 0.7497 0.3476
MDH genotypes
MDHaa 0 0 9 (2.5082) 9 (0.8095)
ab
MDH 0 0 0 (12.9836) 0 (16.3810)
MDHbb 30 34 22 (15.5082) 86 (77.8095)
MDH allele frequencies
MDHa 0 0 0.2903 0.0947
MDHb 1.000 1.000 0.7097 0.9053
Hardy-Weinberg test
d.f. - - 1 1
X2 - - 32.5034 100.1114
P - - 0.0000** 0.0000**
G - - 38.3835 60.5680
P - - 0.0000** 0.0000**
Population samples
Maracarana* Trombetas* Monte Cristo* Total*
N=27 N=33 N=26 N=86
PGM-2 genotypes
PGM-2aa 15 (13.2642) 16 (17.4545) 5 (4.9608) 36 (34.4211)
ab
PGM-2 8 (11.4717) 16 (13.0909) 13 (13.0784) 37 (40.1579)
bb
PGM-2 4 (2.2642) 1 (2.4545) 8 (7.9608) 13 (11.4211)

Journal of Research in Biology (2012) 2(4): 315-337 323

 Teixeira and Moura, 2012

PGM-2 allele frequencies

PGM-2a 0.7037 0.7273 0.4423 0.6395
PGM-2b 0.2963 0.2727 0.5577 0.3605
Hardy-Weinberg test
d.f. 1 1 1 1
X2 2.6086 1.4359 0.00097 0.5390
P 0.1063 0.2308 0.9751 0.4628
G 2.4752 1.6216 0.00097 0.5353
P 0.1156 0.2029 0.9751 0.4644
d.f. = degrees of freedom; *Three, one and five specimens were not genotyped at the PGM-2 locus in Maracarana,
Rio Uatum; Trombetas, Rio Trombetas and Monte Cristo, Rio Tapajs, respectively; **P < 0.001.

alleles (Alba and Albb) at the locus Alb. The theoretically General protein (GP)
bb
expected genotype (Alb ) was not detected in any of the Two bands common to all specimens were seen
population samples analysed. All samples showed a good in the gels, in a zone of electrophoretic activity classified
genetic balance at the intra and inter-population levels, as as post-albumin (Manwell and Baker, 1970) with
2
revealed by the chi-square (X ) and log-likelihood ratio electrophoretic migrations immediately prior to albumin,
(G) tests (Table 2). Homogeneity test for Alb allele and presumably encoded by the fixed loci
frequency distributions among population samples and (GP-1 and GP-2) (Table 1). Some additional bands were
between pairs of population samples revealed no seen in the gels, but these were too weakly or diffusely
statistically significant differences (Table 3). stained for being reliably scored.

Table 3. The allele frequency distributions at four polymorphic loci among the population
samples and between pairs of the population samples of Podocnemis expansa, were analysed by
means of homogeneity tests. Chi-square (X 2), log-likelihood ratio (G) and probability statistical
values were calculated by using contingency tables.
Population samples
Maracarana vs Maracarana vs Trombetas vs
Locus Among samples
Trombetas Monte Cristo Monte Cristo
X2(2) =1.124 X2(1) =1.037 X2(1) = 0.546 X2(1) =0.069
P = 0.570 P = 0.309 P = 0.460 P =0.792
Tf G(2) =1.105 G(1)= 1.035 G(1) = 0.547 G(1) = 0.069
P = 0.576 P = 0.309 P = 0.460 P = 0.792
X2(2) = 0.628 X2(1) =0.042 X2(1) = 0.608 X2(1) = 0.355
P = 0.731 P =0.836 P = 0.436 P = 0.551
Alb G(2) = 0.656 G(1)=0.042 G(1) = 0.613 G(1) = 0.359
P = 0.720 P = 0.837 P = 0.434 P = 0.549
X2(2) =41.050 X2(1)= 20.434 X2(1) = 22.915
P = 0.000*** P = 0.000*** P = 0.000***

MDH G(2) =44.375 G(1) = 27.392 G(1) =29.859
P = 0.000*** P = 0.000*** P = 0.000***
X2(2) =10.315 X2(1) =0.081 X2(1) =6.398 X2(1) =8.634
P = 0.006** P =0.776 P =0.011* P =0.003**
PGM-2 G(2) =10.112 G(1) =0.081 G(1) =6.466 G(1) =8.682
P = 0.006** P =0.776 P =0.011* P = 0.003**
X2(8) =53.117
Grand P = 0.000***
total G(8) =56.248
P = 0.000***
*p < 0.05; **p < 0.01 or ***p < 0.001; fixed locus in the samples.

324 Journal of Research in Biology (2012) 2(4): 315-337

Teixeira and Moura, 2012

Enzyme systems samples and between all pairwise comparisons made

Esterase (EST EC 3.1.1.1) with Monte Cristo sample (Table 3).
The esterase enzyme revealed two zones of Malato desidrogenase (MDH EC 1.1.1.37)
electrophoretic activity, with a monomorphic isozyme The MDH zimogram revealed a zone of
pattern presumably encoded by the same fixed loci electrophoretic activity located in the more cathodic
(EST-1 and EST-2) in all Amazon turtle samples tested region of the gels, supposedly controlled by MDH locus.
(Table 1). The loci (EST-1 and EST-2) were visualized This locus proved to be polymorphic in the Monte Cristo
in the gel, in a more cathodic electrophoretic region. The population sample, with genotypes (MDHaa and MDHbb)
locus EST-1 was weakly stained with an electrophoretic produced by action of codominant alleles (MDHa and
migration immediately ahead of EST-2 locus. The locus MDHb) but with the absence of theoretically expected
EST-2 was strongly stained. genotype MDHab (Fig. 3). In population samples of
Phosphoglucomutase (PGM EC 5.4.2.2) Maracarana and Trombetas this locus proved to be fixed
The phosphoglucomutase zymogram showed two for the allele MDHb (Table 2). Allele MDHa and
loci (PGM-1 and PGM-2 (Fig. 2). PGM-1 locus proved genotype MDHaa were detected only in Monte Cristo
to be monomorphic and PGM-2 polymorphic. PGM-2 sample. Due to the enormous deficiency of heterozygotes
showed three theoretically expected genotypes and the excess of homozygotes (MDHaa) the Monte
(PGM-2aa, PGM-2ab and PGM-2bb) presumably encoded Cristo population sample and the total population sample
by codominant alleles (PGM-2a and PGM-2b). The loci are not in Hardy-Weinberg equilibrium, as shown in the
(PGM-1 and PGM-2) were detected in a more cathodic chi-square (X2) and log-likelihood ratio (G) tests
region of the gel. PGM-1 showed a faster electrophoretic (Table 2). The homogeneity tests revealed high levels of
migration with a slightly stained band. The locus PGM-2 statistically significant differences among population
despite showing zymogram bands that could be samples and all pairwise comparisons made with Monte
genotyped for most specimens examined, nine specimens Cristo sample (Table 3).
could not be genotyped due to the low resolution of the Superoxide dismutase (SOD EC 1.15.1.1)
bands. All samples showed a good genetic balance at the The superoxide dismutase zymogram revealed
intra and inter-population levels, as revealed by the two zones of electrophoretic activity, with a
2
chi-square (X ) and log-likelihood ratio (G) tests monomorphic isozyme pattern presumably encoded by
(Table 2). However, homogeneity tests revealed two fixed loci (SOD-1 and SOD-2) in all samples tested
statistically significant differences among the population (Table 1). The loci (SOD-1 and SOD-2) were detected in
Table 4. Linkage disequilibrium analysis between a more cathodic region of the gels. The locus
pairs of polymorphic loci in the population samples SOD-1 migrated slightly ahead of the SOD-2 locus.
of P. expansa.
Eventually, it was noted the presence of satellite bands
Loci compared X2 d.f P
positioned in front of the locus SOD-1.
Tf and Alb 0.38 1 0.5353
Linkage disequilibrium
Tf and MDH 0.76 1 0.3836
The hypothesis of independent segregation
Tf and PGM-2 0.21 1 0.6505 between pairs of the polymorphic loci detected in
Alb and MDH 2.98 1 0.0841 P. expansa, was tested by means of the chi-square (X 2)
Alb and PGM-2 0.96 1 0.3261 test. Of the six possible combinations between pairs of
MDH and PGM-2 2.03 1 0.1538 loci, there were no statistically significant

Journal of Research in Biology (2012) 2(4): 315-337 325


Fig. 2. Zymogram of phosphoglucomutase showing
the monomorphic locus PGM-1, and the polymorphic
locus PGM-2 in the blood plasma of P. expansa. The
PGM-2 genotypes from left to right are: PGM-2aa
(lanes 3, 5, 6 and 9); PGM-2ab (lanes 1, 2, 7 and 8);
PGM-2bb (lane 4).
differences (Table 4).
Measures of genetic diversity
The proportion of polymorphic loci (P) when
calculated by using the 0.95 criterion (i.e. the common
allele with a frequency equal to or less than 0.95),
showed a P of 27.27% for the population samples of
Trombetas and Maracarana, and of 36.36% for Monte
Cristo and total sample (Table 5). The highest P values
presented by Monte Cristo and total population samples
were due to the polymorpism at the MDH locus detected
only in the Monte Cristo sample. The average number of
alleles per locus in the population samples of Maracarana
and Trombetas was 1.27 and in Monte Cristo 1.36. The
average observed heterozygosity (Ho) in the Maracarana
population sample was 0.0785, Trombetas 0.0869 and
Monte Cristo 0.0771. The average expected
heterozygosity (He) in the Maracarana population
sample was 0.0874, Trombetas 0.0766 and Monte Cristo
0.1211. As one can see, (He) value for the Monte Cristo
sample was much higher as compared to the other two
samples.
326
Teixeira and Moura, 2012
Fig. 3. Zymogram of malate dehydrogenase showing
the polymorphism at the locus MDH in the blood
plasma of P. expansa. The genotypes from left to
right are: MDHbb (lanes 1, 2, 4, 5, 6 and 8); MDHaa
(lanes 3, 7 and 9).
Genetic structure
The estimates of population genetic structure in
the population samples performed by using Wright's F
statistics showed average values of 0.1347 and 0.1912
for FIS and FIT, respectively. The Alb locus was the only
one to show negative values for FIS and FIT, due to the
slight excess of heterozygous individuals in the samples.
The average value of FST was 0.0652 among the
population samples (Table 6). Maracarana vs Trombetas
samples showed an average value of FST = 0.0035,
whereas Monte Cristo vs Maracarana and Monte
Cr i st o vs T r om be t a s, r e vea l ed a ver a ge
FST values = 0.061 and 0.066, respectively (Table 7).
All pairwise comparisons of FST values made with Monte
Cristo sample were significantly different from zero
(P < 0.001).
Gene flow estimation expressed as number of
migrants per generation (Nm) among the population
samples was 3.58 (Table 6).
Corroborated by the results obtained from
homogeneity tests (Table 3), the pairwise comparisons
performed by means of exact tests for population
Journal of Research in Biology (2012) 2(4): 315-337
Teixeira and Moura, 2012

Table 5. Summary of genetic variation at 11 loci in the population samples of P. expansa.

Standard deviations are shown in parentheses.
Population samples
Maracarana Trombetas Monte Cristo Total
Mean number of turtle per locus
29.6 33.9 30.5 94
Number of locus
11 11 11 11
*Proportion of polymorphic loci (P)
27.27 27.27 36.36 36.36
Average number of alleles per locus
1.27 1.27 1.36 1.36

Number of polymorphic loci 3 3 4 4

Average observed heterozygosity (Ho) 0.0785 0.0869 0.0771 0.0811

(0.1442) (0.1668) (0.1553) (0.1533)
0.0874 0.0766 0.1211 0.1004
**Average expected heterozygosity (He)
(0.1632) (0.1439) (0.1933) (0.1631)
**Nei (1973) expected heterozygosity; *P corresponds to 0.95 criterion.
differentiation revealed high statistically significant the higher values detected between these samples and
differences between all comparisons made with Monte Monte Cristo (Table 8), as illustrated by the UPGMA
Cristo sample (Table 7). dendrogram method (Fig. 4). The internal support values
Genetic distance of the nodes obtained by bootstrap methods showed a
The Rogers (1972) genetic distance modified by low node bootstrap value of 27.27% between
Wright (1978) revealed the lowest distance between Maracarana and Trombetas samples. A maximum
Maracarana and Trombetas population samples, unlike bootstrap value of 100% was found in the node that
includes the samples Maracarana, Trombetas and Monte
Table 6. Wrights F-statistics and gene flow (Nm) for
each locus in the P. expansa population samples. Cristo.

Locus FIS FIT FST *Nm

DISCUSSION
Tf 0.0174 0.0233 0.0060 41.65
Linkage disequilibrium
Alb -0.0771 -0.0734 0.0034 73.31
In population genetic studies typically using a
GP-1 - - 0.0000 -
- - 0.0000 - system of multiple polymorphic gene loci, it is necessary
GP-2
EST-1 - - 0.0000 - to subject these loci to the test of linkage disequilibrium,

EST-2 - - 0.0000 - since genetic coadaptation may exist between some

MDH 1.000 1.000 0.2143 0.92 alleles, but not between others within the same

PGM-1 - - 0.0000 - population. In this circumstance, certain alleles of a

PGM-2 0.0198 0.0895 0.0711 3.27 given locus can also be coadapted with others of distinct

SOD-1 - - 0.0000 - loci (Ayala and Kiger, 1980; Allendorf and Luikart,
SOD-2 - - 0.0000 - 2007).
Mean 0.1347 0.1912 0.0652 3.58 Given that, the alleles of the polymorphic loci
*Nm = Gene flow estimated from examined here in the Amazon turtle (P. expansa)
FST= 0.25 (1 -FST) / FST.
population samples, are not associated (Table 4) and

Journal of Research in Biology (2012) 2(4): 315-337 327

 Teixeira and Moura, 2012

Table 7. Exact tests for population differentiation (Raymond and Rousset, 1995) based on the allele frequency
distributions at polymorphic loci were applied for making pairwise comparisons in the P. expansa population
samples (lower half of matrix). In addition, FST values (upper half of matrix) and corresponding Nm values
(between parentheses) are shown. The FST values significantly different from zero are shown in bold.
Population samples
Maracarana Trombetas Monte Cristo
Maracarana 0.0035 0.061
-
(71.15) (3.62)
Trombetas
X2(6)= 2.230 0.066
-
P = 0.897 (3.54)
Monte Cristo
X2(8) = 31.230 X2(8)= 33.023
-
P = 0.0001* P = 0.0001*

*p < 0.001.

presumably are segregating independently (i.e. no
epistasis), they can be used in future population genetic
studies of this species, requiring gametic equilibrium
between loci, as basic premise. Therefore, the use of
these loci as genetic markers for investigating Amazon
turtle probably should not bring about any influence in
the estimation of parameters on the population genetic
structure of this species. Nevertheless, we cannot
completely rule out the possibility that some of these loci
are in linkage disequilibrium with other loci that have not
been examined thus far, due to evolutionary processes,
such as: inbreeding, genetic drift, natural selection,
population bottleneck and Wahlund effect (Ayala and
Kiger, 1980; Durand et al., 2003; Hartl, 2008).
Hardy-Weinberg equilibrium
The Hardy-Weinberg test is a simplified
statistical method commonly used to describe population
genetic characteristics by means of the binomial
expansion: (p + q)2 = p2 + 2pq + q2 = 1, where p and q,
correspond the allelic frequencies of the two different
alleles of a given population presumably in genetic
equilibrium. This method allows one to describe a
population by its allele and genotype frequencies
displayed at each gene locus, and the effects of natural
selection. The main assumption of Hardy-Weinberg Law
is that in an infinitely large population in the absence of
evolutionary processes: mutation, migration, drift and
328
natural selection; gene frequencies will remain constant
across generations. This law also says that if the
crossings occur at random, the genotypic frequencies are
related to the gene frequencies through the
aforementioned binomial expansion (Ayala and Kiger,
1980; Ferguson, 1980; Allendorf and Luikart, 2007).
Of the four polymorphic loci analysed (Tf, Alb,
MDH, PGM-2) only MDH locus showed Hardy-
Weinberg disequilibrium in the Monte Cristo population
sample, with repercussion in the total population sample
(Table 2). Among the factors that normally bring about
genetic imbalance in gene loci, are: 1) adaptive
differences between genotypes (Nevo et al., 1984);
2) possible existence of other loci in linkage
disequilibrium with a particular locus under study
(Futuyma, 1997); 3) selection against heterozygous
individuals (Gillespie, 1998; Beiguelman, 2008);
4) inbreeding (Wright, 1921); 5) Wahlund effect (i.e.
physical mixtures of genetically distinct populations)
(Smith et al., 1981); and 6) production of a non-
functional protein (i.e. a null allele) (Ferguson, 1980).
With the exception of the null allele and Wahlund effect
(factors not observed in the present MDH analysis), one
or more of the aforementioned factors, acting in
combination, could be affecting the genotypic and allelic
distributions in the locus MDH. Therefore, a broader
sampling should be conducted in the occurrence area of
Journal of Research in Biology (2012) 2(4): 315-337
Teixeira and Moura, 2012
Fig. 4. The UPGMA dendrogram based on Rogers
(1972) genetic distance modified by Wright (1978)
between P. expansa population samples.
P. expansa, with analyses including preferably the loci
examined in this study, and extra loci, in order to better
understand this peculiar genetic imbalance detected in
the Monte Cristo population sample. Additionally, it is
necessary to conduct further studies with historical series
of the molecular genetic markers already examined in
P. expansa (Moura et al., 2011, and references therein);
to assess what alleles at certain loci could be increasing
or decreasing in frequency over subsequent generations
of this species.
Measures of genetic diversity
The knowledge of allele frequency distributions
is essential in researches of genetic variability and
population genetic structure. This kind of research
contributes significantly to the knowledge of the
evolutionary history of a given population, therefore,
may indicate the action of certain evolutionary
mechanisms on the population under study, such as
inbreeding, genetic drift, founder effect and population
bottlenecks. Additionally, by comparisons of allele
frequencies it is possible to demonstrate whether certain
populations have similar evolutionary histories (Hartl,
2008).
The genetic variation of a population is usually
measured by the proportion of polymorphic loci (P),
average heterozygosity per locus (H), number of
polymorphic loci and average number of alleles per locus
(Nei, 1975).
Among these measures of genetic variation,
heterozygosity is the most informative, and is therefore
Journal of Research in Biology (2012) 2(4): 315-337
Table 8. Rogers (1972) genetic distance modified by
Wright (1978) between the Podocmenis expansa
population samples.
Population samples
Monte
Maracarana Trombetas
Cristo
Maracarana - - -
Trombetas 0.0240 - -
Monte
0.1195 0.1230 -
Cristo
preferred by most population geneticists, and less
affected by sample size. It is a good measure of variation
because it estimates the probability that two alleles
drawn at random from a population are different. The
polymorphism of a population is, moreover, an imprecise
measure of genetic variation due to the arbitrariness in
the choice of the polymorphism criterion to be used (0.95
or 0.99), i.e. the most common allele with frequency no
greater than 0.95 or 0.99. For example, a certain locus
presenting two alleles with frequencies 0.95 and 0.05,
while another presenting 20 alleles, each with a
frequency of 0.05 are equally considered polymorphic
under the 0.95 criterion of polymorphism (Nei, 1975;
Ayala and Kiger, 1980).
In the present study, the average expected
heterozygosity (He) of P. expansa in the Maracarana
population sample was 0.0874, Trombetas 0.0766 and
Monte Cristo 0.1211. The population sample of Monte
Cristo showed the higher variation between the average
observed heterozygosity (Ho) and the average expected
heterozygosity (He) (0.0771 and 0.1211, respectively)
(Table 5). This was noted due to the level of
polymorphism detected in the MDH locus, where the
allele frequencies of MDHa MDHb were 0.29 and 0.71,
respectively, only in this sample. As a result, a great
variation between these measures Ho and He was also
observed in the total sample. The value for He in the
poplation sample of Monte Cristo (0.1211) was much
higher than the values revealed in the two other samples
analysed in the present study, Maracarana (0.0874) and
329

Trombetas (0.0766); and in the population samples
examined by Bock et al., (2001), from Peru (0.043) and
Brazil (0.072- 0.078). However, these values are within
the range estimated for the group of reptiles, where, in
studies of protein-isozyme markers, this measure ranged
from 0.050 to 0.124 (Chakraborty et al., 1980;
Ferguson, 1980; Allendorf and Luikart, 2007).
The polymorphism (P) in P. expansa was
estimated to be 0.17 (Bock et al., 2001). This value is
well below the values estimated in the P. expansa
population samples examined in the present study, which
was 0.27 in Maracarana and Trombetas, and 0.36 in
Monte Cristo. It should be emphasized that the Monte
Cristo sample is showing a polymorphism 33% higher, in
relation to the samples of Maracarana and Trombetas,
and 122% higher in relation to the samples examined by
Bock et al., (2001). In addition, the samples of the
present study showed polymorphism values above that
shown in reptiles (P = 0.23) (Ferguson, 1980; Ayala and
Kiger, 1980; Hartl, 2008).
The Monte Cristo population sample of
P. expansa with an average number of alleles per locus
equal to 1.36, exceeded the value of 1.27 detected in the
Trombetas and Maracarana samples.
This prominence in the measures of genetic
variation of P. expansa displayed by Monte Cristo
(i.e. population sample collected from the Rio Tapajs)
in relation to the other population samples investigated in
the present study, is consistent with the results of
microsatellite and mitochondrial DNA of this species
presented by Sites et al., (1999) and Pearse et al., (2006).
Sites et al., 1999 found a higher allelic richness and a
larger number of haplotypes in the P. expansa population
sample from Rio Tapajs in relation to the population
sample from Rio Araguaia. Pearse et al., (2006)
confirmed this much higher genetic diversity of Tapajs
compared to most other Brazilian population samples
collected from Amazon areas.
330
Teixeira and Moura, 2012
Genetic structure
In order to generate information on the genetic
structure of natural populations for breeding and genetic
conservation of species, many researchers have used
Wright's F statistics (Dias, 1998). The parameter used to
estimate the gene fixation indexes resulting from
biparental inbreeding is the F coefficient (FIS, FIT and
FST) of Wright (1951) defined as the correlation between
the alleles in the gametes that form a zygote (Robinson,
1998). FIS is known as the inbreeding index of
individuals relative to its subpopulation (it estimates the
deviation from Hardy-Weinberg genotypic frequencies in
the subpopulation); FIT is the inbreeding in the
population (estimated deviation from Hardy-Weinberg
equilibrium in the entire population); and FST is the
inbreeding due to the differentiation among
subpopulations in relation to the total population
(divergence of allele frequencies among populations)
(Frankham et al., 2008).
According to Wright (1978) F equal to 0.25 is
considered an arbitrary value above which there is very
great genetic differentiation among populations; values
in the range 0.15 to 0.25 indicate moderately great
genetic differentiation; 0.05 to 0.15 indicate moderate
genetic differentiation; and genetic differentiation is,
however, by no means negligible if F is as small as 0.05
or even less. By using Wright (1978)'s criterion the
average value of FIS = 0.1347 obtained in P. expansa in
the present study indicates a moderate inbreeding within
the population samples of this species (Table 6). The
average value of FIT = 0.1912 indicates that inbreeding in
the total sample population was moderately great. The
average value of FST = 0.0652 points to a genetic
differentiation moderate among population samples. The
loci that contributed most to genetic differentiation
among the samples were MDH and PGM-2. The MDH
locus in particular stood out as having a great deficiency
of heterozygous individuals in the population sample of
Monte Cristo, which reflected in the total population
Journal of Research in Biology (2012) 2(4): 315-337
Teixeira and Moura, 2012
sample (Table 2). In addition, statistically significant
differences were detected in the allele distributions of
these loci, as demonstrated in the homogeneity
tests (Table 3).
The values of FIS, FIT and FST in population
studies of Pseudemys scripta using allozymes were
0. 183, 0.142 and 0.048, r esp ecti vel y
(Scribner et al., 1986), and approach those values
observed in the present work for Podocnemis expansa.
According to Wright (1969 cited in Frankham et
al., 2002, p. 328) one migrant per generation would be
enough to avoid a complete genetic differentiation
among idealized populations. Theoretically, the number
of migrants Nm 4 means the occurrence of panmixia
among populations. While, Nm 2 indicates a moderate
genetic divergence; however, considerable variation
exists for these values of Nm among organisms,
providing a considerable scope for genetic divergence
that results from random genetic drift (Hartl, 2008).
Sites et al., (1999) found a value of Nm = 1.67
in P. expansa, reflecting a moderate genetic divergence
among the population samples studied.Valenzuela (2001)
estimated a value of Nm 4.60, revealing a moderate
genetic divergence among population samples of Rios
Caquet (Colombia), Tapajos and Araguaia (Brazil). In
the present study an average value of Nm = 3:58, was
estimated in the total population sample of this species
(Table 6), which resulted in a moderate genetic
divergence as shown above (FST = 0.0652). In the
pairwise population sample comparisons, Maracarana vs
Trombetas, the detected value of Nm = 71.15 provides
strong evidence for panmixia between these two samples
examined. In contrast with the Tf locus data presented by
Teixeira et al., (1996), a limited gene flow was detected
in the pairwise population sample comparisons, Monte
Cristo vs Maracarana and Monte Cristo vs Trombetas
(Nm = 3.62 and 3:54, respectively). This gene flow
limitation also led to a moderate genetic differentiation
detected in the aforementioned pairwise sample
Journal of Research in Biology (2012) 2(4): 315-337
comparisons, Monte Cristo vs Maracarana and Monte
Cristo vs Trombetas, with FST values = 0.061 and 0.066,
respectively (Table 7). In this context, the following
question comes up: what is the Nm in the natural
populations of P. Expansa. The data presented here,
based only on a limited number of loci and population
samples examined, plus the paucity of data available in
literature for this species regarding genetic structure, do
not allow answering this question. Currently what can be
clearly seen is that, the greatest genetic differentiation
revealed in the present study when Monte Cristo (i.e.
population sample collected from the Rio Tapajs) was
compared to the other samples, shows to be congruent
with the DNA data published by Pearse et al., (2006),
who compared Tapajs with other population samples
from the Amazon basin.
Genetic distance
Genetic distance measuments have long been
applied as one of the major tools for investigating the
genetic differentiation between populations. These
measurements that use gene and genotypic frequencies
obtained through protein electrophoresis technique from
individuals taken at random in natural populations, are
able to provide an estimate of intra and interspecific
differentiation. These frequencies can be transformed
into a series of indexes that allow estimating the degree
of similarity or genetic distance between species and
populations (for review see Nei, 1975; Wright, 1978;
Ayala and Kiger, 1980; Ferguson, 1980; Solferini and
Selivon, 2001).
When genetic distance, genetic identity, or other
measurements of genetic similarity have been compiled
for all possible pairs of populations or species, the data
are best presented in the form of a matrix. Then, for
visual display of the results, dendrograms can be
constructed. One of the most useful dendrograms for
electrophoretic data is the Unweighted Pair-Group
Method with Arithmetic Average (UPGMA) (Ferguson,
1980). This method is mainly applied because it assumes
331

an evolutionary constant rate in each branch of the tree
and works poorly when this assumption is violated
(Hartl, 2008).
The matrix of the estimated genetic distances
between population samples of P. expansa, showed the
lowest value in the pairwise comparison Maracarana vs
Trombetas (Table 8). These findings suggest that these
samples are presumably part of a large panmictic
population, i.e. belonging to the same stock, with this
fact being supported by the large estimated number of
migrants per generation between them (Nm = 71.15). It is
noteworthy that tagged specimens of Amazon turtle,
from the Rio Trombetas were already found near the
town of Itacoatiara, in the state of Amazonas (Alfinito et
al., 1976). This fact could explain a possible flow of
animals from the Rio Trombetas towards Rio Uatum
(river from where the Maracarana population sample
was caught). Considering that these rivers are tributaries
of the same bank (i.e. left bank) of the Amazon River,
such a flow would be facilitated, resulting in a greater
homogeneity between these sampled areas (Table 3,
Table 7).
It also should be highlighted that the greatest
distance values were detected between the pairwise
population sample comparisons made with Monte Cristo,
meaning restricted flow of genes between Monte Cristo
vs Maracarana (Nm = 3.62) and Monte Cristo vs
Trombetas (Nm = 3.54) (Table 7, Table 8). In fact,
statistically significant differences were detected in these
comparisons (Table 3).
The dendrogram (Fig. 4) generated from the
data of Table 8, in a way, seems to represent graphically
the ancestry relationships and genetic structure of the
P. expansa population samples examined.
CONCLUSION
1) The present electrophoretic data based on
plasma protein markers point out a possible existence of
distinct subpopulations stocks of P. expansa in the
332
Teixeira and Moura, 2012
sampled area, where Monte Cristo (population sample
from the Rio Tapajs), was always genetically different
compared to the other two population samples examined.
2) Due to the reasonably high degree of
polymorphism revealed in plasma proteins of P. expansa,
an extension of the present research is highly
recommended in order to detect a greater number of
polymorphic loci and apply them as genetic markers in
population studies of this species.
3) Even with the advent of modern DNA based
markers, the protein and isozyme markers separated by
electrophoresis technique, remain a powerful and
effective tool in studies of population genetic structure,
and, therefore, it should never be underestimated. The
relative simplicity, speed and low cost of protein
electrophoresis in relation to many other molecular
techniques, still makes it very popular and useful in
many laboratories worldwide.
ACKNOWLEDGMENTS
This study was partially financed by the
Instituto Nacional de Pesquisas da Amaznia (INPA),
Manaus, AM, Brasil, through the Institutional Project
Programme (PRJ12.01). Special thanks to the
Coordenao de Aperfeioamento de Pessoal de Nvel
Superior (CAPES), Braslia, DF, Brasil and Fundao de
Amparo Pesquisa do Estado do Amazonas (FAPEAM),
Manaus, AM, Brasil, for the Master of Science (MSc)
scholarship given to Arlisson Silva de Moura. The
authors are indebted to Mr Paulo Henrique Guimares de
Oliveira, Centro de Preservao e Pesquisas de
Quelnios Aquticos (CPPQA), Presidente Figueiredo,
AM, Brasil, Mr Gilmar Nicolau Klein, Instituto Chico
Mendes de Conservao da Biodiversidade (ICMBio),
Porto Trombetas, PA, Brasil, and Mr Nicola Sebastio
Tancredi, Instituto Brasileiro do Meio Ambiente e dos
Recursos Naturais Renovveis (IBAMA), Santarm, PA,
Brasil, for their logistical support, guidance and
extensive help during the catch of the turtle specimens
Journal of Research in Biology (2012) 2(4): 315-337
Teixeira and Moura, 2012
in the field. The authors are also most grateful to Mrs
Cristina Alcntara da Silva for her generous technical
assistance in the laboratory during the electrophoresis
analyses.
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 Journal of Research in Biology An International Scientific Research Journal

Original Research

Biofiltration Efficiency of Three Sea Weed Species

Journal of Research in Biology

Authors: ABSTRACT:
Nirmala A, Manjari P,
Sunita Seepana. Rising global demand for seafood and declining catches have resulted in the
volume of aquaculture doubling each decade, a growth expected by the FAO to persist
in the decades to come. The use of technologies with economical and environmental
sustainability would influence aquaculture growth. In aquaculture, feed accounts for
about half the cost in current high-volume fed mono-species culture such as in fish net
Institution:
Department of pens or shrimp/fish ponds with most of this feed going as waste or uneaten. As a
Biotechnology, Vinayaka result, immense impact on the environment, hampers the further growth of
Missions University, aquaculture. In traditional polyculture systems, the nutrient-assimilating
Aarupadi Veedu photoautotrophic plants use solar energy to turn nutrient-rich effluents into profitable
Institute of Technology, resources and improve to a large extent the aquatic environment. Additionally some
Chennai-603104, of these aquatic plants are commercially exploited for the preparation of various
Tamilnadu, India. products. Thus the dual role of some of these aquatic plants can be effectively used in
shrimp aquaculture ponds in the maintenance of good water quality through
regulating toxic gases such as ammonia and nitrite and hence improve the shrimp
production. A study was conducted to evaluate different marine algal species with
respect to good water quality maintenance. The study showed that Gracilaria crassa
Corresponding author: can improve water quality more effectively compared to Ulva reticulate and
Nirmala A. Enteromorpha in all the three water systems namely sea water, pond water and
shrimp culture effluent water.

Email: Keywords:
nimmi_aruna@yahoo.com. Seaweed, Waste water, Aquaculture, Biofilteration, Gracilaria crassa.

Phone No: Article Citation:

9841681587. Nirmala A, Manjari P, Sunita Seepana.
Biofiltration Efficiency of Three Sea Weed Species.
Journal of Research in Biology (2012) 2(4): 338-347
Web Address: Dates:
http://jresearchbiology.com/ Received: 14 Aug 2011 Accepted: 27 Aug 2011 Published: 31 May 2012
documents/RA0084.pdf.

This article is governed by the Creative Commons Attribution License (http://creativecommons.org/

licenses/by/2.0), which gives permission for unrestricted use, non-commercial, distribution and
reproduction in all medium, provided the original work is properly cited.

338-347 | JRB | 2012 | Vol 2 | No 4

Journal of Research in Biology
An International
Scientific Research Journal www.jresearchbiology.com

INTRODUCTION
Aquaculture, also known as aquafarming, is the
farming of aquatic organisms such as fish, crustaceans,
mollusks and aquatic plants. Aquaculture involves
cultivating freshwater and saltwater populations under
controlled conditions. On the other hand it refers to
aquaculture practices in marine environments such as the
farming of marine fish in enclosed pens or nets,
mariculture, and also is the farming of marine
crustaceans, molluscs and seaweed. The other methods
include, which integrates fish farming and plant farming
in water. Thus the output, as reported, from aquaculture
would supply one half of the fish and shellfish that is
directly consumed by humans (FAO. 2006). Further, in
current aquaculture practice, products from several
pounds of wild fish are used to produce one pound of a
fish like salmon. One drawback of many methods is the
discharge of nutrient rich effluent in to the environment.
This discharge includes feed derived wastes composed
of dissolved components such as nitrogen, phosphorus
and suspended solids (Losordo and Westers 1994).
So, aquaculture probably being the fastest
growing food-producing sector and has high potentials to
fulfill the nutritional needs of a growing population, it
now accounts for almost 50% of the worlds food and is
perceived as having the greatest potential to meet the
growing demand for aquatic food. It is estimated that at
least an additional 45 million tones of aquatic food will
be required by 2030 to maintain the current per capita
consumption (FAO. 2009). Further, in view of ensuing
food crisis there is a call for turning to aquatic plants for
human food needs (Shpigel et al., 1993). China produces
nearly 10.7 million tones of plants from aquaculture most
of which are used as human food (FAO. 2006).
Need of Seaweed in aquaculture
On noticing the increasing pollution in our rivers
and streams, the pollutants are ever increasing which are
affecting most of the industrial and domestic systems
functioning. So there is a need to identify agents other
339
Nirmala et al., 2012
than manual or chemical treatments with more cost
affective simple technology. Today, seaweed and
mussels as a possible "natural cleaning system" (similar
to what aquaponics does...). Thus, Extractive
Aquaculture has come to the rescue wherein aquatic
plants like seaweed have significant positive role. Some
of the basic seaweeds functions are:
Seaweeds farms acts as nutrient sinks
Seaweeds farms increase the primary productivity
The farms act as habitat for certain fish and shell fish
Seaweeds farming provides a sustainable lively hoods
Many old people are engaged in tying and drying of
seaweeds
Since it is a sustainable and lucrative business, it
prevents migration
Since seaweeds are cash crops it gives instant money
to the farmers
In many island nations, these seaweeds have become
the crops with highest export earnings, limited usage
having higher priority.
There are two main areas where seaweeds have
the potential for use in wastewater treatment.
The first is the treatment of seawage and some
agricultural wastes to reduce the total nitrogen- and
phosphorus-containing compounds before the release of
these treated waters into rivers or oceans.
The second is for the removal of toxic metals
from industrial wastewater (Schramm. 1991).
The aim of this project was the development of a
seaweed biofilter system to reduce the environmental
pollution impact of marine eco system. Specifically, we
sought to evaluate three species of seaweeds (Ulva
reticulate, Gracilaria crassa, Enteromorpha comprassa)
with their performance for a biofilter. The pH,
Temperature, salinity of water samples, and also check
the capability of sea weeds in reducing ammonia and
nitrite content were considered here.
MATERIALS AND METHODS
Journal of Research in Biology (2012) 2(4): 338-347
Nirmala et al., 2012
Environmental concerns and limitations in water
availability are some of the factors that make
recirculation an important option for the aquaculture
industry. However, water reuse is limited by the
accumulation of waste product excreted by fish such as
carbondioxide, ammonia nitrogen and dissolved faecal
solids. In addition, nitrate and phosphate levels
accumulate in the water of recirculation system as a
result of biofilteration at a rate dependent on fish density
and water replacement flow rate.
Study location
The present experimental studies on shrimp
using locally available algae were carried out at Central
Institute of Brackish water Aquaculture (CIBA) in
Muttukadu experimental station, Chennai.
Experimental species
Ul v a re ti cul at a, Gracil ari a c rassa,
Enteromorpha species of seaweeds obtained from
Kovalam and coastal areas of Chennai
Experimental set up
The experiment was conducted for 36 days in 3
different water samples namely sea water, pond water
and shrimp culture effluent water with four groups
namely group1 ,group2, group3 and group -4 (control )
with three replicates each. Where group-1 contain
seaweeds ulva reticulata , group - 2 Graccilaria craasa ,
group- 3 Enteromorpha and group-4 control with no
seaweeds. Uniform sized Fibreclass Reinforced
Polypropylene (FRP) tanks of 100L capacity were used.
Each were cleaned and disinfected with bleaching
powder before starting the experimental trial. The tanks
were filled with 40L of sea water, pond water and shrimp
culture effluent water and continued aeration was
provided throughout the experimental period. The
seaweeds were weighed approximately as 20g and
stocked into the tanks.
Maintenance
Water was not exchanged in tanks through out
the process. Every day, checked the aeration; a biotic
Journal of Research in Biology (2012) 2(4): 338-347
parameters like salinity, pH and temperature were
recorded. The estimation of Ammonia and Nitrite were
done in every alternate day. Once in 15 days DO value
was calculated. And it is observed that the seaweeds
stocked in the tanks were alive and maintained.
Water quality analysis
Before stocking seaweeds, the abiotic parameters
including water, temperature, salinity and pH were
recorded and after stocking the seaweeds the abiotic
parameters were maintained everyday. Temperature was
recorded using a mercury thermometer; salinity was
recorded using a refractometer. After standardizing with
distilled water and pH was recorded using a portable pH
meter, every alternate day, water samples were collected
from each tank and analysed for the ammonia and nitrite.
Ammonia content was estimated by the method of
(Solorzano.1969). Nitrite was estimated by the
Nitroprusside method by (Strickland and Parsons,
1972).and expressed in ppm. Dissolved Oxygen given by
Winklers method, Lipid by (Bligh and Dyer, 1959)and
protein by (Bradford, 1976).
RESULTS AND DISCUSSION
Annual consumption of seafood has increased
dra-matically over the past three decades worldwide, but
supply from wild capture fisheries appears to have
reached an uppermost limit (FAO. 2007). Accordingly,
aqua-culture production has been growing by more than
10% annually and will reach 50% of worlds seafood
supply in 2030 (FAo, 2007). However, intensive fish
aquaculture has caused serious ecological problems, such
as coastal eu-trophication due to the release of excess
nutrients (Read and Frenandes 2003). Moreover, this
release may negatively influence the aquaculture activity
itself by increasing the ammonium toxicity and water
turbidity (Troell et al., 1999). Accordingly, various
approaches are being taken by government authorities to
reduce excess nutrients in effluents, including effluent
regulations that limit maximum allowed nutrient
340

concentrations in effluents discharged by aquaculture
facilities (Tacon and Foster 2003).
The 30 days experimental trail with different
species of seaweed stocked in tanks showed better results
in water quality management than in the controls.
Hence the, effectivity of different seaweeds in
different water samples are as follows
Sea water ammonia & nitrite content:
It is identified that all the three species
(Ulva reticulata, Gracilaria crassa, Enteromorpha) can
reduce Ammonia and Nitrite content from Seawater
(Table1,2) Pond water (Fig1,2 ) and most effectively
from Effluent water (Table-3,4). And among all the three
Algal species Gracilaria crassa is identified to remove
ammonia nitrite content more effectively in all three
water samples.
The use of seaweeds as biofilters to remove
dissolved nitrogen from fish pond effluents has been
widely studied (De Boer and Ryther, 1977).
(Harlin et al., 1979) used Gracilaria sp. to remove the
ammonium produced by the fish Fundulus heteroclitus,
removing lightly more ammonium than was produced by
an equal biomass of fish. Similar results were reported
by (Haglund and Pedersen 1993).in a system of
Gracilaria tenuistipitata. (Jimnez del Ro et al., 1996)
described that Ulva spp. not only show a higher
N-removal capacity than Gracilaria spp. but also a
higher resistance to epiphytes.
Our results are similar to that of (Seema and
Reeta Jayasankar, 2005). who have reported that the
green seaweed Ulva reticulata used as co-culture species
for monitoring the changes in toxic nitrogenous wastes in
the shrimp culture system was found to efficiently
remove ammonical nitrogen from 249.5 to 17.39 pmol
nitrogen (94%). The nitrate nitrogen reduced from 28.39
to 24.21 pmol nitrogen (5%) and nitrite nitrogen from
14.51 to 9.03 pmol nitrogen (22%). The removal of total
nitrogen from the aquaculture system was found to be
45% when treated with seaweeds .The concentration of
341
Nirmala et al., 2012
toxic nitrogen wastes was found to be always at a lower
level in the integrated system when compared to the
monoculture system. Seaweed of economical importance
can be used in aquaculture system to improve water
quality and generate revenue for the industry.
According to (Neori et al., 1996) and
(Ahn et al., 1998). ammonia is assimilated as much as
two to three times faster than the oxidized nitrate by
many types of seaweed. (Rosenberg and Ramus 1984).
Cohen and Neori (1991). who have reported that the
highest growth rates were recorded in Ulva reticulata
followed by Eucheuma denticulata and Gracilaria
crassa at the same stocking densities. This may be
attributed to high surface area of Ulva reticulata. The
difference in the ratio of surface area to volume (S:V)
may lead to difference in nutrient uptake rates among
macro algae. Although the macro algae were grown at a
low irradiance their high ability for photoacclimation
might have contributed to the high growth rate and
nutrient uptake rates. It is also possible that light
limitation had negative effects on growth rates and
nutrient uptake rates.
Water from shrimp aquaculture ponds were
circulated through a mangrove woodlot, Oyster beds, and
seaweed ponds in biofilter experiments designed to
reduce environmental loads (Toru et al., 2006).
Dissolved oxygen content:
Presence of Algal species also helps in
improving DO content in water medium, thereby
improving water quality. Ulva reticulata and
Gracilaria crassa improves Oxygen content in different
water samples (Fig-3,4,5).
Associated with the presence of seaweed as an
natural filters in these sedimentation tank, where the
seaweed, naturally have a function as a filter of carbon
dioxide, which are absorbed and converted into the
oxygen element. So the measurement of dissolved
oxygen concentrations was done at the fish culture tank
with the combination biofilter system and control.
Journal of Research in Biology (2012) 2(4): 338-347
 Table-1 Seawater Ammonia
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Control 0.031 0.043 0.049 0.091 0.089 0.042 0.043 0.065 0.202 0.221 0.175 0.213 0.173 0.184 0.171 0.183 0.189 0.185

Group I 0.031 0.034 0.032 0.032 0.03 0027 0.025 0.04 0.044 0.085 0.046 0.073 0.032 0.014 0.02 0.061 0.042 0.05

Group II 0.031 0.033 0.031 0.028 0.033 0.025 0.019 0.042 0.033 0.061 0.038 0.051 0.03 0.018 0.019 0.02 0.056 0.048
Nirmala et al., 2012

Group
0.031 0.03 0.029 0.03 0.027 0.026 0.03 0.034 0.042 0.066 0.042 0.048 0.05 0.024 0.019 0.031 0.042 0.059
III

Table-2 Sea water Nitrite

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Control 0.08 0.081 0.83 0.84 0.089 0.091 0.95 0.95 0.098 0.098 0.99 0.136 0.158 0.194 0.143 0.379 0.394 0.039

Group I 0.072 0.071 0.071 0.07 0.064 0.063 0.063 0.065 0.079 0.068 0.07 0.069 0.068 0.027 0.074 0.062 0.076 0.087

Group II 0082 0.049 0.044 0.046 0.041 0.044 0.045 0.045 0.042 0.043 0.041 0.041 0.039 0.041 0.038 0.043 0.058 0.045

Journal of Research in Biology (2012) 2(4): 338-347

Group III 0.083 0.053 0.046 0.046 0.043 0.042 0.045 0.044 0.042 0.04 0.039 0.041 0.0042 0.045 0.039 0.041 0.041 0.045

Table-3 Effluent water Ammonia & Nitrite

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Control 0.038 0.036 0.041 0.048 0.031 0.044 0.074 0.097 0.104 0.1 0.147 0.145 0.106 0.111 0.095 0.092 0.101
0.039
Group I 0.038 0.033 0.03 0.031 0.036 0.042 0.048 0.049 0.055 0.062 0.05 0.074 0.06 0.061 0.072 0.076 0.076 0.071

Group II 0.038 0.039 0.036 0.029 0.038 0.03 0.034 0.036 0.038 0.042 0.049 0.06 0.03 0.031 0.024 0.028 0.05 0.058

Group III 0.038 0.039 0.035 0.031 0.037 0.029 0.024 0.037 0.046 0.055 0.05 0.062 0.039 0.041 0.036 0.043 0.053 0.065

Table-4 Effluent Water Nitrite

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Contro l 0.065 0.066 0.052 0.054 0.052 0.063 0.062 0.075 0.079 0.086 0.095 0.107 0.118 0.123 0.129 0.147 0.173 0.177

Group I 0.059 0.056 0.056 0.051 0.048 0.051 0.054 0.071 0.071 0.073 0.081 0.082 0.087 0.087 0.094 0.101 0.014 0.126

Group II 0.059 0.051 0.047 0.039 0.043 0.044 0.046 0.044 0.047 0.053 0.055 0.062 0.061 0.064 0.071 0.072 0.071 0.06

342
Group III 0.055 0.045 0.051 0.046 0.043 0.052 0.061 0.069 0.061 0.073 0.095 0.089 0.091 0.085 0.086 0.089 0.1 0.108

FIGURE-1.
The result showed that DO levels that exist in the fish
culture tanks with filtration system are 5.0 to 6.9mg/l,
while the DO concentration at control tank were
4.7 to 5.3mg/l.
One of the main benefits of integrating animals
and plants is the improvement in water quality by the
plant component. Many seaweeds prefer nitrogen in
ammonium form and all produce oxygen and remove
carbon dioxide through the process of photosynthesis.
With the exception of solids removal, these are the main
functions of water treatment in a recirculating
aquaculture system. Unfortunately, the contribution of
seaweeds to improved water quality mainly occurs
during light hours, as these functions which include
oxygen production, carbon dioxide and ammonia uptake
are predominantly correlated to photosynthesis. In a
RAS, each kg of Ulva produces enough oxygen daily for
2 kg of fish stock (Neori et al., 2004). Based on a lower
metabolic rate, 100 kg of Ulva could maintain enough
FIGURE-3
343
Nirmala et al., 2012
FIGURE-2.
oxygen for 500 kg of abalone. Therefore, to improve
water quality parameters such as oxygen levels, a
relatively low quantity of seaweed is required as a ratio
to the abalone component.
Lipid deposition:
The lipid deposition indicates that Algae is
reducing ammonia and nitrite content from water
samples (Table-5,6,7). All the three species can deposit
lipid where the average lipid percent in tissues of
Ulva reticulata is 6.89, Gracilaria crassa is 7.14,
Enteromorpha is 5.54. Hence Gracilaria crassa is
consider a bit more effective.
All the algae were low in lipid (22.4% DW) but
within the range of other species of red seaweeds
(13% DW) reported previously (Mabeau and Fleurence,
1993). (Wong and Cheung, 2000). Nevertheless, abalone
species show low lipid requirement, typical of herbivore
molluscs and fish (Mai et al., 1995). This low lipid
requirement has been associated by some authors
FIGURE-4
Journal of Research in Biology (2012) 2(4): 338-347
Nirmala et al.,2012

FIGURE-5

Table-5 Lipid Deposition in Seawater PLATE -1 Protein deposition in Sea water

Species Initial Final
Group I 4.31 4.869
Group II 4.96 5.67
Group III 4.48 5.44

Table-6 Lipid Deposition in Pond water

Species Initial Final
Group I 4.31 6.09
Group II 4.96 7.89
Group III 4.48 5.54

Table-7 Lipid Deposition in Effluent water

Species Initial Final
Group I 4.31 9.72 PLATE -2 Protein deposition in Pond water
Group II 4.96 7.87
Group III 4.48 5.66

(Durazo-Beltran et al., 2004). with the low use of

dietary lipids as energy source by abalone based upon its
low metabolic rate. Indeed, high levels of dietary lipid
seem to negatively affect abalone growth
(Thongrod et al., 2003).
Protien deposition:
The protein deposition indicates that Algae is
reducing ammonia and nitrite content from water
samples (Plate-1,2,3). All the three species can deposit PLATE -3 Protein deposition in Effluent water

protein where the average protein content in tissues of
Ulva reticulata is 13.95,Graccilaria crassa 14.82 and
Enteromorpha 13.97. Hence, Graccilaria crassa is
considered a bit more effective. Our results are similar to
that of (Neori et al., 2000). Cultured Ulva and Gracilaria
can be rich in protein and so can be a good food source
for shellfish, such as abalone or as an ingredient in fish
feed as used at the Makoba integrated system
(Mmochi et al., 2002).

Journal of Research in Biology (2012) 2(4): 338-347 344

 Nirmala et al.,2012

Physical parameters: utilizing the principle of protein-dye binding. Analytical

pH (all the three species maintain the pH Biochemistry 72:248-254.
constantly in all these water samples). Temperature
Chen S, Ling J and Blancheton JP. 2006. Nitrification
(25 to 27C). Salinity (26-31ppm for seawater,
kinetic of biofilm as affected by water quality
15-17ppm for pond water, 26-29ppm for effluent water).
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Our results are similar to that of
(Rijin and Rivera, 1990). (Sato et al., 2000). (Chen et al., Cohen I and Neori M. 1991. Ulva lactuca biofilter for
2006). who have reported that physicochemical marine fishponds effluents and Ammonium uptake
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De-Boer J and Ryther J. 1977. Potential yields from a
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Plant Biomass of the Pacific Northwest Coast, R.W.
Temperature, Alkalinity, Salinity, turbulence level,
Krauss (ed.). Oregon State University Press, Oregon .
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231-248.

CONCLUSION Durazo-Beltran E, Viana MT, DVAbramo LR,

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347 Journal of Research in Biology (2012) 2(4): 338-347

 Journal of Research in Biology
Nutritive value and utilization of Baobab (Adansonia digitata) seed meal as
plant protein source in the diet of juveniles of Clarias gariepinus
(Burchell, 1822) (Pisces: Clariidae)
Journal of Research in Biology
Authors:
Afamdi Anene1, Olivia C.
Afam-Anene2 and Chisom
Onyekachi1.
Institution:
1. Animal Nutrition
Laboratory, Department of
Animal Science/Fisheries
Abia State University,
Umuahia Campus.
2. Department of Nutrition
and Dietetics Imo State
University, P. M. B. 2000,
Owerri, Imo State, Nigeria.
Corresponding author:
Afamdi Anene.
Email:
afamanene31@yahoo.com
Phone No:
+2348037107726.
Web Address:
http://jresearchbiology.com/
documents/RA0160.pdf.
Journal of Research in Biology
An International
Scientific Research Journal
An International Scientific Research Journal
Original Research
ABSTRACT:
This paper aimed at evaluating the potentials of boiled Baobab
(Adansonia digitata) seed meal as plant protein source in the diet of juveniles of
Clarias gariepinus. A sixty-day feeding trial was conducted in 40L plastic aquaria tanks.
Boiled baobab seeds meals were incorporated in diets at 0, 25, 50, 75 and 100%
designated as diets D1 (Control), D2, D3, D4 and D5 respectively. All diets were
iso-nitrogenous and iso-caloric. Juvenile Clarias gariepinus with initial mean weight
101.2434g were fed ad lib on allotted diets twice per day for 60 days. Mean body
weight gain in fish fed on D1 was highest with a value 901.22g and lowest in Diet 5
with a value of 250.39g. The mean daily weight gain in fish was highest in Diet 1 with a
value of 15.4% and lowest in Diet 5 with a value of 4.17%. Specific Growth rate (SGR)
of experimental fish was highest with a value of 3.62% in Diet 1 and lowest in Diet 5
with a value of 2.09%. Proximate composition of fish fillets fed on test diets show that
the protein level in diet 21.880.11% in Diet 3 and 21.001.00% in D4 were
significantly higher than the other diets while the crude fiber was highest in Diet 4
with a value of 2.900.17% and lowest in Diet 3 with a value of 1.930.12%. This study
shows that up to 25% of boiled baobab seeds can be incorporated into the diets of
juvenile C. gariepinus without any negative effects on growth of fish.
Keywords:
Nutritive value, Adansonia digitata, Clarias gariepinus.
Article Citation:
Afamdi Anene, Olivia C. Afam-Anene and Chisom Onyekachi.
Nutritive value and utilization of Baobab (Adansonia digitata) seed meal as plant
protein source in the diet of juveniles of Clarias gariepinus (Burchell, 1822) (Pisces:
Clariidae).
Journal of Research in Biology (2012) 2(4): 348-354
Dates:
Received: 03 Dec 2011 Accepted: 12 Dec 2011 Published: 31 May 2012
This article is governed by the Creative Commons Attribution License (http://creativecommons.org/
licenses/by/2.0), which gives permission for unrestricted use, non-commercial, distribution and
reproduction in all medium, provided the original work is properly cited.
348-354 | JRB | 2012 | Vol 2 | No 4
www.jresearchbiology.com
 Anene et al.,2012

INTRODUCTION performance.
Catfish species are very popular to fish farmers Assessing the quality of fish fillets fed on the
and consumers, they command a very good commercial different experimental diets.
value in Nigerian markets (Ezenwaji, 1985).
Clarias species is one of the commercially important MATERIALS AND METHODS
catfish. This is because, the fish is good as food, has the The experiment was conducted inside the Animal
capability to grow fast especially in intensive culture Nutrition Laboratory at the Department of Agriculture,
system, produce thousand of eggs in a breeding period, Abia State University, Umuahia Campus. Two hundred
withstand both handling and environmental stress. (200) juvenile (one month old) C. gariepinus fish were
It can withstand diseases and many other adverse obtained from a private fish farm in Owerri, Imo State,
conditions that can kill fish. Ayinla (2003) reported that Nigeria. They were then transported in two 50 liter
feed accounts for at least 60% of the total cost of fish plastic containers to the Laboratory. They were
production in Africa, which to a large extent determines acclimatized for 21 days during which period they were
the viability and profitability of fish farming enterprise. fed ad-lib on Coppes a commercial feed.
Fish production is facing many problems including the Preparation and Analysis of experimental diets:
availability of suitable and economical feed for rearing Raw seeds of baobab were sourced from a local
young and adult fish (Adeyemo et al., 1994). market in Katsina State, Nigeria. The seeds were soaked
Conventional protein sources for fish feed for 24 hours in water and boiled for 1 hour at 105C
production include soybean, groundnut cake and cotton without change of water. The baobab seeds were oven
seed meal and these constitute the major vegetables dried at 60C before milling which was achieved using
protein sources. These protein sources are scarce, hammer miller. Each of the diet was compounded and
expensive and highly competed by man and other mixed separately as shown in Table 1. The mixture was
animals. These legumes are produced by rain fed manually made into pellets using a machine, sun dried
agricultural systems which make them scarce and very and stored in sack bays for further use.
expensive, thereby leading to high cost of fish feeds and Ash, crude lipid, crude fiber and protein of
consequently fish production (Ayinla, 2003). baobab seeds, fruits and experimental diets were
Adansonia digitata is a desert crop which is Table 1: Level of replacement of baobab
seed meal in fish diets.
resistant to draught and produce all year round. The
D1 D2 D3 D4 D5
seeds are not consumed by man who rely on their leaves Ingredient 0% 25% 50% 75% 100%
BBSM BBSM BBSM BBSM BBSM
for food, and are not subjected to various other uses. Maize 49.80 49.80 49.80 49.80 49.80
A. digitata seeds are unknown in fish feeding and Soybean 28.50 24.50 14.24 4.00 0.00
BBSM 00.00 4.00 14.25 24.50 28.50
generally underutilized. With this background, the study Wheat offer 11.00 11.00 11.00 11.00 11.00
aims at: Palm kernel
5.10 5.10 5.10 5.10 5.10
cake
Investigating the nutritional quality of baobab seeds Fish meal 3.00 3.00 3.00 3.00 3.00
Investigating the effect of boiled baobab seed meal Bone meal 1.00 1.00 1.00 1.00 1.00
Vitamin
on the growth performance of C. gariepinus. premix 0.25 0.25 0.25 0.25 0.25
Salt 0.25 0.25 0.25 0.25 0.25
Determining the optimal replacement levels of
Methionine 0.10 0.10 0.10 0.10 0.10
boiled baobab seed meal in the diets of Palm oil 1.00 1.00 1.00 1.00 1.00
C. gariepinus fingerlings based on growth Total 100 100 100 100 100
349 Journal of Research in Biology (2012) 2(4): 348-354
Anene et al.,2012

similarly determined using AOAC methods (1995). The satiation for 60 days daily in the morning (6.00am) and
carbohydrate content was calculated by difference. in the evening (18.00pm). All fishes were subjected to a
Nitrogen Free Extract (NFE) was calculated as the 12:12 light and dark cycle using a natural day and night
remainder of crude protein +crude fat +ash and assuming regime.
crude proteins 5.85=N (Gnaiger and Bitterlich, 1984). Water Management
All reagents used for the analyses were of analytical There was 50% exchange of water in all the
grades and were not subjected to further purification. tanks daily. Raw borehole water was used for the study.
At the end of the feeding trial, fish fed on the Water was temporally stored in 500L plastic containers
different experimental feeds were analyzed for their from where it was transferred into the experimental tanks
proximate composition using AOAC methods (1995). every morning.
To obtain fish fillets, each fish was cut along its full Measurement of Weight
length. Fish fillet from each treatment were blended into Before stocking and before the feeding trial each
a smooth paste in a 3.8 L kitchen-type blender (Warning experimental fish was weighed (W1) and after the
Products, New Hartford, CT) which was thoroughly feeding trial (W2). Increase in body weight the
cleaned and dried between samples. Triplicate difference in final body weight (W2) and initial body
determination was made for each treatment. weight (W2) in grams was used as the major indicator
Feeding regime for growth.
Juvenile fish were stocked seven in each Computation of Data
container (40L capacity) where they were fed for 60 days Mean weight gain (g) was estimated as, mean
on diets containing various levels of inclusion of boiled final weight mean initial weight. Mean Daily weight
baobab seeds meal with varying crude protein levels in gain (MDWG) was calculated as W2-W1/T2-T1,
triplicates. Fishes were fed to satiation daily at 7:00 Specific growth rate was calculated as Specific Growth
hours and 18:00 hours. Rate = (LogW2 - LogW1) 100 / T2 - T1, where W2
Fish in each tank were individually weighed at and W1 = final and initial weight respectively; T2 and
the beginning (W1) and at the end (W2) of the feeding T1 = final and initial time (Zaid and Sogbesan, 2010).
experiment. Dead fish were removed and recorded daily. The mean, standard deviation and analysis of variance
Mortality during the experiment was 8%. Unconsumed (ANOVA) were computed using Statistical Package for
feed and excreta were siphoned out daily (one hour after Social Sciences (SPSS) version 15. Means were
feeding) after which water level was topped to maintain separated using Duncan Multiple Range Test and
the same water level. Stocking/Feeding Regime: significance was judged at p<0.05.
All fish were starved for 24 hours prior to the RESULT AND DISCUSSION
commencement of the experiment. The feeding trail was Table 2: Proximate Composition of Baobab
Seed and Fruit
carried out in five transparent plastic tanks of 40L
Composition Seed (%) Fruits (%)
capacity with water depth of 0.40m. The tanks were Moisture 4.30.1 10.4 0.4
placed on wooden table with a height of 1.4m. Protein 18.40.5 3.2 0.1
Fat 12.20.1 0.30.0
Experimental fish (juvenile C. gariepinus) were Ash 3.80.1 4.50.2
randomly distributed into five treatment groups and each Crude fiber 16.20.9 5.40.3
Carbohydrate 491.7 76.21.0
group had 10 replicates. The feeding trial was carried out
Metabolizable
in a 40L transparent plastic tank. The fish were fed to 363.89.7 320.34.4
Energy

Journal of Research in Biology (2012) 2(4): 348-354 350

 Anene et al.,2012
Table 3: Proximate composition of experimental diet of C. gariepinus at varying
replacement levels of boiled baobab seed meal.
D1 D2 D3 D4 D5
Constituents
0%BBSM 25%BBSM 50%BBSM 75%BBSM 100%BBSM
Moisture 9.44ab0.41 9.05b 0.06 10.18a0.01 10.13a1.01 9.39ab0.38
a a a a
Protein 41.13 1.63 40.25 1.59 42.88 1.66 40.38 1.65 42.00a0.38
a a a a
Ash 10.50 1.39 12.00 2.00 11.0 1.73 11.80 0.90 10.00a1.73
a a a a
Crude fiber 1.90 0.00 1.91 0.00 2.39 0.76 2.00 1.00 1.50a0.10
b a b c
Fat (ether extract) 9.00 0.86 10.97 1.17 8.95 0.79 6.97 1.01 9.98ab0.67
ab ab b a
Carbohydrate 28.07 1.00 25.82 2.54 25.05 2.61 29.34 0.67 27.13ab1.73
NFE 37.471.01 34.871.14 34.780.98 38.850.99 36.521.08
Value are mean standard deviation of triplicate samples abc means SD with similar superscripts in the
same column are not significant different (p>0.05).

The result of the proximate composition of fruits agree with literature reports of the same seed
and boiled baobab seeds are presented in Table 2. The (Aduku, 1993; Ezeagu, 2005; Nkafamiya et al., 2007).
moisture content of baobab seeds was 4.30.1%, crude Composition of feed ingredient and experimental
protein 18.400.5%, cruder fiber 16.200.9%, either diets
extracts 12.200.1%, and ash 3.80%. The moisture level The proximate composition of experimental diets
in baobab fruit was 10.4 0.4%, protein 3.20.1%, fat is presented in Table 3. The result revealed diets were
0.30.0%, ash 4.50.2% and crude fiber 5.40.3%. generally iso-nitrogenous. The protein content ranged
Moisture content in fruits was generally higher than in from 40.381.65 to 42.881.66% were not significantly
seeds. Protein levels in seeds of baobab in this study are different (p>0.05) and conform to the recommendation
not in accord with Nkafamiya et al., (2007) who reported of optimum dietary protein of the fish (Luquet, 1991).
protein levels of 21.750.12 g/100g. However, the crude The results also show that there was a significant
protein of the seeds of baobab were compared favourably difference (p>0.05) in moisture, fat and carbohydrates of
with that published by Ezeagu, (2005). However, the the experimental diets.
amount of protein recorded in this study is considered to Growth performance of C. gariepinus
be moderate Aduku, (1993) and Ezeagu, (2005) qualifies The growth performance of C. gariepinus fed on
the boiled baobab seeds as commendable protein various experimental diets was evaluated in terms of
supplement in fish feed formulations. Similarly, ash, fat body weight gain (g), mean daily body weight gain (g)
and carbohydrate contents of baobab seed in this study and specific growth rate is presented in Table 4.
Table 4: Effect of replacement of Boiled Baobab seeds (BBMS) as protein source in C. gariepinus diet.
D1 D2 D3 D4 D5
Parameters SEM
0%BBSM 25%BBSM 50%BBSM 75%BBSM 100%BBSM
Mean Initial body
101.03a 101.67a 102.11a 101.67a 101.03a 43.12
weight (g)
Mean Final body
1002.11a 803.35b 651.65c 483.16d 354.88e 34.76
weight (g)
Mean Body weight
901.22a 700.69b 550.19b 380.94c 250.39d 22.8
gain (g)
Mean Daily weight
15.4a 11.67b 10.76b 6.33c 4.17c 17.9
gain (MDWG)
Specific Growth rate
3.62a 3.55a 3.08b 2.59c 2.09d 11.6
(SGR) (%)
Value are mean standard deviation of triplicate samples abc means SD with similar superscripts in the
same column are not significant different (p>0.05).

351 Journal of Research in Biology (2012) 2(4): 348-354

Anene et al.,2012

Tables 5: Proximate Composition of C. gariepinus Fed Diets with various Level of Boiled Baobad Seed Meal.
D1 D2 D3 D4 D5
Constituents
0%BBSM 25%BBSM 50%BBSM 75%BBSM 100%BBSM
Moisture 73.77a1.96 74.21a9.65 70.21c2.65 73.77a5.39 71.67b1.96
c d a a
Protein 19.29 0.01 17.50 0.50 21.88 0.11 21.00 1.00 20.13b0.03
ab a bc a
Crude fiber 2.70 0.30 2.90 0.17 2.39 0.09 1.93 0.12 2.00cd0.30
a a a a
Ash 1.00 0.00 1.00 0.00 1.20 0.09 1.15 0.12 1.20a0.10
ab a cd cd
Fat (ether extract) 1.05 0.30 2.90 0.17 1.36 0.24 1.72 0.24 2.88a0.42
a a a a
Carbohydrate 2.19 0.03 2.29 0.49 2.45 0.49 2.13 0.84 2.12a0.25
Value are mean standard deviation of triplicate samples abc means SD with similar superscripts in the
same column are not significant different (p>0.05).

Experimental fish utilized the five (5) different diets at
varying levels that solicited significant differences in
some parameters and did not in some others. The Table
shows that there was a significant difference (p>0.05) in
the mean final body weight of C. gariepinus fed with
different levels of BBSM.
The results indicated that there were significant
differences (p>0.05) in mean body weight gains of fish
groups fed Diet D1 (0% BBSM) and the other diets. This
group of fishes had significantly higher (p>0.05) mean
body weight gain. Mean body weight gain of fish fed
Diets D2 and D3 were not significantly different
(p>0.05). Fish fed on Diets D2 and D3 had significantly
higher mean body weight gain when compared with
those fed with diet D4 and D5. Legumes have been
variously used as a protein source in the diet of fish
(Olvera et al., 1988; Viola, et al., 1988; De-Silva and
Gunasekera, 1989; Hughes, 1991). The specific growth
rate of C. gariepinus fingerlings fed Diet 1 (0% BBSM)
is not significantly different (p>0.05) from those fed with
25% BBSM but significantly different from the
remaining diets. Differences in SGR of C. gariepinis
between feeds significantly declined (p>0.05) as the
level of substitution of boiled baobab seed meal
progressed. However, such significant declines in mean
daily weight gain occurred only beyond 25%
substitution. Gomes et al., (1995) had similarly observed
that the inclusion of plant protein made from rapeseed
and field peas had no effect at up to 15% replacement of
the protein but at 45% inclusion, growth performance of
rainbow trout was significantly lower than the control
diet.
Proximate analyses of fish fillets fed with the
experimental diets
The result of fillet composition of C. gariepinis
fed with the experimental diets is presented in Table 5
and it revealed a significant difference (P<0.05) in fillet
concentrations of moisture, protein, crude fiber and ether
extract while there were no significant differences in the
concentrations of ash and carbohydrates.
As presented in this table, fillets of C. gariepinus
fed diets containing 0% BBSM, 25% BBSM and 100%
BBSM had significantly higher (p<0.05) moisture
contents compared to other diets. Protein content in the
diets containing 50% BBSM (21.88a0.11%) and 75%
BBSM (21.00a1.00%) were significantly higher than for
other test diets. Ether extract levels in fish fillets fed diet
0% BBSM was significantly lower (P<0.05) than the
other treatment groups (Table 5).
CONCLUSION
In this study, boiled baobab seed meals were
incorporated at various levels into the diet of juvenile
C. gariepinus. Analyses of the seeds of A. digidata
indicate that the proximate composition is comparable
with those of plant protein sources. The diets so
formulated were nutritionally acceptable and biologically
available to juvenile C. gariepinus under laboratory
conditions. The protein, fat, crude fibre and moisture
values of the diets used for this study could satisfy the

Journal of Research in Biology (2012) 2(4): 348-354 352


growth requirements of C. gariepinus at this stage of its
development and are comparable to the quality of other
feed ingredients particularly seeds. A similar result was
obtained when Tilapia (Oreochromis niloticus)
fingerlings and juvenile C. gariepinus were fed on
different grains (Solomon 2007; Zaid and Sogbesan,
2010). Using the mean daily weight gain and specific
growth rate as indicators of feed utilization, a 25%
inclusion level is most desirable for juvenile
C. gariepinus.
REFERENCES
Adeyemo AA, Oladosu GA, Ayinla OA. 1994. Growth
and survival of fry of African catfish species
C. gariepinus (burchell) Heterobranchus bidorsalis S.
(geognosy) and heterclarias reared on motional Dubai in
compares with other first feed sources. Aquaculture
119:41-44.
Aduku AO. 1993. Tropical feedstuff analysis table.
Department of Animal science, Faculty of Agriculture,
Ahmadu Bello University, Zaria, Nigeria.
AOAC. 1995. Official Methods of analysis of the
association of official analytical chemists. In: P. Cunniff
(Ed.), International, VA. Vol. II Chapter 49, Arlington,
Virginia, United States. 1-49.
Ayinla OA. 2003. Integrated fish farming: A veritable
tool for poverty alleviation/Hunger eradication in the
Niger Delta Region. In, Eyo A. A and Atanda, J. O
(eds.). Conference Proceedings of Fisheries Society of
Nigeria, Owerri, Nigeria. 40-41
De-Silva SS and Gunasekera RM. 1989. Effect of
dietary protein level and amount of plant ingredient
(Phaseofas attreus) incorporated into the diets on growth
per formance and carca ss compositi on in
Oreochromis aureus fry. Aquaculture (80):123-133.
Ezeagu IE 2005. Baobab (Adansonia digitata l.) seed
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protein utilization in young albino rats I: Biochemical
Ingredients and performance characteristics. Animal
Research International 2(1):240-245.
Ezenwaji HMG. 1985. African Clarias Taxonomy
implication for field work processing of the 4th annual
conference of the Fisheries Society of Nigeria (FISON)
Held at Port Harcourt 26th -29th Nov. 1985. 191-195.
Gnaiger E and Bitterlich G. 1984. Proximate
biochemical composition and caloric content calculated
from elemental CHN analysis: a stoichiometric concept.
Oecologia (Berlin) 62,289-298.
Gomes EF, Rema P and Kaushik SJ. 1995.
Replacement of fish meal by plant proteins in the diet of
rainbow trout (Oncorhynchus mykiss): digestibility and
growth performance. Aquaculture 130(2-3):177-186.
Hughes SG. 1991. Use of lopin flour as a replacement
for fullfat soy in diets for rainbow trout. Aquaculture
93:57-62.
Luquet P. 1991. Tilapia (Oreochromis Spp) in R.P
Wilson (ed). CRC handbook of fin fish. CRC Press, Inc.
Florida. 161-179.
Nkafamiya II, Aliyu BA, Manji AJ and Modibbo UU.
2007. Degradation properties of wild Adansonia digitata
(Baobab) and Prosopsis africana (Lughu) oils on storage.
African Journal of Biotechnology 6(6):751-755.
Olvera NMA, Martinez PCA, Galvan CR and Chavez
SC. 1988. The use of the leguminous plant sesbania
grand flora as a partial replacement for fish meal in diets
for tilapia (Oreochromis mossambicus). Aquaculture
(71):51-60.
Solomon SG, Tiamiyu LO, Agaba UJ. 2007. Effect of
feeding different grain sources on the growth
performance and body composition of Tilapia
(Oreochromis niloticus) fingerlings fed in outdoor hapas.
Pakistan J. Nutr., 6(3):271-275.
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Viola S, Arielity S and Zohar G. 1988. Unusual

feedstuff (tapioca and lopin) as ingredients for carp and
tilapia feed in intensive culture, Bamidgeh, 40:29-34.

Zaid AA and Sogbesan OA. 2010. Evaluation and

potential of cocoyam as carbohydrate source in catfish,
(C. gariepinus [Burchell, 1822]) juvenile diet. African
Journal of Agricultural Research 5(6):453-457.

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Journal of Research in Biology (2012) 2(4): 348-354 354

 Journal of Research in Biology
Purification of phenylalanine ammonia lyase (PAL) and peroxidase (POX) enzymes obtained from
lucerne (Medicago sativa L. cv. Vertus) following inoculation with Verticillium albo-atrum
Journal of Research in Biology
Authors:
Murat Dikilitas.
Institution:
Department of Plant
Protection, Faculty of
Agriculture, Harran
University, S. Urfa, Turkey.
Corresponding author:
Murat Dikilitas.
Web Address:
http://jresearchbiology.com/
documents/RA0220.pdf.
Journal of Research in
Biology
An International Open Access Online
Research Journal
An International Online Open Access
Original Research Publication group
ABSTRACT:
Phenylalanine ammonia lyase (EC 4.3.1.24, PAL) and peroxidase (EC 1.11.1.7,
POX) enzyme activities were determined in seedlings of lucerne
(Medicago sativa L. cv. Vertus) after the inoculation of V. albo-atrum (V2, a weak
pathogenic isolate to lucerne). PAL and POX activities of lucerne inoculated with V2
were found significantly higher than that of the control group after 48 h inoculation.
Partial purification of enzymes with 0-50% (NH4)2SO4 precipitation in control groups
resulted in 2.74 and 2.42 fold increases. On the other hand, 1.78 and 1.74 fold
increases in PAL and POX activities, respectively were evident in V2-inoculated plants.
This study showed that the purification of the enzymes with little effort enabled us to
see the differences between the treatments clearly. This type of work could be
suggested where the protein concentration of the sample is low or a time-scale
experiment is to be carried out to see the enzyme changes or to characterize the
pathogen-related proteins.
Keywords:
Ammonium sulphate precipitation, Verticillium, lucerne, PAL, POX.
Article Citation:
Murat Dikilitas.
Purification of phenylalanine ammonia lyase (PAL) and peroxidase (POX)
enzymes obtained from lucerne (Medicago sativa L. cv. Vertus) following
inoculation with Verticillium albo-atrum.
Journal of Research in Biology (2012) 2(4): 355-361
Dates:
Received: 21 Mar 2012 Accepted: 15 Apr 2012 Published: 01 Jun 2012
Ficus Publishers.
This Open Access article is governed by the Creative Commons Attribution License (http://
creativecommons.org/licenses/by/2.0), which gives permission for unrestricted use, non-
commercial, distribution, and reproduction in all medium, provided the original work is properly
cited.
355-361 | JRB | 2012 | Vol 2 | No 4
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INTRODUCTION
V. albo-atrum is a major problem in lucerne,
cotton and vegetable grown areas. Various cultivars have
different responses to the various isolates of
V. albo-atrum. For example, various levels of resistance
to V. albo-atrum were found to vary from one plant to
another within the same cultivar (personal
communication with Prof. Barbara W. Pennypacker,
USA). Defense responses such as peroxidase
(EC 1.11.1.7, POX) and phenylalanine ammonia lyase
(EC 4.3.1.24, PAL) have been reported to change during
infecion stage. These enzymes are a good criteria to
measure the responses of the crop plants. However,
during infection process, it is difficult to differentiate the
control plants from the infected ones by measuring the
enzyme activities in early stages through crude extract of
plants since the level of enzymes in both control and
infected groups show similar patterns due to similar
protein profiles or similar molecular weights, therefore,
it is difficult to judge about the defence responses at this
stage. Under these circumtances, it would be better to
purify the proteins and measure their enzymatic activities
if very low concentration of proteins exist.
Peroxidase (EC 1.11.1.7, POX) enzymes, in
general, are divided into three groups differing in
molecular weight and in absorption spectra including
ascorbate type, fungal derived and plant derived enzymes
(Das et al., 2011). Of them, plants are the richest sources
of peroxidases and primarily found in roots and leaves of
higher plants, which play important roles in lignifying
cell walls after infection, and thus increasing plant
resistance (Tang, 2001; Dikilitas, 2003; Boka and Orban,
2007; Renugadevi et al., 2011). Plant derived peroxidase
enzymes were isolated, purified and characterized from a
various plant species including horseradish, soybean,
beet, tomato, carrot, wheat, apricot, pears, bananas and
tea (Silva and Franco, 2000; Lavery et al., 2010).
Among them, horseradish peroxidase was the most
commonly used enzyme for the standard preparation and
356
Murat Dikilitas, 2012
characterization of the protein profiles. Isolation of
peroxidase enzymes from microorganisms also led to
increased research interest in this area. Many fungal
agents have been known to produce peroxidase enzymes
(Tsujimura et al., 1994; Hamid and Rehman, 2009).
The role of peroxidase in metabolism is not clear due to
the large number of reactions involved and the
considerable number of isoenzymic species (Kim and
Lee, 2005) although it is clearly involved in the
enzymatic reactions for removal of H2O2 and oxidants in
cells.
Phenylalanine ammonia lyase (EC 4.3.1.24,
PAL), synthesized by a multistep phenylpropanoid
biosynthesis pathway, is the entry-point enzyme
catalysing the deamination of phenylalanine to
transcinnamic acid. The induction of PAL is associated
with the accumulation of secondary pruducts such as
flavonoids and lignins involved in defence responses
providing specific means to protect the plant from stress.
PAL is also involved in phenolics synthesis, including
phytoalexins and suberin (Tang, 2001; Agrios, 2002).
Studies with several different species of plants showed
that PAL activity increases with the biotic and abiotic
stresses (Hammerschmidt, 1999). The resistance of
plants to the pathogen may depend on the speed and the
extent of synthesis of the enzymes. Therefore, PAL
shows the level of resistance of the organism against
stress. For example, Dunn et al., (1998) reported that
after 30 days of high salinity (0.1 M NaCl), citrus plant
grew more slowly and produced lower PAL activity and
as a result of that they became more susceptible to
nematode attack (Tylenchulus semipenetrans). Similarly,
Dikilitas (2003) stated that increase in PAL activity
after the inoculation of elicitor derived from
Verticillium albo-atrum on cell cultures of lucerne
(Medicago sativa cv. Maris Kabul) decreased when the
culture was grown in Murashige and Skoog (M&S)
medium containing 50 mM NaCl. As a result, the
resistant cultivar became more susceptible to the effect
Journal of Research in Biology (2012) 2(4): 355-361
Murat Dikilitas, 2012
of pathogen when exposed to salinity.
Partial prufication of enzymes was reported by
many research people (Altunkaya and Gkmen, 2011;
Hu et al., 2012) to characterize and further investigate
their properties. In this study, partial purification of the
enzymes obtained from the lucerne plants after the
pathogen attack was achieved by (NH4)2SO4
precipitation method to differentiate the enzyme levels at
similar end-points with little effort. Via this method, we
would then be able to see the differences between the
severity of stress factors such as salinity and the
pathogens in crop plants by demonstrating their
combined and seperate effects of each stress factor. Data
of this study would also enable us to establish other
purification systems for further purifications.
MATERIALS AND METHODS
Inoculation of plants
V. albo-atrum, isolate V2 were isolated from
tomato plants and maintained in PDA and Dox agar
media. The fungal culture was originally obtained from
the culture collection of Dr Chris J. Smith (University of
Wales, Swansea-UK). The isolate was characterized as a
weak pathogen according to studies carried out by
Dikilita s (1997 and 2003). Lu cer n e
(Medicago sativa cv. Vertus) plants were inoculated with
the root dipping method with the spores of
V. albo-atrum, V2 (1x107 conidia ml-1). For this, plants
were removed from pots and the soil was removed by
shaking the plants gently, followed by washing in tap
water. The root of the plants were then placed in the
spore suspension for 10 min. The inoculated plants were
replanted using the same soil. Distilled water was used
for the control plants.
Preparation and purification of tissue homogenate for
POX
Ten grams of fresh lucerne leaves were
homogenized at 4C with 100 ml of 50 mM Na
phosphate buffer (pH 7.0). The homogenate was
Journal of Research in Biology (2012) 2(4): 0355-361
centrifuged at 10,000 g for 15 min at 4C.
The supernatant obtained at this stage was designated as
crude extract.
The crude extract was then partially purified by
adding solid ammonium sulphate (calculated through the
web site http://www.encorbio.com) to give 0-50%
saturation and the precipitate was collected by
centrifugation at 10,000 g for 90 min at 4C.
The precipitate was then suspended in about 10 ml of the
same buffer which was used for homogenization and
dialyzed overnight at 4C against 40 mM Na phosphate
buffer (pH 7.0) to remove ammonium sulphate and other
minerals. The supernatant at this stage was designated as
purified enzyme.
The activity of POX in the supernatant was
determined before and after partial purification.
Determination of POX activity
POX activity was assayed with guaiacol
according to the modified method of Meriga et al.,
(2004). A 3 ml reaction mixture containing 13 mmol l -1
guaiacol, 5 mmol l-1 H2O2, 50 mmol l-1 Na phosphate
buffer (pH 6.5) and 500 ml of enzyme solution was
prepared. Oxidation of guaiacol was followed by the
increase of absorbance at 470 nm, after initiating the
reaction with H2O2. One unit of POX activity was
defined as 0.01 A470 per min.
Preparation and purification of tissue homogenate for
PAL
To assess the early defence responses of lucerne
leaves, PAL activity was measured. Ten grams of fresh
leaves were homogenized at 4C with 100 ml of 50 mM
Tris-HCl, pH 8.4, containing 4 mM Na2EDTA, 10 mM
mercaptoethanol, 2 mM ascorbic acid and 1 mM PMSF
(phenylmethylsulfonyl fluoride) and 1 g acid-washed
sand with a cold pestle and mortar. The homogenate was
filtered through two layers of wet Mira Cloth
(Calbiochem), and the resulting filtrate was centrifuged
(20,000 g, for 20 min at 4C).
The supernatant was then dialyzed overnight
357

against two litres of dialysis buffer (50 mM Tris-HCl, pH
8.4 containing 10 mM mercaptoethanol, 4 mM
Na2EDTA and 0.5 mM PMSF).
The crude extract was then partially purified as in
that of POX except that the buffer was changed into
Tris-HCl (pH 8.8).
Determination of PAL activity
PAL activity was assayed using a method
described by Bolwell et al., (1985) with slight
modifications (Dikilitas, 2003). The reaction mixture
contained the following components: 1.5 ml of 50 mM
Tris-HCl (pH 8.4) containing 4 mM Na2 EDTA, 10 mM
mercaptoethanol, 5 mM ascorbic acid and 1M PMSF
and 1 ml of 10 mM L-phenylalanine
concentration). The reaction was then started by the
addition of enzyme extract (0.5 ml) containing
approximately 500 mg protein to the mixture, which was
incubated at 40C for two hours. Assays were performed
in duplicate and the control incubation was prepared
using D-phenylalanine (10 mM final concentration) in
place of the L-isomer. Changes in absorbance was
recorded at 30 min intervals at 290 nm using UV-1700
Shimadzu Spectrophotometer.
Activities have been calculated from the molar
absorption coefficient of trans-cinnamic acid at 290 nm,
-1
which was determined to be 10, 900 litre mol cm
under the conditions of assay. PAL activity was
-1 -1
expressed as: nmol cinnamic acid mg protein h . The
change in absorbance was converted to nmol of
trans-cinnamic acid using the following equation;
A Ecl
A : Absorbance at 290 nm.
c : Concentration of cinnamic acid in Moles/L.
l : Path length of light (1 cm).
E: The molar absorption coefficient of cinnamic acid at
290 nm, which was determined to
10,900 litres mol-1 cm-1
The rate of formation of trans-cinnamic acid was
taken as a measure of enzyme activity. One unit of PAL
358
Murat Dikilitas, 2012
was defined as 0.01 A at A290 per hour, which was
calculated as 2.74 nmol of trans-cinnamic acid.
Protein determination
Protein in the samples was determined in a
Coomassie blue dye-binding assay by the method of
Bradford (1976) with standard curves prepared using
bovine serum albumin (BSA, Sigma).
Chemicals
All chemicals were from Sigma and Fisher Co.
except where specified.
RESULTS AND DISCUSSION
POX and PAL enzymes from lucerne plants
(final inoculated with the weak pathogen V. albo-atrum, isolate
V2 showed higher activities than that of control group.
The activities from control and inoculated plants in
respect to both enzymes were found much higher
following purification with ammonium sulphate
precipitation, 0-50% (Table 1). Ammonium sulphate
precipitation was done by using the finely ground
ammonium sulphate. The rich form of proteins and
enzymes after precipitation were subjected to dialysis to
remove extra salts and minerals. Civello et al., (1995)
reported an increase in POX activity in strawberry fruit
following purification of ammonium sulphate. Similarly,
-1
Zia et al., (2011) stated that 1.16 and 4.14 fold
purification was achieved in apple and orange seeds,
respectively following ammonium sulphate precipitation.
Again, Mohamed et al., (2008) reported the peroxidase
activity of orange and apple seed extracts were increased
as compared to crude enzyme extract up to 30.64 and
8.34 fold with their specific activity of 18.16 and
9.20 U/mg, respectively following purification.
The increase in enzyme activities were reported
in many plant & abiotic and plant & microorganism
be interactions (Chutia et al., 2012). An increase in enzyme
activities are regarded as a general defense response of
the organism to the effect of stress (Dikilitas, 2003). In
our study, the effect of V. albo-atrum, isolate V2 led to
Journal of Research in Biology (2012) 2(4): 355-361
Murat Dikilitas, 2012

Table 1. Purification and activities of POX and PAL of lucerne inoculated with V. albo-atrum, isolate V2.
Total Protein Total Total Specific
Enzymes- Purification Activity Yield Fold
Volume Content protein activity Activity
Treatments Steps (EU ml-1) (%) purification
(ml) (mg ml-1) (mg) (EU) (EU mg-1)

POX
Control Crude 16 90 0.50 45.0 1440 32 100 1
(NH4)2SO4 48 11 0.62 6.82 528 77.42 37 2.42
V2 Crude 136 92 0.49 45.08 12512 277.55 100 1
(NH4)2SO4 265 11 0.55 6.05 2915 481.81 23 1.74
PAL
Control Crude 22 90 0.45 40.5 1980 48.88 100 1
(NH4)2SO4 87 11 0.65 7.15 957 133.84 48 2.74
V2 Crude 150 94 0.47 44.18 14100 319.14 100 1
(NH4)2SO4 329 12 0.58 6.96 3948 567.24 28 1.78

increase in activities of enzymes such as POX and PAL CONCLUSIONS

after 48 h of inoculation. The increased activities of
enzymes and their mechanisms which led to various
defense responses were discussed in detail in the works
of (Tang, 2001; Eldeen et al., 2010). The main issue here
is to evaluate the purification of enzymes by determining
their activities. In this study, the purification procedure
allowed us POX and PAL to be purified 2.42- and 2.74-
fold with 37 and 48% recovery, respectively as compared
to those of the crude extracts of controls. Similarly,
POX and PAL enzymes were purified 1.74- and
1.78-fold with 23 and 28% recovery, respectively in
V2-incoulated plants as compared to their crude extracts.
In POX enzyme, the ratio between control and V2 was
0.11 (32/277) in crude extracts while this ratio increased
up to 0.16 (77/481) in ammonium precipitated extracts.
Similar case was also observed in PAL in which the ratio
between control and V2 was 0.15 (48/319) in crude
extracts while the ratio between control and V2 was
0.23 (133/567) in precipitated extracts. Although the
ratios between control and V2 showed similar trends in
both enzymes in respect to their activities before and
after purification, however, purification enabled us to see
the differences more clearly through the higher
absorbance values. This type of work could well be
suggested in similar absorbance values obtained in which
the control and treatment groups do not differ from each
other.
In this work, POX and PAL enzymes from
lucerne leaves were extracted and partially purified with
ammonium sulphate precipitation. According to the
results of analysis, the purified enzymes gave higher
absorbance values and clear cut-offs between treatments
were evident when compared to those of crude extracts.
Purification could be helpful to find out the differences
between control and treatment groups as well as in
determining the primary and secondary infections in
which the induced enzyme activities might not differ in
crude extracts.
ACKNOWLEGEMENTS
The use of trade names in this document does not
imply endorsement by the Harran University nor
criticism of similar ones not mentioned. A part of this
study was conducted at Central Science and Plant
Pathology Laboratories of Harran University. The author
would like to express his gratitude the technical staff of
the laboratories.
REFERENCES
Agrios GN. 2002. Plant Pathology. Academic Press.
4th ed.
Altunkaya A, Gkmen V. 2011. Purification and
characterization of polyphenol oxidase, peroxidase and

Journal of Research in Biology (2012) 2(4): 355-361 359


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 Journal of Research in Biology An International Scientific Research Journal

Original Research

The effect of MRET activated water on plants

Journal of Research in Biology

Authors: ABSTRACT:
Igor Smirnov.
This particular article relates to the study of the effect of MRET Activated
water on growth and development of higher plants. It provides some evidence that
MRET Activated water with the modified physical and electrodynamic characteristics
Institution: may enhance specific molecular mechanisms in living cells of botanical origin. In
Global Quantech, Inc.,
particular, the discovered change (a reduction) of the viscosity of activated water
Encinitas, California, USA.
should influence essentially the movement of cellular juice in the vessels of xylem and
phloem. Change of the conductivity and the dielectric permittivity should render a
strong influence on the movement and the characteristics of ions in water. The living
Corresponding author: cells of botanical origin have rather complex structure, consisting of the folding
Igor Smirnov. membranes, the specialized connections, and organelles. The localization of ions is
particularly important, since each of the complex structures must be expected to have
a specific role in the electrical function of the cells and as a result it may affect plants
germination and development. To verify the validity of the proposed hypothesis the
Email: study on higher plants was conducted at the Institute of Cellular Biology and Gene
igor@gqusa.com Engineering of the National Academy of Sciences of Ukraine.

Keywords:
Viscosity, dielectric permittivity, germination; plant, root system, MRET
Fax No:
Water.
(760)652-5173.

Web Address: Article Citation:

http://jresearchbiology.com/
documents/RA0232.pdf.
Igor Smirnov.
The effect of MRET activated water on plants.
Journal of Research in Biology (2012) 2(4): 362-369
Dates:
Received: 13 Apr 2012 Accepted: 14 May 2012 Published: 01 Jun 2012

This article is governed by the Creative Commons Attribution License (http://creativecommons.org/

licenses/by/2.0), which gives permission for unrestricted use, non-commercial, distribution and
reproduction in all medium, provided the original work is properly cited.

362-369 | JRB | 2012 | Vol 2 | No 4

Journal of Research in Biology
An International
Scientific Research Journal www.jresearchbiology.com

INTRODUCTION:
Water makes up about 90% of all the contents of
plant cells. Alongside macro- and microelements, it is a
necessary part of any soil mixture for the cultivation of
plants under natural conditions, as well as the cultural
medium for the cultivation of plants under sterile
conditions. In the vessels of xylem and phloem and in the
lacticifers of higher plants, there is a kind of vegetative
juice, which is analogous in function to blood plasma of
higher animals and even human. The composition of this
juice includes a big complex of organic substances and
inorganic elements. The composition and concentration
of these substances and elements differ strongly not only
in different plants, but also in different parts of the same
plant. The main characteristic that unites all kinds of
cellular juice is the large content of water which reaches
98% in relative concentration. With the lack of water, all
growing processes are violated, which can cause the
death of plants. We may say without exaggeration that
water is the main component of any plant, and its
presence is the main condition for the plants existence
as a living being. This implies at once that water is the
main element of a biological system, whose influence
allows one to realize the strongest influence on this
system. Thus, the introduction of water with any 3
particular characteristics into soil (irrigation) or into a
cultural medium will definitely influence the basic
growing parameters of plants. Such water can render a
positive or negative influence on all developmental
phases of a plant, including the peculiarities of the
germination of seeds (speed of germination, percentage
of germinating capacity), the growth of the over ground
part of a plant e.g. a sprout (the growth of its height and
weight), and the growth of a root system. It can also
influence the size of leaves (the area of a leave surface),
and can cause the appearance of a typical (by their form,
size, and color) leaves.
The studies of the influence of MRET activated
water on plants were carried out under the supervision of
363
Smirnov, 2012
N.A. Matveeva, Ph.D. at the Institute of Cellular Biology
and Gene Engineering of the National Academy of
Sciences of Ukraine.
In addition to the results of studying the
influence of MRET activated water on the growth of
plants under natural conditions, we also discuss here its
influence on the growth of sterile cultures under
conditions of a special cultural medium.
The usage of sterile cultures for the study of the
influence of MRET activated water is caused by certain
circumstances, among which the most important is the
increase of reliability and authenticity of the obtained
results. The point is that a sterile culture does not
practically contain any bacteria.
Therefore, the influence of extraneous factors
(for example, infection or contamination) is practically
excluded. This circumstance allows us to judge the direct
effect rendered by the additional factors connected, in
particular, the usage of activated water. The results of
studies of the physico-molecular properties of MRET
activated water stated below allow us to predict the
influence of water activation on the development of
plants. In particular, the discovered change (a reduction)
of the viscosity of activated water should influence
essentially the movement of cellular juice in the vessels
of xylem and phloem. Change of the conductivity and
the dielectric permittivity should render a strong
influence on the movement and the characteristics of
ions in water (Smirnov, 2007, 2008).
Thus, investigating some key parameters, which
characterize the growth of plants, allows us to make
certain conclusions about the influence of some
particular properties of MRET activated water on plants
and, eventually, to find the optimum ways of using such
water.
METHOD AND MATERIALS:
The different kinds of plants were used in the
experiments to investigate effects of MRET activated
Journal of Research in Biology (2012) 2(4): 362-369
Smirnov, 2012
water. These systems can be represented by two
hierarchical levels:
higher plants grown in a nutritious medium;
higher plants grown in a soil;
The influence of MRET activated water
(activated for 30 minutes and 60 minutes respectively)
on the growth of plants in soil and under conditions of a
sterile cultural medium was investigated in these
experiments. Water was activated directly before its
usage or it was kept after the activation in a cooler at a
temperature of 4C no longer than one day. Control
plants were irrigated with similar, non-activated water.
To prepare sterile culture, a sterile concentrate of
the medium was prepared beforehand, which was then
diluted with distilled water and which was later
activated. In order to study the influence of activated
water on higher plants, sterile higher plants such as
Solanum tuberosum of grade Lugovskoi and
Solanum rickii capable of growth in sterile cultural
media (in sterile test tubes) were used as the object of
study. The MurashigeSkoog standard sterile cultural
medium (Murashige and Skoog, 1962) was prepared for
the growth of plants. For the preparation of an agar-
based medium, we took one part of ordinary distilled
water and four parts of the corresponding MRET
activated water.
Firstly, a concentrated solution of the cultural
medium dissolved in a small amount of ordinary distilled
water was prepared. After sterilization and cooling, this
solution was diluted with the corresponding fraction of
activated water in the ratio of 1:4. In such a way, the
cultural medium was enriched with a specific fraction of
activated water by 80%. For the use of the liquid cultural
medium, it was activated for 0.5 h or 1 h after the
sterilization process directly in sterile test tubes under
conditions of a sterile box. To preserve the sterility of
experiments, special measures were undertaken. In
particular, in the case of a liquid cultural medium, it was
activated after the sterilization directly in sterile test
Journal of Research in Biology (2012) 2(4): 362-369
tubes. Control and experimental plants were cultivated
under identical conditions of illumination and at the
same temperature. In test tubes with the sterile medium,
the parts of plants (shoots) having one bud were sown.
Plants were cultivated in the presence of light at a
temperature of 20C with the following light/dark period
within each day: 16 h of light - 8 h of darkness. In three
weeks after the sowing, the evaluated plants were taken
from test tubes, and the measurement of their major
parameters: the 5 height of plants, weight of the over
ground part, and weight of leaves was carried out, and
the surface area of leaves were evaluated as well
(Vysotskii et al., 2009).
In addition, after the completion of each
experiment, the following coefficients of action of
activated water which characterize the average
parameters of evolved plants were determined:
1. Coefficient of inhibitory action of activated water
on the growth of plants in the sterile culture
K1 = N1a : N1c. Here, N1a is the amount of segments
survived from the cultivation in the medium with
MRET activated water, and N1c is the amount of
segments survived from the cultivation in the control
medium.
2. Coefficient of stalk formation K2 = N2a : N2c.
Here, N2a is the amount of segments, of which
sprouts in the medium with MRET activated water
were formed, and N2c is the amount of segments, of
which sprouts in the control medium were formed.
3. Coefficient of the length of formed sprouts
K3 = La : Lc. Here, La is the average length of the
sprouts formed on one segment cultivated with
activated water, and Lc is the average length of a
sprout formed on one segment in the control.
4. Coefficient of a change of the coloring of leaves
K4 = N4a : N4c. Here, N4a is the amount of plants
with atypical coloring of leaves in the medium with
MRET activated water, and N4c is the amount of
plants with atypical coloring of leaves in the control
364
 Smirnov, 2012

medium. we carried out each experiment simultaneously

5. Coefficient of root formation K5 = N5a : N5c.
Here, N5a is the amount of segments, on which roots
were formed in 3 weeks of the cultivation in the
medium with MRET activated water, and N5c is the
amount of segments on which roots in the control
medium were formed.
For the study the influence of different fractions
of MRET activated water on the germination of seeds
and the formation of leaves, seeds of the following
vegetable crops were used: radish Red giant and
Krasa rannyaya, peas Alpha, string beans asparagus
Valentino, cabbage of grade Dymerskaya and
pumpkin of grade Zhdana.
Test Results
The growth of plant Solanum tuberosum
(one plant in a separate test tube for each fraction of
MRET activated water and for the control one) was
investigated to study the 6 influence of MRET activated
water. To get reliable data and the necessary statistics,
(in parallel) as a series of 11 recurrences. In general, 33
plants were studied. For a plant Solanum rickii, the study
was carried out according to an analogous scenario (one
plant in a separate test tube for each fraction of MRET
activated water and for the control one) with the use of a
series of 20 recurrences. In total, 60 plants were studied.
The data of experiments, the metrological parameters of
evolve plants Solanum tuberosum of grade Lugovskoi,
and the general view of evolve plants are presented in
Figs. 1 - 2, and in Tables 1-2 respectively.
To study the influence of different fractions of
MRET activated water on the germination of seeds and
the formation of leaves, the seeds of the following
vegetable crops were used: radish Red giant and
Krasa rannyaya, peas Alpha, string beans asparagus
Valentino, cabbage of grade Dymerskaya and
pumpkin of grade Zhdana. These seeds were sown into
compositionally similar soil and were periodically
irrigated with a certain fraction of water. The study of the

Fig 1. The view of sterile higher plants Solanum tuberosum in test tubes in the sterile cultural medium based
on MRET activated water and regular control) water after three weeks cultivation period of time.

Table 1 Influence of MRET activated water on the growth of sterile higher plants solanum tuberosum.

Average height Average weight of the Average Weight Average area

Fraction of Water over ground part of a plant
of a plant of leaves of a plant of leaves of a plant
tact = 1.0 h 7.8 cm 0.072g 0.013g 0.93 cm2
tact = 0.5 h 6.8 cm 0.068g 0.014g 1.00 cm2
Control 4.4 cm 0.058g 0.013g 0.93 cm2

365 Journal of Research in Biology (2012) 2(4): 362-369

Smirnov, 2012

Fig.2. Sterile higher plants Solanum tuberosum taken from test tubes after three weeks cultivation period of
time in the sterile cultural medium based on MRET activated water and regular (control) water.

Table 2 Coefficients of action of MRET activated numbers of sprouts using activated and 10 control
water on the growth of sterile higher plants
solanum tuberosum (non-activated) water was insignificant. It is found that
water activated for 0.5 h increased the number of sprouts
Fraction of Water K1 K2 K3 K4 K5
in comparison with the control for radish and peas. In 10-
tact = 1.0 h 1 1 1.77 0 1
20 days of the cultivation, the effect of MRET activated
tact = 0.5h 1 1 1.54 0 1 water (tact = 0.5 h) on the development of plants was
germination of above-mentioned vegetable seeds similar to that of water activated for one hour.
irrigated with two different fractions of MRET activated The photos given in Figs 3-5 and the data
water and regular water has shown that, practically for presented in Table 3-4 allows to conclude that the
all tested plants (except for string beans), irrigation with activation of water renders essential stimulating
water activated for 60 min promoted a much faster influence and promotes a much 11earlier germination of
germination of seeds at the initial stage. The example of radish seeds. The strongest effect of stimulation
such stimulating influence of MRET activated water on corresponds to MRET water activated for 1 hour.
the germination of seeds of radish Red giant for the
first 10 days is presented in Figs. 3-4. Thirty seeds were CONCLUSION:
sown in a Petri dish. The first shoots of this plant have MRET-activated water is produced with the help
appeared in all variants in four days after sowing. The of a patented in the USA Molecular Resonance Effect
sequence and characteristics of the seed germination are Technology (MRET). The MRET water activator is the
presented in Table 3-4. stationary source of a subtle, low frequency, resonant
It is clear that the usage of MRET water electromagnetic field with a composite structure. The
activated for one hour renders a very large stimulating origin of the low frequency composite electromagnetic
effect on vegetable crops at the beginning of the field is the intensive electrical activity inside the
cultivation period. In 10-20 days after the beginning of nano-circles formed by linear molecular groups of the
the cultivation, the effect of the stimulation was MRET polymer compound when the polymeric body is
noticeably reduced, and the final difference between the exposed to the external electromagnetic fields.
Table 3 Germination of seeds of radish Red giant irrigated with MRET activated and control (regular) water
Fraction of water Number of sprouts Number of sprouts Number of sprouts Number of sprouts
for irrigation in 4 day after sowing in 5 day after sowing in 6 day after sowing in 9 day after sowing
tact = 1.0 h 15 23 25 28
tact = 0.5 h 12 20 22 25
Control 5 14 16 24

Journal of Research in Biology (2012) 2(4): 362-369 366

 Smirnov, 2012

MRET Activated water with the modified physical and The localization of ions is particularly important, since
electrodynamic characteristics may enhance specific each of the complex structures must be expected to have
molecular mechanisms in living cells of botanical origin. a specific role in the electrical function of the cells and as
In particular, the discovered change (a reduction) of the a result it may affect plants germination and
viscosity of activated water should influence essentially development. The analysis of results of the plants
the movement of cellular juice in the vessels of xylem cultivation experiments allows to make the following
and phloem. Change of the conductivity and the conclusions:
dielectric permittivity should render a strong influence For 25 days of the observation of the growth of
on the movement and the characteristics of ions in water. sprouts of radish Red giant, a gradual and

Fig. 3 Shoots of radish Red giant in 4 days after
sowing into soil. Immediately after the sowing of
plants, the soil was irrigated with ordinary (control)
or MRET activated water with the duration of
activation of 1 h and 0.5 h.
367
Fig. 4 Shoots of radish Red giant in 9 days after
the sowing in to soil (soil was irrigated with control
and MRET activated water).
Journal of Research in Biology (2012) 2(4): 362-369
Smirnov, 2012

Table 4 Characteristics of radish Red giant irrigated with MRET activated water compared
with control (regular) water in 25 days after the appearance of shoots

Average height of the over Average weight of the over Average area of the surface
Fraction ground part of a plant in 25 ground part of a plant in 25 of leaves of a plant in 25
of water days after the appearance of days after the appearance of days after the appearance of
shoots shoots shoots

tact = 1.0 h 121.9% 157.1% 137.6%

tact = 0.5 h 106.9% 103.5% 94.2%

Control 100% 100% 100%

accumulating change of the intensities of the growth of

plants was registered for those irrigated with MRET
water activated for 1 h. At the end of the observation
period, the plants which were irrigated with such
activated water exceeded substantially the control plants
in all parameters (as for the increment of the average
height of the overground part of a plant by 21.9%, the
average weight of the over-ground part of a plant by
57.1%, and the average area of the surface of leaves of a
plant by 37.6%). At the same time, the differences
between the plants irrigated with MRET water activated
for 0.5 h and control plants turned out to be insignificant.
MRET activated water is non toxic for plants of
the sterile culture. The survivability of the sowed
material in all variants made 100% and did not differ
from that in a cultural medium prepared with regular
water.
MRET activated water does not interfere with the
normal growth of the over-ground part of plants and the
root system and does not result in the appearance of
atypical coloring of leaves.
The average increase in the height of plants by
77.2% and the weight of their over-ground part by 24.1%
was observed for plants Solanum tuberosum for the
growth in the medium with MRET activated water
compared to the control samples.
Thus, studying some key parameters, which

Fig. 5 Plants of radish Red giant in 25 days characterize the growth of plants, allows to make certain
after the sowing. conclusions about the influence of MRET activated

Journal of Research in Biology (2012) 2(4): 362-369 368

 Smirnov, 2012

water on plants and, eventually, to find the optimum

ways of using such water.

REFERENCE:
Murashige T and Skoog F. 1962. A revised Medium
for Rapid Growth and Bioassays with Tobacco culture,
Physiol. Plant, 15:473-497.

Smirnov I. 2007. The Anomalous Low Viscosity and

Polarized-Oriented Multilayer Molecular Structure of
MRET Activated Water, Explore Magazine, Vol.16,
No.4:37-39.

Smirnov I. 2008. The Anomalous Electrodynamic

Characteristics and Polarized-Oriented Multilayer
Molecular Structure of MRET-Activated Water,
International Journal of Nanoscience, Vol. 7, Nos. 4 & 5,
223227.

Vysotskii V, Kornilova A, Smirnov I. 2009.

APPLIED BIOPHYSICS OF ACTIVATED WATER:
The Physical Properties, Biological Effects and Medical
Applications of MRET Activated Water, World
Scientific Publishing Co., Singapore.

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 Journal of Research in Biology
Journal of Research in Biology
Authors:
Rajesh Sawhney.
Institution:
Bhojia Dental College and
Hospital, Budh, Baddi. Distt.
Solan (H.P) India.
Corresponding author:
Rajesh Sawhney.
Email:
sawhneyrajesh@yahoo.com front to fight the nuisance of drug resistance.
Web Address:
http://jresearchbiology.com/
documents/RA0234.pdf.
Journal of Research in Biology
An International
Scientific Research Journal
An International Scientific Research Journal
Overview
Drug resistance: a social perspective
ABSTRACT:
Drug resistance is a defensive strategy developed by the microorganisms to
evade the detrimental effects of antimicrobial agents. Over the years microorganisms
have successfully counteracted the action of antimicrobials using both genetic
methods such as random mutations, chromosomal or plasmid mediated transfer of
genetic information and biochemical mechanisms like decreased permeability of the
organism to the drug, inactivation of the inhibitor by enzyme produced by the
resistant organism, modification of the properties of the drug receptor site, increased
synthesis of an essential metabolite antagonistic for the drug. However, the human
governed factors viz. faulty prescriptions, misdiagnosis, self medication, incomplete
medication, reservation for antibiotic sensitivity tests, supplementation of antibiotics
in cattle feed and toiletries seem to have taught and prompted the microorganisms,
the so called miniature industries, to gear up to develop antibiotic resistant
mechanisms. As a consequence, multi-drug resistant strains have posed a challenge to
the humanity. The costly inputs and hardships of scientists in the laboratories unravel
the mysteries and present a valuable product and technologies for human well being.
However, the judicious use of the process, product or technology remains the joint
responsibility of common masses, technocrats and the government. Thus, our roles to
curb the factors that lead to drug resistance need to be given key priority.
The approach could shun the burden of scientists and open new and well executed
Keywords:
Drug resistance, Antimicrobials, Medications.
Article Citation:
Rajesh Sawhney.
Drug resistance: a social perspective.
Journal of Research in Biology (2012) 2(4): 370-376
Dates:
Received: 23 Apr 2012 Accepted: 14 May 2012 Published: 04 Jun 2012
This article is governed by the Creative Commons Attribution License (http://creativecommons.org/
licenses/by/2.0), which gives permission for unrestricted use, non-commercial, distribution and
reproduction in all medium, provided the original work is properly cited.
370-376 | JRB | 2012 | Vol 2 | No 4
www.jresearchbiology.com
 Sawhney, 2012

INTRODUCTION: Loss of enzymes involved in drug activation

Drug resistance is a defensive mechanism (Rosolan et al., 2002).
developed by the microorganisms to evade the Biofilm formation: The biofilms have been reported
detrimental effects of antimicrobial agents. Over the to be less susceptible to antimicrobial agents and has
years number of microorganisms such as Staphylococcus reduced sensitivity to inhibitors, thereby adding to
(MRSA and VRSA), Mycobacterium tuberculosis their survival (Jabra-Rizk et al., 2006). The findings
(multidrug resistant strains), vancomycin resistant have shown delayed penetration of Ciprofloxacin
enterococci, Streptococus pneumoniae, H. influenzae, into Pseudomonas aeruginosa biofilms (Suci et al.,
Vibrio cholerae, multidrug resistant Acinetobacter, 1994). E. coli biofilms exhibited decreased
Pseudomonas, Serratia, Stenotrophomonas sp, susceptibility to cetrimide (Evans et al., 1990).
Escherichia coli and Klebsiellae.,Shigella, Neisseria Similar r ep or t s are available in
gonorrhoeae, Neisseria meningitidis have successfully Staphylococcus aureus exposed to tobramycin
counteracted the action of one or the other antimicrobial (DuGuid et al., 1990). The resistance shown by
(Raghunath, 2008). Microorganisms exhibit genetic, these biofilms in general has been attributed to
biochemical and phenotypic mechanisms to develop this factors such as poor penetration of antimicrobials,
resistance. nutrient limitation, accumulation of toxic
Mutations give rise to drug resistant mutants with metabolites and decreased oxygen tension (Tresse et
altered susceptibility to the drug (Russell, 2002). al., 1995).
Chromosomal or plasmid mediated transfer of Sa li cyl a t e-In duced anti bi ot i c r esi st an ce
genetic information pertaining to drug resistance (Cohen et al., 1993).
from resistant organisms to the susceptible one. Obviously, to be fit for survival, the
Such transfer is accomplished through conjugation, microorganisms have to learn ways to counteract the
transformation or transduction (Lacey,1975; Sheehy, effects of their killing agents. However, a number of
and Novick, 1975; Elwell et al., 1978; Brunton, factors might have tempted and given sufficient room to
1986; Dowson et al., 1989). these miniature industries to device the mechanisms for
Decreased permeability of the organism to the drug evading the detrimental effects of the antimicrobials.
(Aires et al., 1999; Poole, 2001). Drug resistance: the tempting factors
Inactivation of the inhibitor by enzyme produced by The researchers have come up with the
the resistant organism (Handsfield, 1982; Aronoff, conclusive evidence to the reasons that felicitate the
1989; Tomasz, 1990). organisms to gear up for drug resistance.
Modification of the properties of the drug receptor Faulty medical prescriptions: About 50% of the
site (Handwerger and Tomasz, 1986; antibiotic prescriptions in the hospitals are given without
Jabes et al.,1989). clear evidence of infection or adequate medical
Altered metabolic pathway, as shown by indication (Jain, 1996).
sulphonamide resistant bacteria that utilize Misdiagnosis of certain infections have led to wrong
preformed folic acid and do not require presence of administration or unwanted /undue prescription of
PABA in extracellular fluid (Chakraborty, 1996). antibiotics (Willey et al., 2007). Physicians have
administered antibacterial drugs to patients with cold,
influenza, viral pneumonia and other viral diseases.
371 Journal of Research in Biology (2012) 2(4): 370-376
Sawhney, 2012
However, it has been documented earlier that the patients
diagnosed with colds and upper respiratory tract
infections are given antibiotics in spite of the fact that
90% of these cases are caused by viruses (Willey et al.,
2007).
Self medication: Drugs are consumed without
consulting the qualified medical practitioners. This leads
to the wrong choice as well as under dose of the drug.
Thus, giving an opportunity to the microorganism to
learn the drug resistance strategies; Self-medication with
antibiotics may increase the risk of inappropriate use and
the selection of resistant bacterial strains (Chalker, 2001;
Grigoryan, 2007; Nalini, 2010).
Incomplete medication course: the situation is made
worst by patients not completing their course of
medication. When antibiotic treatment is ended too early,
drug resistant mutants may survive. The under or over
prescribing may lead to drug resistance
(Akkerman et al., 2005).
Use of antibiotics in feed supplements: Use of
antibiotics as growth promoters in animal feed is another
reason for emergence of resistant bacteria (Little et al.,
1986). The addition of low levels of antibiotics to
livestock feeds do raise the feed efficiency and growth in
cattle, pig, chicken (Pond et al., 2005). There is an
evidence of Salmonella Newport infection resulting from
eating hamburger from beef cattle fed sub-therapeutic
doses of chlortetracycline for growth promotion
(Cromwell, 2001; Hay, 2005). The use of quinolone
antibiotic enrofloxacin in swine herds appear to have
promoted ciprofloxacin resistance in pathogenic strain of
Salmonella.
Antibiotics in daily toiletries: The spread of antibiotic
resistance could be due to quite subtle factors eg.
products such as soaps, deodorant, moth washes, cutting
boards, baby toys often now contains triclosan and other
germicides. There is an increasing evidence that
widespread use of triclosan actually favours an antibiotic
Journal of Research in Biology (2012) 2(4): 370-376
resistance (Bamber and Neal, 1999).
Besides all the above mentioned points, skipping
culture/sensitivity tests might be a factor leading to
development of drug resistant strains. Antibiotics are
administered without culture and sensitivity test. The
broad spectrum drugs are given as substitute for culture
sensitivity test with consequent risk of dangerous side
effects, super infections and selection of drug resistant
mutants.
Microbial preparedness, present scenario & future
perspective:
Numerous reports have sprung up on emergence
of drug resistant strains (Overturf et al., 1974; Crossley
et al., 1979; Koornhof, 1980; Peacock et al., 1981;
Saravoltz et al., 1982; Weinstein et al., 1982; Hawkey,
1984; Archer et al., 1985; Craven et al., 1986; Cohen
and Tauxe, 1986; Warren, 1986; Henderson et al., 1988).
The advent of newer drugs, the drug resistance pattern
has also changed drastically with evolution of multi-drug
resistant strains (Varaiya and Gogate, 1998). This change
has posed a new challenge to the scientists across the
world in respect of discovering new combat weapons for
the microbial entities.
The miniatures have exhibited wonderful warfare
strategies by developing new defense mechanisms. The
ultimate goal seems to be nonetheless their survival and
continuance of generation. A cold war between
pathogenic microorganisms and the mankind is on.
Scientists are busy with discovering effective
antimicrobial molecules and on the same time the
miniatures are excelling fast in evolving efficient
virulence factors. Undoubtedly, scientists have done
commendable job by discovering agents of control and
elimination of these dreaded microorganisms. However,
we could hardly boost of eradicating only a couple of the
noxious microbial entities. Moreover, the expense per
eradication or control in terms of resources, finance,
manpower and time is beyond calculation.
372

Thus, there is a need to introspect and redraw the
strategies for control and elimination of pathogenic
organisms. Scientists have stressed the need for
surveillance of isolates for antibiotic resistance
(Adebayo et al., 2012). Besides pumping our energies
and resources in evolving new molecules, processes and
technologies, the outcome of highly expensive researches
need to be used judiciously to the benefit of mankind.
Prevention is better than cure needs to be practiced in
its true sense to nip the pathogen in the bud before it
exhibits itself in uncontrolled proportions.
microorganisms are pretty smart and active in terms of
multiplication, reproduction and emergence.
Neisseria gonorrhoeae could learn to evade the effect of
sulphonamides in just six years and took only sixteen
years to resist the action of penicillin (Willey et al.,
2007). This implies that either the development of new
antimicrobial molecules to be faster or existing
molecules rendered effective for longer periods through
our practices. Moreover, there is a need to view the drug
resistance from social perspective and sincerely
implement and practice the guidelines that are enforced
as a result of proven research outcomes. Further, the ill
effects arising due to non-implementation need to be
undone. This would be a real payback to the hard earned
innovations.
CONCLUSION
From social perspective point of view, curbing
the menace of drug resistance could be viewed as a joint
social responsibility. The government organizations,
non-government organizations, clinicians, practitioners
and each individual need to come forward to contribute
for curbing the practices that lead to the emergence of
drug resistant strains. It would not be absurd to state that
the righteous approach towards the subject and sincere
efforts along with major budget allocation
implementation of the research findings could shun the
373
Sawhney, 2012
burden of scientists and open up new and well executed
front to fight the nuisance of drug resistance.
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 Journal of Research in Biology An International Scientific Research Journal

Original Research

Microbial contamination of Indian currency notes in circulation

Journal of Research in Biology

Authors:
Pradeep NV1, Anupama1,
Marulasiddaiah BS2,
Chetana M2, Gayathri P2,
Maduri SN2.
Institution:
1. Asst Professor,
Department of
Biotechnology, Ballari
Institute of Technology
and Management,
Bellary-583101, India.
2. B.E Scholar, Department
of Biotechnology, Ballari
Institute of Technology and
Management,
Bellary-583101, India.
ABSTRACT:
Paper currency, an exchangeable fomite, is constantly subjected to
contamination. The objective of this study was to identify the micro-organisms
present on the currency notes circulating in India. A total of 12 currency notes
(Rs.10, Rs.50 and Rs.500) were randomly collected from bank, Municipal Corporation,
food sellers, butchers, hospital. Persons handling the notes were asked to deposit
them in sterile envelopes. The notes were taken to the laboratory immediately and
microorganisms were identified using standardized microbiological techniques. All the
notes collected during this study were contaminated by micro-organisms. Species
isolated were Escherichia coli, klebsiella pneumonia, Pseudomonas aeruginosa and
Staphylococcus aureus. The currencies used by public (bank, hospital, Municipal
Corporation) in India were found to be extremely contaminated with various
pathogenic bacteria followed by the currency used by butchers and food sellers.
Infected currency was identified as a potential public health hazard, as pathogens
could spread by circulating the contaminated notes. We recommend that currency
notes must be handled with caution.

Corresponding author: Keywords:

Pradeep NV. Indian currency notes, pathogenic microorganisms, contamination.

Email: Article Citation:

nagamallivpradeep@gmail.com Pradeep NV, Anupama, Marulasiddaiah BS, Chetana M, Gayathri P, Maduri SN.
Microbial contamination of Indian currency notes in circulation.
Journal of Research in Biology (2012) 2(4): 377-382
Phone No:
07760251535. Dates:
Received: 18 May 2012 Accepted: 25 May 2012 Published: 07 Jun 2012

Web Address:
http://jresearchbiology.com/
documents/RA0243.pdf.
This article is governed by the Creative Commons Attribution License (http://creativecommons.org/
licenses/by/2.0), which gives permission for unrestricted use, non-commercial, distribution and
reproduction in all medium, provided the original work is properly cited.

Journal of Research in 377-382 | JRB | 2012 | Vol 2 | No 4

Biology
An International Scientific
Research Journal www.jresearchbiology.com

INTRODUCTION
Microorganisms are known to spread via air,
water, food etc. an important mechanism of the spread of
pathogens by formites. Paper currency is widely
exchanged for goods and services in countries worldwide
(Felgo and Nkansah 2010; Alwakeel and Naseer, 2011).
It is used for every type of commerce. Accumulated data
obtained over the last 20 years on the microbial status
and survival of pathogen on coins and currency notes
indicate that this could represent a potential cause of
sporadic cases of food borne illness. The paper/polymer
currency notes and coins may harbor various deadly
pathogenic microorganisms. Currency in the form of
notes represent a universal medium for the transmission
of bacteria in the environment and among humans. There
is a possibility that currency notes might act as
environmental vehicles for the transmission of potential
pathogenic microorganisms (Prasai T et al., 2009).
An individual living in unhygienic conditions
having unhygienic habits will contaminate the notes with
bacteria e.g. habits such as using saliva to count the
paper notes also leads to the contamination and these
notes will act as a vehicle delivering bacteria to
contaminate the hands of the next user. The currencies
acts as a tool for easy transfer of bacterial and thus cross
contamination takes places (Sushil Kumar et al., 2011).
Research has shown that paper currency offers a
larger surface area as a breeding ground for pathogens
(Ayandele and Adeniyi, 2011). Microbes may persist on
it for longer periods. The older the paper note the more
accumulation of microbes occurs (Ghamdi AL et al.,
2011).
The possibility that currency notes might act as
environmental vehicles for transmission of potential
pathogenic microorganisms was suggested in 1970s.
Various pathogens related with throat infection,
pneumonia, tonsillitis peptic ulcers, urino-genital tract
infection, gastro enteritis and lung abscess had been
reported (Saeed and Rasheed, 2011).
378
Pradeep et al., 2012
Researchers at the Regional Sophisticated
Instrumentation Center (RSIC) at the North Eastern
University in Shilong, India, who examined Indian
banknotes, found germs which can cause tuberculosis,
meningitis, tonsillitis, peptic ulcers, throat infections,
genital tract infections etc (Nagesh Bhat et al., 2010).
Studies of the contamination of money with
microbial agents is lacking in most developing countries.
Shortage of information may contribute to the absence of
public health policies regarding currency usage,
handling, and circulation (Ghamdi-AL et al., 2011).
Lower-denomination notes receive the most
handling because they are exchanged more often. Money
may serve as an unrecognized reservoir for pathogenic
and non-pathogenic bacteria. One type of pathogenic
bacteria that represents a threat is enteric bacteria
(Oo et al., 1989).
MATERIALS AND METHODS
Sample collection
A total of 12 currency notes were randomly
collected from people like butchers, food sellers, bank,
hospital and municipal corporation workers. The
currency collected and used were Rs.10, Rs.50 and
Rs.500. To collect the currency notes, the individuals
were asked to drop currency or money into a sterile
plastic packet, which were sealed and immediately
transported to the lab for microbial analysis (Saeed and
Rasheed, 2011).
Isolation of Microbes
A sterile cotton swab was dipped in the sterile
distilled water and rubbed on both the surfaces of
currency note and used to inoculate onto the nutrient agar
and potato dextrose agar (PDA) for each note. The plates
were incubated at 37C for 24 hours. After 24 hours
the plates were observed for bacterial colonies
(Kawo et al., 2009).
Journal of Research in Biology (2012) 2(4): 377-382
Pradeep et al., 2012

Morphological and biochemicalcharacterization of Table No.1: Butcher sample.

Bucher Gram Microscopic
the isolates
Sample character morphology
The bacterial isolates were characterized on the Rs.10 -ve Bacilli
basis of their morphology, staining and biochemical Rs.50 +ve Cocci
Rs.500 -ve Cocci
tests. These tests were carried out according to the
Table No. 2: Municipal corporation sample.
methods prescribed by Cappucino and Sherman (2007).
Municipal Gram Microscopic
Grams staining was carried out to ascertain Sample character morphology
themorphology and Grams reaction-behaviour of the Rs.10 +ve Bacilli
bacterial isolates. In addition, the followingbiochemical Rs.50 -ve Bacilli

tests were carried out: MR (methyl red) test, Table No. 3: Food sellers sample.
Food Sellers Gram Microscopic
VP (Voges-Proskauer) test, Indole production test, Sample character morphology
Catalase test, Citrate utilization test, Starch hydrolysis Rs.10 +ve Bacilli
Rs.50 -ve Bacilli
test and Gelatin hydrolysis test.
Table No. 4: Hospital sample.
Hospital Gram Microscopic
RESULTS Sample character morphology
Rs.10 +ve Bacilli
Microbial examination was carried out for 12
Rs.500 -ve Cocci
currency notes in which all the currency notes were
contaminated with microorganisms. Bacterial Table No. 5: Bank sample.
Bank Gram Microscopic
concentration was found to be high in butcher and Sample character morphology
municipal corporation samples when compared to bank Rs.10 -ve Bacilli
samples. Rs.50 -ve Bacilli
Gram staining results for Butcher, Municipal Rs.500 -ve Bacilli
Corporation, Food seller, Hospital and Bank samples are carry money in wallets and squeezing of currency notes
as shown in Table 1, 2, 3, 4 and 5. is a common occurrence. Women, especially among the
Biochemical characterization for Butcher, unenlightened, often place money underneath their
Municipal Corporation, Food sellers, Hospital, and Bank brassieres, while men place it in their socks. These
samples are shown in Table 6, 7, 8, 9 and 10. activities not only enhance currency contamination but
Bacteria isolated from bank, butcher, Municipal may also increase the risk of infection from
Corporation, hospital and food seller sample are contaminated notes.
pathogenic. Based upon the morphology, gram In a similar study con duct ed by
staining and biochemical characterization, the Ghamdi-AL et al., (2011) a total of 176 samples of the
micro-organisms found may be Staphylococcus aureus, examined 200 one Riyal notes (100 of the 4th version
Klebsiellapneumoniae, E.coli and Pseudomonas and 100 of the 5th version) had mixed ( 2 types)
aeruginosa. bacterial growth. One hundred percent of the 4thversion
notes had bacterial contamination, of which 60% were
DISCUSSIONS potentially pathogenic bacteria: Staphylococcus aureus,
In India, poor-currency-handling culture is Klebsiella species, Pseudomonas species and
widespread, and there is an indiscriminate abuse of Escherichia coli. The bacteria isolated from the 4th
currency notes. A great majority of the populace does not version notes were: gram-positive bacilli,

Journal of Research in Biology (2012) 2(4): 377-382 379

 Pradeep et al., 2012

Table No. 6: Butcher sample.

Bucher Sample Catalase MR VP Citrate Gelatin Starch Indole
Rs.10 +ve +ve -ve -ve +ve -ve -ve
Rs.50 +ve +ve -ve -ve +ve -ve -ve
Rs.500 +ve +ve -ve -ve +ve -ve -ve

Table No. 7: Municipal corporation sample.

Municipal Sample Catalase MR VP Citrate Gelatin Starch Indole
Rs.10 +ve +ve -ve -ve +ve -ve -ve
Rs.50 +ve +ve -ve -ve +ve -ve -ve

Table No. 8: Food sellers sample.

Food Sellers Sample Catalase MR VP Citrate Gelatin Starch Indole
Rs.10 +ve -ve +ve -ve -ve -ve -ve
Rs.50 +ve -ve +ve +ve -ve -ve -ve

Table No. 9: Hospital sample.

Hospital Sample Catalase MR VP Citrate Gelatin Starch Indole
Rs.10 +ve -ve +ve -ve -ve -ve -ve
Rs .500 +ve +ve -ve +ve -ve -ve +ve

Table No. 10: Bank sample.

Bank Sample Catalase MR VP Citrate Gelatin Starch Indole
Rs.10 +ve +ve -ve -ve -ve -ve +ve
Rs.50 +ve +ve -ve +ve -ve -ve -ve
Rs.500 +ve +ve -ve +ve -ve -ve +ve

coagulase-negative staphylococci, viridans
streptococci (VGS), and non-hemolytic streptococci.
Janardan et al., (2009) in their study isolated
bacteria from Nepal currency notes. The microorganisms
wer e c oa g u l a s e - n e g a t i ve St a p hy l oc oc c u s,
alpha-hemolytic Streptococcus, Enterobacter species,
Acinetobacter species, non-aeruginosa species
Pseudomonas, Bacillus species, Alcaligenes species,
diphtheroids, and Escherichia vulneris, which do not
typically cause infections in healthy people rather they
were known to cause significant infections in those with
depressed immune systems, including those infected with
HIV, undergoing cancer chemotherapy, or taking other
medications that depress the immune system. Those
bacteria may also cause infection in hospitalize patients.
Igumbor et al., (2007) tested 240 banknotes for
microbial contamination. All the notes
contaminated by bacteria or fungi. 84-100% of all the
banknotes obtained from the various sources were
380
group contaminated with bacteria. 12 different bacterial species
were isolated, with the most common isolates being
Staphylococcus epidermidis, Klebsiella species,
Staphylococcus aureus and E.coli. Only one fungus,
Candida albicans, was isolated.
Ahmed et al., (2010) suggested that the
of Bangladesh paper currency commonly contaminated
with pathogenic microorganisms and this contamination
may play a significant role in the transmission of
potentially harmful microorganisms or different diseases
such as cholera, diarrhea, skin infections and also poses
antibiotic resistant.
CONCLUSION AND RECOMMENDATIONS
From this study it can be concluded that Indian
currency is commonly contaminated with pathogenic
were bacteria and this contamination may play a significant
role in the transmission of infectious diseases. Compared
to butcher, municipal, hospital and food sellers sample,
Journal of Research in Biology (2012) 2(4): 377-382
Pradeep et al., 2012
the bank sample was less contaminated and the
recommendations are as below:
It is recommended that currency notes must be
handled with caution and great care especially
during the preparation and handling of food to avoid
contamination.
Personal hygiene to reduce risk of infection is
recommended especially for those who
simultaneously handle food and money.
Food sellers and butchers, and other common people
should be educated to avoid possible cross
contamination between currency notes and food.
There should be public awareness of the fact that
currency notes could be a source of infection and
could be dangerous to health.
Regular microbial testing of currency notes and
establishment of method for large scale replacement
of contaminated currency should be employed.
Introduction of plastic currency notes which can be
washed easily as in Australia can serve as an
alternate.
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382 Journal of Research in Biology (2012) 2(4): 377-382

 Journal of Research in Biology An International Scientific Research Journal

Original Research

Cell surface properties of Phenol-Utilizing Bacteria Isolated from

petroleum refinery wastewater
Journal of Research in Biology

Authors: ABSTRACT:
Nwanyanwu CE1, Alisi CS2, Cell surface hydrophobicity of six phenol-utilizing bacteria isolated from Port
Nweke CO1 and Orji JC1. Harcourt Petroleum refinery wastewater was assessed via bacterial adhesion to
hydrocarbon (BATH), salt aggregation test (SAT) and Congo red binding (CRB) assays.
The test organisms exhibited high to moderate hydrophobicity with BATH assay
Institution:
respectively when n-octane and p-xylene were employed. Bacillus sp. RBD,
1. Department of
Microbiology, Federal Escherichia coli. OPWW, Corynebacterium sp. DP, Citrobacter sp. RW and
University of Technology, Pseudomonas sp. SD showed moderate hydrophobicity in SAT assay. On the other
P.M.B. 1526, Owerri, hand, Pseudomonas sp. RWW showed high hydrophobicity in SAT assay.
Nigeria. Similar results of moderate hydrophobicity were obtained with CRB except
Corynebacterium sp. DP that exhibited high hydrophobicity value of 14.701.00g.
2. Department of The results obtained in this study showed that the isolates are mainly moderately
Biochemistry, Federal hydrophobic which make them good candidates in the clean up activity of organic
university of Technology, pollutants in polluted sites.
P.M.B. 1526, Owerri,
Nigeria.
Keywords:
Phenol-utilizing bacteria, Hydrophobicity, SAT, Congo red binding.

Corresponding author: Article Citation:

Nwanyanwu CE. Nwanyanwu CE, Alisi CS, Nweke CO and Orji JC.
Cell Surface Properties Of Phenol-Utilizing Bacteria Isolated From Petroleum Refinery
Wastewater.
Email: Journal of Research in Biology (2012) 2(4): 383-391
cnwanyanwu2000@yahoo.com
Dates:
Received: 23 Apr 2012 Accepted: 28 May 2012 Published: 08 Jun 2012
Web Address:
http://jresearchbiology.com/
documents/RA0233.pdf.
This article is governed by the Creative Commons Attribution License (http://creativecommons.org/
licenses/by/2.0), which gives permission for unrestricted use, non-commercial, distribution and
reproduction in all medium, provided the original work is properly cited.

383-391 | JRB | 2012 | Vol 2 | No 4

Journal of Research in Biology
An International
Scientific Research Journal www.jresearchbiology.com

INTRODUCTION
Large amounts of water are used in the
petroleum refinery activity and, consequently, significant
volumes of wastewater are generated reaching about
0.4-1.6 times the volume of processed oil
(Coelho et al., 2006). The wastewater generated by the
refinery is characterized by the presence of organic and
inorganic pollutants that gave it its toxic nature. The
toxic effects of refinery effluents to bacteria have
been reported (Nwanyanwu and Abu, 2010;
Krishnakumar et al., 2007). Bacteria are unable to
insulate themselves from the toxic nature of their habitat
because of their large surface area that is exposed to
these harsh environments. These pollutants in the
effluent exert their toxic effects on the bacterial surfaces
thereby inhibiting their attachment to substrates and
other surfaces. The most important mechanism of toxic
action of these pollutants is the destabilization of cell
membrane (Walsh et al., 2003). This effect results in
adaptation and changes in effluent autochthonous
microbial physiological functions and moreso can have
serious impact on the cell surface hydrophobicity and
biodegradation processes (Nwanyanwu et al., 2012;
Kaczorek et al., 2008; Leung et al., 1997)
. The hydrophobicity of the outermost bacterial
surface has been cited as a factor in partitioning of
microorganisms at the airwater interface and in the
adherence of bacteria to wide variety of surfaces as well
as growth of cells on insoluble hydrophobic substrates
such as hydrocarbons (Rosenberg, 1981). Cell surface
hydrophobicity has been found to be involved in
interfacial interactions of microbial cells with other
microbial cells (flocculation) and with air (flotation)
(Rosenberg and Kjelleberg, 1986). The hydrophobic
nature of bacterial surfaces has long been blamed for the
formation of stable foams in wastewater treatment plants.
Hydrophobicity appears to be imparted by different
chemical components of the cell wall in different
bacteria; these components include lipoteichoic
384
Nwanyanwu et al., 2012
acids and proteins.
The utilization of hydrocarbon and other aromatic
pollutants found in wastewater treatment plants are aided
by contact between hydrophobic organic compounds and
the cells. The ability to adhere on to hydrocarbon is
correlated with cell surface hydrophobicity. Many types
of microorganisms are found in wastewater treatment
plants. Hydrophobic microorganisms, in particular, are
capable of adhering to the oil-water interface thereby
utilizing oil components as well as other organic
compounds in wastewater treatment plants as a source of
energy for growth and metabolism. This process is of
great interest for the preservation of the natural
environment by reducing the amount of oil-related
contaminants (Pijanowska et al., 2007).
Not much work has been done to assess the cell
surface hydrophobicity of bacteria strains isolated from
refinery effluent treatment plants. This study is therefore
aimed at investigating the cell surface hydrophobicity of
bacteria strains found in Port Harcourt petroleum
refinery effluent treatment plants and water body
receiving petroleum refinery effluent.
MATERIALS AND METHODS
Sample collection and characterization
Physicochemically treated raw wastewater
(addition of additives, flocculation, sedimentation and
filtration) (RWW), biologically treated wastewater
(Rotary biodisk) (RBD), observation pond treated
wastewater (oxidation pond) (OPWW) and discharge
pipe wastewater (DP) samples of Port Harcourt
petroleum refinery were collected in sterile polyethylene
containers. Also river water (RW) and sediment (SD)
samples from Okrika River in Port Harcourt Nigeria that
receives the petroleum refinery wastewater were
collected. The containers were rinsed twice with the
samples at the point of collection. To avoid deterioration,
the samples were taken to the laboratory in icebox within
6h of collection for analyses. pH, biological oxygen
Journal of Research in Biology (2012) 2(4): 383-391
Nwanyanwu et al., 2012
demand (BOD), chemical oxygen demand (COD),
phosphate (PO4) and sulphate (SO4) were determined
according to APHA (1985) whereas lead (Pb), zinc (Zn),
copper (Cu) were determined by atomic absorption
spectrophotometer (Perkins Elmer 3110) respectively.
Oil and grease of the samples were determined using
partition-gravimetric method (Noweco, 1997). Phenol
content was determined as described by Folsom et al.,
(1990). Electrical conductivity was measured with
electrical conductivity meter (HACH conductivity
meter).
Isolation and identification of bacterial strains
Phenol-utilizing bacteria were isolated from the
samples by spreading 0.1ml of decimally diluted (10-4)
wastewater samples on mineral salt agar plates amended
with 2.5mM phenol as described by Hill and Robinson
(1975). The medium has the following composition
(mg/l) of KH2PO4, 840; K2HPO4, 750; (NH4)2SO4, 400;
MgSO4.7H2O, 60; NaCl, 60; CaCl2, 60; FeCl3, 60 and
15 g agar added to solidify the medium. Ketoconazole at
50g/ml was added to the medium to exclude fungi and
pH adjusted to 7.2. The plates were incubated for 72 h
and the developed colonies were purified on freshly
prepared nutrient agar plates. The purified isolates were
characterized biochemically following standard
microbiological methods. Identification to the genus
level followed the schemes of Holt et al., (1994).
The isolates were maintained on nutrient agar slants.
Preparation of inoculum and culture condition
The bacterial isolates for the assay were grown in
100 ml of BushnellHaas (BH) broth consisting of (g/l):
KH2 PO4 , 1.0; K2 HPO4 , 1.0; NH4 NO3 , 1.0;
MgSO47H2O, 0.2; FeCl36H2O, 0.085; CaCl22H2O,
0.02 and phenol, 0.05 contained in 250 ml Erlenmeyer
flasks. The flasks were incubated on a rotary shaker
o
(150 rpm) for 24 h at room temperature (282 C). The
cells were recovered by centrifugation (6,000 rpm for
10 min) and washed twice in phosphate buffered saline
(PBS, 0.02M; pH 7.2 for BATH assay and Congo red
Journal of Research in Biology (2012) 2(4): 383-391
assay and pH 6.8 for SAT). The washed cells were
resuspended in the buffer medium and the turbidity
adjusted spectrophotometrically to give an optical
density of 1.0 at 540nm.
Cell surface hydrophobicity assays
Cell surface hydrophobicit y of the
phenol-utilizing bacteria was assessed using the bacterial
adherence to hydrocarbon (BATH), modified salt
aggregation test (SAT) and Congo red binding.
Bacterial adherence to hydrocarbon
BATH was performed as described by
Rosenberg et al., (1984). The cell suspensions were
dispensed in 4ml aliquots into sterile 20ml screw capped
culture tubes. The tubes received different volumes viz
0.1, 0.2, 0.3, 0.4 and 0.5ml of either n-octane or
p-xylene (Sigma Chemical Co., St. Louis, Mo., USA).
The mixtures were vortexed for 2 min and allowed to
stand for 15 min for the completion of biphasic
formation. After phase separation, the aqueous phase was
carefully recovered and the OD540 was determined (A1).
Values were then expressed as the percentage of bacteria
adhering to the hydrocarbons (A) compared with the
control suspension (A0) as follows:
(A0 - A1)
A (%) = 100
A0
The reference value for the BATH assay is the
percentage of bacteria from 4ml of suspension that
partition into 0.5ml of n-octane or p-xylene. Strains were
considered strongly hydrophobic when values were
>60%, moderately hydrophobic when values were in the
range of 40 - 60% and hydrophilic when values were
<40% (Basson et al., 2008).
Salt aggregation test
The SAT assay was carried out as described by
Lindahl et al., (1981) with little modifications. The assay
is based on bacterial precipitation in the presence of
salts. Isolates were salted out (aggregated) by combining
25l volumes of the cell suspension with equal (25l)
385
 Nwanyanwu et al., 2012

volumes of a series of varying molarities (M) of
(NH4)2SO4 solution (0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6,
1.8, 2.0, 2.2, 2.4, 2.6, 2.8, 3.0 and 4.0M) in different
wells in a microplate. Addition of 400 l of 0.1% w/v
methylene blue solution to 10ml volumes of (NH4)2SO4
solution facilitates better visualization of aggregation
(Rozgonyi et al., 1985). The plate was rocked for 4 min
after which it was visually examined and scored against a
white background for cell aggregation. Concentration of
(NH4)2SO4 solution causing aggregation was considered
positive whereas the absence of aggregation was
considered as negative. Hydrophobicity was expressed as
the lowest salt concentration in the mixture that produced
visual clumping. Classification was expressed as: < 1.0
M = strongly hydrophobic, 1.0-2.0 M=Hydrophobic,
> 2.0 M = Hydrophilic.
Congo red binding assay
This assay is used to study the pigment binding
ability of the strains as well as a marker of
hydrophobicity. The experiment was performed as
described by Qadri et al., (1988). Aliquot (1.0ml) of
bacterial suspension were transferred into screw capped
test tubes containing 4ml of PBS amended with 25g/ml
of congo red dye and were incubated at room
temperature for 15min. Thereafter, the Congo red bound
to cells are removed by centrifugation at 6,000 rpm for
10min. The supernatant (cell free Congo red solution)
was collected in separate tubes and its absorbance
determined spectrophotometrically at 480nm against a
PBS blank. The amount of congo red dye that bind to the
cells were calculated from a standard curve as the
difference between the amount added to the mixture and
the amount remaining in the cell free Congo red solution.
Uptake of Congo red greater than 10 g was scored as
strongly hydrophobic (Payne and Finkelstein, 1977).
RESULTS AND DISCUSSION
The physicochemical properties of the petroleum
refinery wastewaters and Okrika river water and
sediment are shown in Table 1. The level of oil and
grease, Biological Oxygen Demand (BOD), Chemical
Oxygen Demand (COD) and phenol content of the
petroleum refinery wastewater samples are indication
that the samples are polluted with organic compounds.
This also showed that autochthonous microorganisms
isolated from these samples are subjected to pollutant
stress (Sarala and Sabitha, 2012). The organisms isolated
from the refinery wastewater samples include
Pseudomonas sp. RWW, Bacillus sp. RBD,
Escherichia coli OPWW and Corynebacterium sp. DP
while Citrobacter sp. RW and Pseudomonas sp. SD were
isolated from Okrika river water and sediment samples

Table 1 Characteristics of the petroleum refinery wastewater and okrika river

Sample source
Parameter/unit RWW RBD OPWW DP RW SD
pH 7.64 8.18 7.45 8.87 8.97 6.80
Temperature ( oc) 26.4 26.1 26.8 26.7 - -
Electrical Conductivity.(s/cm) 845 443 926 643 364 615
Oil and grease (mg/l) 17.5 15.0 21.0 16.0 16.0 100
BOD (mg/l) 32.0 8.0 12.8 12.8 - -
COD (mg/l) 112.0 76.0 114.0 84.0 - -
PO4 (mg/l) 0.22 0.14 0.13 0.12 0.12 0.07
SO4 (mg/l) 37.63 13.52 35.3 11.8 117 115
Phenol (mg/l) 71.2 13.6 10.1 9.4 8.6 15.5
Pb (mg/l) <0.01 <0.01 <0.01 <0.01 <0.01 <0.01
Zn (mg/l) 0.13 0.02 0.06 0.08 0.05 38.6
Cu (mg/l) <0.01 <0.01 0.01 0.01 <0.01 0.061
Key : RWW = Raw wastewater, RBD = Rotary biodisk OPWW = Observation pond wastewater,
DP = Discharge pipe RW = River water, SD = Sediment

386 Journal of Research in Biology (2012) 2(4): 383-391

Nwanyanwu et al., 2012

respectively. The bacterial strains represent
preponderant morphotypes in their respective sources.
These bacteria have been reported to grow on
hydrocarbon and aromatic compounds as well as other
organic pollutants in the environment (Akpoveta et al.,
2011; Okerentugba and Ezeronye, 2003; Mo et al., 2000;
Zhang and Miller, 1994)
Different results were obtained, depending on the
method adopted to estimate cell surface hydrophobicity
of the organisms. Cell surface hydrophobicity using
BATH was evaluated in terms of change in absorbance at
540 nm. Figure 1 showed a progressive increase in the
adhesion to hydrocarbon with increasing volume of
n-octane and p-xylene. This indicated that the cells were
partitioned into the hydrocarbon phase and the cells
exhibited relative high surface hydrophobicity in the
BATH assay. The loss of bacteria from the aqueous
phase was approximately proportional to the volume of
n-octane and p-xylene added to the cell suspension. The
values from the BATH assay recorded for the
partitioning of the organisms is the percentage of
bacteria suspension that partitioned into 0.5ml of
n-octane and p-xylene as indicated in Table 2.
Sorongon et al., (1991) and Lachica and Zink (1984)
reported that loss of bacteria from aqueous phase was a
function of hydrocarbon concentration employed. The
organisms partitioned more in n-octane than in p-xylene
the classi fi ed as strongl y hydrophobic while
Corynebacterium sp. DP, Citrobacter sp. RW and
Pseudomonas sp. SD are moderately hydrophobic.
Similarly, p-xylene indicated that all the organisms are
moderately hydrophobic.
Cell surface hydrophobocity estimated by
salt aggregation test (SAT) assay showed that majority of
the test isolates aggregated at 1.0M (NH4)2SO4
(Table 2). This indicated that the bacterial strains found
in Port Harcourt refinery effluent are moderately
hydrophobic. The differences in SAT values between
Pseudomonas sp. RWW that showed hydrophobic and
other test organisms that showed moderate SAT values
may be as a result of differences on cell surface charges.
This indicates that SAT values may be dependent on the
charge on microbial surface as well as the age of the
culture. This is in agreement with the results obtained by
Qadri et al., (1988) in which they found that aggregation
of bacterial strains increase with old cultures as charges
on microbial surfaces increases.
Adherence (%)

as shown in Table 2. This may be as a result of

devastating effect of p-xylene on the surface of the
organisms than n-octane.
The adherence of the organisms correlated with
volume of hydrocarbons with R2 values greater than 0.90
(0.9044 R2 0.9922) for n-octane and greater than
0.94 (0.9455 R2 0.9795) for p-xylene in all the
bacterial strains (Figure 2). The high R2 values observed
in all the bacterial strains indicated that volume of
hydrocarbon was a strong determinant of the adherence.
Hydrocarbon (ml)
Using n-octane in BATH assay, Pseudomonas sp.
Figure 1: Effect of hydrocarbon and aqueous
RWW, Bacillus sp. RBD and Escherichia sp. OPWW are phase ratios on hydrophobicity of bacteria

Journal of Research in Biology (2012) 2(4): 383-391 387

 Nwanyanwu et al., 2012

Adherence (%)

Hydrocarbon (ml)
Figure 2: Correlation of hydrocarbon (octane/xylene) volume with percentage
of adherence in cell surface hydrophobicity of The bacteria. The solid line
(n-octane) and dotted line (p-xylene) are predicted adherence values. The
closed and open squares represent data obtained from n-Octane and p-Xylene
respectively

The Congo red binding, commonly used as a
marker of hydrophobicity (Majtan and Majtanova, 2001)
was employed in this test and the test organisms
exhibited distinct uptake of congo red in solution
(Table 2). Previous studies (Haider et al., 1990;
Qadri et al., 1988) have shown that congo red binding
may reflect an arrangement of cell surface components.
388
Corynebacterium sp. DP showed highest Congo red
uptake of 14g while the least Congo red uptake of
10.5g was recorded for Escherichia coli. OPWW.
Congo red binding test indicated that all the test
organisms are strongly hydrophobic.
BATH and SAT hydrophobicity tests sometimes
fail to correlate and this has also been observed in the
Journal of Research in Biology (2012) 2(4): 383-391
Nwanyanwu et al., 2012

Table 2 Hydrophobicity characteristics of phenol-utilizing bacterial strains

% Adherence SAT Congo red binding
Bacteria n-octane p-xylene M (NH4)2SO4 (g)
Pseudomonas sp. RWW 60.33 2.59 56.78 5.03 0.40 0.00 11.80 1.00
Bacillus sp. RBD 61.33 1.92 54.83 2.00 1.40 0.00 10.90 0.00
Escherichia coli. OPWW 60.06 2.34 46.27 2.87 2.00 0.00 10.50 5.00
Corynebacterium sp. DP 51.22 1.02 51.94 2.17 1.60 0.00 14.70 1.00
Citrobacter sp. RW 53.44 1.95 53.44 1.05 1.20 0.00 12.10 2.00
Pseudomonas sp. SD 45.22 3.04 45.22 3.07 1.00 0.00 12.40 3.00
BATH expressed as the percentage of bacteria from 4 ml of cell suspension that partition into 0.5 ml of
n-octane or P-xvlene. SAT expressed as the lowest M(NH4)2SO4 concentration in the reaction mixture that
produced visual clumping Uptake of Congo red dye greater than 10g was scored as strongly hydrophobic

present study. Lack of correlation between SAT
and BATH test results had been observed by
Soto-Rodriguez et al., (2003) and Lee and Yii (1996).
Also the BATH and CRB, SAT and CRB sometimes fail
to correlate with one another. Even within BATH assay
using different hydrocarbons n-octane and p-xylene the
hydrophobicity did not correlate as shown in Table 2.
Pseudomonas sp. RWW, Bacillus sp. RBD and
Escherichia coli. OPWW have relatively higher
adherence to n-Octane than p-Xylene. The failure of
BATH and SAT to correlate might be explained by the
SAT assay measuring the hydrophobicity of the outer
surface as a whole while the BATH assay measured it in
terms of adhesion (Mattos-Guaraldi et al., 1999).
In addition, hydrophibicity and surface charge of bacteria
may differ between species, strains, and changes with
variation in physiological state of cells and composition
of suspension media or might involve variable
expression of surface-associated proteins between
strains. Collectively, this might account for the diversity
of BATH, SAT and CRB indices obtained in this work.
A cell surface hydrophobicity influences the
direct contact of cell with hydrocarbons and is hence,
one of the major factors affecting the degradation of
organic compounds. Bioavailability of organic
compounds, a physicochemical parameter critical in the
overall rate of degradation, is a function of phase
solubility and solution transport processes, hence, phenol
is therefore, easily biodegraded. Therefore, phenol
degrading bacteria with hydrophobic cell surface
properties could be potentially applicable in the
treatment of phenolic wastewater contaminated with
hydrocarbons.
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Journal of Research in Biology (2012) 2(4): 383-391 391

 Journal of Research in Biology An International Scientific Research Journal

Original Research

Kinetics of dose-response relationship of heavy metals with

dehydrogenase activity in wastewater bacteria
Journal of Research in Biology

Authors:
Nweke CO1,2 and
Okpokwasili GC2.
Institution:
1.Department of
Microbiology, Federal
University of Technology,
P. M. B. 1526, Owerri,
Nigeria.
2. Department of
Microbiology, University of concentration ranged from 0.0183 mM (Cd against Pseudomonas sp. DAF1) to
Port Harcourt, P. M. B.
5323, Port Harcourt, Nigeria. the IC50, thus they are suitable parameters for kinetic analyses of metal toxicity against
ABSTRACT:
Toxicity of Zn2+, Cd2+ and Co2+ to Escherichia coli, Pseudomonas and Bacillus
species isolated from petroleum refinery effluent was assessed using
dehydrogenase activity (DHA) inhibition test. Exposure of the cells to the metal ions
resulted in inhibition of dehydrogenase activity. The median inhibitory concentration
of the metal ions ranged from 0.0554 to 0.3883 mM (Zn2+), 0.0279 to 0.3004 mM
(Cd2+) and 0.0013 to 0.2778 mM (Co2+). The trends of the inhibitory effects could be
mathematically described with logistic and sigmoid dose-response models and in a
manner similar to the non-competitive inhibition of enzymes. The threshold
concentration above which toxic effect is observed ranged from 0.0013 mM
(Zn2+ against Pseudomonas sp. RWW2) to 0.05 mM (Zn2+ against Escherichia coli).
In terms of non-competitive inhibition of dehydrogenase activity, the threshold
2+
2+
0.05 mM (Zn against Escherichia coli). The coefficients of inhibition Ki correlated with
bacteria.

Corresponding author: Keywords:

Nweke CO. Dehydrogenase activity, heavy metals, dose-response models, toxicity.

Email: Article Citation:

xrisokey@yahoo.com.
Web Address:
http://jresearchbiology.com/
document/RA0200.pdf.
Nweke CO and Okpokwasili GC.
Kinetics of dose-response relationship of heavy metals with dehydrogenase
activity in wastewater bacteria.
Journal of Research in Biology (2012) 2(4): 392-402
Dates:
Received: 18 Feb 2012 Accepted: 03 Apr 2012 Published: 08 Jun 2012

This article is governed by the Creative Commons Attribution License (http://creativecommons.org/

licenses/by/2.0), which gives permission for unrestricted use, non-commercial, distribution and
reproduction in all medium, provided the original work is properly cited.

Journal of Research in 392-402| JRB | 2012 | Vol 2 | No 4

Biology
An International Scientific
Research Journal www.jresearchbiology.com
 Nweke and Okpokwasili, 2012

INTRODUCTION Dehydrogenase activity assay is also an effective

Heavy metals are widely distributed and primary test for assessing toxicity of phenols
persistent environmental pollutants that are introduced (Nweke and Okpokwasili, 2010a, b; Nwanyanwu and
into the environment through industrial activities. Heavy Abu, 2011) and plant extracts (Alisi et al., 2011) against
metals contaminate natural habitats and alter macro and bacteria. However, the kinetics of toxicity has not been
microbiological communities (Purves, 1985; widely studied. The objective of this study was to
Davies et al., 1991; Binning and Baird, 2001; Horsfall compare the effects of zinc, cadmium and cobalt on the
and Spiff, 2002; Nweke et al., 2006; 2007a). Some heavy dehydrogenase activity in petroleum refinery effluent
metals (e.g Cd and Hg) have no known physiological bacteria, using kinetic analysis.
function and are toxic even at low concentrations. Others
(e.g Cu, Ni, Zn and Co) are essential trace elements MATERIALS AND METHODS
required for normal physiological function of Bacterial strains
microorganisms. The toxic effects of metals include; Bacterial strains were isolated from wastewater
blocking of functional groups, displacement or and wastewater treatment system of Port Harcourt
substitution of essential metal ions from biomolecules, petroleum refinery, Port Harcourt, southeastern Nigeria.
conformational modifications, denaturation and The method of isolation and identification of the
inactivation of enzymes and disruption of membrane bacterial strains was as described elsewhere
integrity (Gadd, 1993; Doelman et al., 1994; Li and Tan (Nweke and Okpokwasili, 2010a).
1994). Dehydrogenase assay
Microorganisms are vital for efficient Dehydrogenase activity was determined using
functioning of any ecosystem, thus factors affecting their 2,3,5-triphenyltetrazolium chloride as the artificial
metabolism and diversity are of great concern. Microbes electron acceptor, which is reduced to red-coloured
respond promptly to environmental pollution and triphenylformazan (TPF). The assay was done in 3-ml
monitoring microbial responses have been recommended volume of nutrient broth-glucose-TTC medium
as an early warning indicator of ecosystem stress supplemented with varying concentrations of Zn 2+, Cd2+
(Griffiths, 1983; Odum, 1985). Microbial parameters and Co2+ as ZnSO4, CdCl2 and CoCl2 respectively in
including growth rate (Juliastuti et al., 2003b), biomass separate screw-capped test tubes. Portions (0.3 ml) of
measurement (Guckert, 1996), inhibition of washed bacterial suspensions (A420 = 0.5) were
bioluminescence (Ren and Frymier, 2003) and activity of inoculated into triplicate glass tubes containing 2.5 ml of
specific and non-specific enzymes (Bitton et al., 1992) phthalate-buffered (pH 6) nutrient broth glucose medium
may be used to evaluate toxic effects of metals on amended with Zn 2+, Cd2+ and Co2+ and 0.2 ml of 0.4%
bacterial populations. (w/v) TTC in deionized distilled water was added to each
Enzymes are key catalysts of metabolic reactions tube to obtain final concentrations of 0.0.05 - 1.4 mM.
in cells and their inhibition by metals has been explored The final concentrations of nutrient broth and glucose in
as basis for ecotoxicity testing. A wide range of enzymes the medium were 2 mg/ml each. The controls consisted
has been considered with special emphasis on of the isolates and the media without metal. The reaction
dehydrogenases (Bitton and Koopman 1986; Obst et al., mixtures were incubated under static conditions at room
1988; Montuelle et al., 1994; Codina et al., 1994; temperature (28 2C) for 24 h. The TPF produced was
Nweke et al., 2006, 2007a,b; Orji et al., 2008). extracted in 4 ml of amyl alcohol and determined
393 Journal of Research in Biology (2012) 2(4): 392-402
Nweke and Okpokwasili, 2012

spectrophotometrically. The dehydrogenase activities as

100
INH (% ) 100 6
g TPF/mg of dry cell wt/h and percentage inhibitions I
c

1
were computed. b

Kinetic modelling Rearranging equation-6 yields:

The enzyme activity (EA) and inhibition of the
1
INH (% ) 1 100 7
enzyme activity (INH) relative to controls are given by 1
I
c

b
the expressions:
where Equation-7 can be rewritten as:
TA
EA (% of control) 100 1
CA 1
INH (% ) 1 100 8
Ic
1
bc
C A TA
INH (% ) 100 2
CA
Equation 8 is equivalent to equation 9 reported in
CA: is absorbance of TPF produced in control test literature (Kroiss et al., 1992; Juliastuti et al., 2003a).
(without metal) Where, c =KI and bc = Ki. Ki is the coefficient of
TA: is absorbance of TPF produced in the test with inhibition (mM).
different concentrations of metal
The enzyme activity (% of control) can be 1
INH (% ) 1 100 9
I KI
described by the logistic function: 1
Ki

am
EA (% of control) c
3 Incorporating the threshold concentration a
I
1 (concentration of metal above which toxic effect is
b
Where observed) in equation 8 and 9 yields equation 10 and 11
am: is maximum enzyme activity (% of control) respectively. Equations 10 and 11 are similar to equation
b: is slope parameter indicating the inhibition rate, 12 originally proposed by Ren and Frymier (2003) to
IC50 (mM) describe inhibition of bioluminescence by metals.
c: is a dimensionless inhibition parameter
I: is inhibitor concentration (mM)
1
But: INH (% ) 1 c
100 10
I a
1
INH (% ) 100 EA (% of control) 4 bc

Substituting equation-3 into equation-4 yields:

1
INH (% ) 1 KI
100 11
am I a
INH (% ) 100 c
5 1
I
1 Ki
b

Assuming there is no stimulation of enzyme

1
activity at low concentrations of metal ion, am becomes INH (% ) 1 100 12
I a
1
100 %, thus equation-5 becomes: Ki Ki

Journal of Research in Biology (2012) 2(4): 392-402 394

 Nweke and Okpokwasili, 2012

Equation 12 assumes that metal ions repress
bacterial dehydrogenase activity by inhibiting the
rate-determining step in the formation of triphenyl
formazan in a manner similar to non-competitive
inhibition of enzymes. Inhibitions of enzyme activity
data derived from equation 2 are fitted into equations 9,
11 and 12. The kinetic parameters were estimated by
iterative minimization of least squares using Levenberg-
Marquardt algorithm of Table Curve 2D. Regression was
done using the mean data and standard deviations. The
toxicity thresholds, IC20, IC50 and IC80 which are the
concentrations of metals that inhibited dehydrogenase
activity by 20, 50 and 80% respectively were estimated
from equation
correlation and analysis of variance (ANOVA) were
done using Microsoft Excel 2003.
RESULTS AND DISCUSSION
The effect of zinc, cadmium and cobalt on the
production of triphenyl formazan in test bacteria is
Dehydrogenase activity (g TPF/ mg cell dry weight/h)
shown in Figure-1. The relative effects of these metals in
terms of percentage inhibition of dehydrogenase activity
in the bacterial isolates are shown in Figures 2 - 5. In all
the bacterial strains, cobalt, zinc and cadmium inhibited
dehydrogenase activity. The inhibitions increased with
increase in the concentration of metal. The inhibitions
were relatively less pronounced at lower concentrations
of zinc than with other metals. However, at 0.05 mM,
zinc inhibited dehydrogenase activity in Bacillus sp.
DISK1 by 43.666 1.523%. Generally, cadmium and
cobalt are more toxic to bacterial dehydrogenases at
0.05 mM than zinc. However, it is noteworthy that
cadmium stimulated dehydrogenase activity in
9. The Pearson product-moment Pseudomonas sp. RWW2 by 6.613 4.675% at
0.05 mM. Cobalt inhibited dehydrogenase activity in
Pseudomonas sp. DAF1, Pseudomonas sp. RWW2,
Bacillus sp. DISK1 and Escherichia coli by
45.669 1.181, 17.679 2.337, 43.666 1.523 and
66.410 8.442 respectively at 0.05 mM. Cadmium
exhibited similar levels of inhibition in the bacterial

Zn2+
Cd2+
Co2+

Bacillus sp. DISK 1

Metal ion (mM)

Figure 1: Production of triphenyl formazan in response to metal
toxicity in pure cultures of wastewater bacteria

395 Journal of Research in Biology (2012) 2(4): 392-402

Nweke and Okpokwasili, 2012

120
100
a Pseudomonas sp. DAF1 a Pseudomonas sp. RWW 2
100
80
80
In hibition of dehydrogenase activity (%)

60

Inhibition of dehydrogenase activity (%)

60

40
40
Zn2+
Zn 2+ Cd2+
Cd2+ Zn2+
Zn 2+ Cd2+
Cd2+
Co2+ 2+
Zn Eq9
20 Co2+
2+
Zn2+
2+
Eq9FitFit 20 Co 2+
Co2+
2+
Zn Eq9
Zn2+ Eq9 Fit
Fit
Cd Eq9 Fit Co Eq9 Fit Cd2+ Eq9 Fit 2+
Cd2+ Eq9 Fit Co2+ Eq9 Fit Cd2+ Eq9 Fit Co Eq9
Co2+ Eq9 Fit
Fit
0
120 0
100
b Pseudomonas sp. DAF1
100 b Pseudomonas sp. RWW 2
80
80

60
60

40 40 2+
2+ Cd2+ Zn
Zn2+ Cd2+
Cd2+
Zn
Zn2+ Cd2+ Co2+ Zn2+Eq12Fit
Co 2+
Co2+
Zn2+Eq12
Zn2+
Fit
Eq12 Fit
Co2+ Zn2+ Eq12 Fit
Cd2+ Eq12
Eq12FitFit Co2+Eq12Fit
20 Cd 2+
Cd2+ Eql 2 FitFit
Eq12 Co2+Eq12
Zn2+ Eq11 Fit
Fit 20
Cd2+ Co2+2+
Eq12 Fit
2+ Zn2+ Eq11Fit
Fit Cd Eq11Fit
Cd2+ Eq11 Fit
Co 2+
Co2+Eql 1 FitFit
Eq11 Cd Eq11Fit
Cd2+ Eq11 Fit Zn2+ Eq11
Co2+2+ Eq11Fit
Co Eq11 Fit
0
0.0 0.4 0.8 1.2 1.6 0
0.0 0.4 0.8 1.2 1.6

Metal ion (mM) Metal ion (mM)

Figure 2: Effects of metals on dehydrogenase
activity in Pseudomonas sp. DAF1. Experimental
data mean 1SD (n = 3) as data points and bars are
shown with predicted values derived from equation 9
(a) and equations 11 and 12 (b).
strain at 0.05 mM with Bacillus
Pseudomonas sp. DAF1 and Escherichia coli having
percentage inhibitions of 66.91 1.533, 68.504 0.000
and 23.077 6.654 % at 0.05 mM respectively. At 0.8
mM, zinc totally inhibited dehydrogenase activity in
Bacillus sp. DISK1 and Escherichia coli. Total inhibition
of dehydrogenase activity occurred at higher zinc
concentration of 1.6 mM in Pseudomonas sp. DAF1. In
all the bacteria except Pseudomonas sp. RWW2, total
inhibition of dehydrogenase activity by cadmium
occurred at either 0.6 or 0.8 mM. In Pseudomonas sp.
DAF1, cobalt totally inhibited dehydrogenase activity at
1.4 mM. Cobalt inhibited dehydrogenase activity by
96.752 0.703 and 88.846 1.692 in Bacillus species
and Escherichia coli respectively.
The inhibitory effects of the metal ions on
dehydrogenase activity are consistent with reported toxic
effects of metals on microbial metabolic processes
Figure 3: Effects of metals on dehydrogenase
activity in Pseudomonas sp. RWW2. Experimental
data mean 1SD (n = 3) as data points and bars are
shown with predicted values derived from equation 9
(a) and equations 11 and 12 (b)
sp. DISK1, (Ji and Silver, 1995; Nies, 1999; Coello Oviedo et al.,
2002; Wang and Crowley, 2005). Although some heavy
metals (e.g. Cu, Fe, Zn, Co, Mn and Ni) are trace
elements and are essential at low concentrations for
normal cellular metabolism, they are toxic at high
concentrations. For instance, zinc ion concentrations at
0.0001-0.01 mM are required for optimal growth in most
microrganisms (Sugarman, 1983). However, at
concentration beyond physiologically required level, zinc
inhibits respiratory activities in microorganisms
(Kleiner, 1978; Kasahara and Anraku 1974; Prez-Garcia
et al., 1993; Beard et al., 1995). Cobalt occurs in
co-factor B12 and nitrile hydratases (Nies, 1999;
Kobayashi and Shimizu, 1998). At high concentrations,
cobalt interacts with Fe2+ and inhibits its physiological
function (Nies, 1999). On the other hand, cadmium is a
non-essential metal and is toxic to living cells even at
very low concentrations. Cd2+ displaces Ca2+ or Zn2+ in

Journal of Research in Biology (2012) 2(4): 392-402 396

 Nweke and Okpokwasili, 2012

120

100

80
2+
Cd

In hibition of dehydrogenase activity (%)

60 Zn2+
Zn 2+ Cd2+
In hibition of dehydrogenase activity (%)

2+
Co 2+ Zn Eq9 Fit
Co2+ Zn2+ Eq9 Fit
40 2+
Cd Eq9 Fit Co2+Eq9 Fit
Cd2+ Eq9 Fit Co2+ Eq9 Fit Zn2+ Data Cd2+ Data
20 Co2+ Data Zn2+ Eq9 Fit
a Bacillus sp. DISK1 Cd2+ Eq9 Fit
Co2+ Eq9 Fit
0

120

100

80
2+
Zn
Zn2+ Cd 2+
Cd2+
60 Co2+
Co2+ Zn 2+
Zn2+ Eq12
Eq12Fit
Fit
Cd2 + Eq12Fit Co2+Eq12 Fit
Cd2+ Eq12 Fit Co2+
2+ Eq12 Fit
Zn2+Eq11Fit Cd Eq11Fit Zn2+ Data Cd2+ Data
40 Zn2+ Eq11 Fit Cd2+ Eq11 Fit
Co2+Eq11Fit
Co2+ Eq11 Fit Co2+ Data Zn2+ Eq12 Fit

20 Cd2+ Eq12 Fit Zn2+ Eq11 Fit

Cd2+ Eq11 Fit Co2+ Eq11 Fit
b Bacillus sp. DISK1
0
0.0 0.2 0.4 0.6 0.8 1.0

Metal ion (mM) Metal ion (mM)

Figure 4: Effects of metals on dehydrogenase
activity in Bacillus sp. DISK1. Experimental data
mean 1SD (n = 3) as data points and bars are
shown with predicted values derived from
equation 9 (a) and equations 11 and 12 (b).
proteins and can cause oxidative stress (Stohs and
2+
Bagchi, 1995; Goyer, 1997). Zn , Cd
reported to inhibit the growth of Pseudomonas
aeruginosa, Bacillus thuringiensis and Escherichia coli
K12 (Hassen et al., 1998a,b). Although the mechanism
of the inhibition of dehydrogenase activity was not
investigated in this study, metals have been reported to
disrupt integrity of cell membrane (Gadd, 1993) which is
the site of dehydrogenase activity in bacteria. Cd2+ was
able to alter the outer membranes of bacteria and disturb
the proton flux through the membrane (Bitton et al.,
1988). However, Surowitz et al., (1984) showed that the
effects of Cd2+ on respiration of sensitive strains seemed
to involve metabolic mechanisms prior to the entry of
electrons in the electron transport system rather than on
the transport system itself.
The effect of metal ions on the dehydrogenase
397
Figure 5: Effects of metals on dehydrogenase
activity in Escherichia coli. Experimental data
mean 1SD (n = 3) as data points and bars are
shown with predicted values derived from equation 9
(a) and equations 11 and 12 (b).
activity in the bacterial strains can be described
2+ 2+
and Co were mathematically with kinetic logistic functions and in the
form similar to non-competitive inhibition of enzyme
activity. Equation 9 was used to determine the inhibition
coefficient (Ki) and predict the toxicity threshold
concentrations (IC20, IC50 and IC80). The toxicity
threshold concentration a, which is the concentration of
metal ions above which there is inhibition, was predicted
from equations 11 and 12. The values of ICs, Ki and a
are shown in Tables 1 and 2. Bacillus sp. DISK1,
Pseudomonas sp. DAF1 and Escherichia coli are most
sensitive to the toxicity of Zn 2+, Cd2+ and Co2+
respectively. Based on the IC50, the order of sensitivity is
Bacillus sp. DISK1 > Escherichia coli >
Pseudomonas sp. DAF1 > Pseudomonas sp. RWW2 (for
Zn2+), Pseudomonas sp. DAF1 > Bacillus sp. DISK1 >
Escherichia coli > Pseudomonas sp. RWW2 (for Cd2+)
Journal of Research in Biology (2012) 2(4): 392-402
Nweke and Okpokwasili, 2012

and Escherichia coli > Bacillus sp. DISK1 >

0.987
0.982
0.918
0.982
0.963
0.988
0.977
0.999
0.828
0.051
0.517
Pseudomonas sp. DAF1 > Pseudomonas sp. RWW2

adj
R2

-
(for Co2+). Cd2+ (as CdCl2) was reported to inhibit
dehydrogenase activity in sediment bacteria by 20 % at
Equation 12

60 ppm (0.327 mM). The inhibitory effect of Cd2+ on

0.3731
0.0147
0.3461
0.6273
0.2293
0.3251
0.0041
0.0005
0.0125
0.0220
0.0071
Ki

-
Bacillus species was detected at 0.05 mM. This
inhibition was marked at 0.10 mM and lethal at 0.15 mM
(Montuelle et al., 1994). According to Kleiner (1978),
Zn2+, Cd2+ and Co2+ inhibited the oxidation of NADH by
-0.2489
-0.0251

-0.0215
0.0085
0.0183

0.0490

0.0468
0.0490
0.0206
0.0500
0.0481
Table 1: Parameter estimates from heavy metal inhibition of dehydrogenase activity data.

-
50% at 0.003, 0.004 and 0.025 mM respectively.
Similarly, oxidation of NADPH and succinate was
inhibited by 50 % at 0.008 and 0.012 mM (Zn2+), 0.025
0.997
0.999
0.988
0.988
0.980
0.993
0.997
0.999
0.841
0.957
0.995
0.547
adj
R2

and 0.012 mM (Cd2+) and 0.1 and 0.4 mM (Co2+)

(Kleiner, 1978). On the basis of respiration inhibition in
Pseudomonas fluorescens, EC50s of CdCl2.2.5H2O and
0.3886
0.0093
0.3072
0.6263
0.2271
0.3270
0.0026
0.0007
0.0236
0.0028
0.0006
0.1232
Ki

ZnCl2 were 52.6 mg/l (0.230 mM) and 74.3 mg/l

Equation 11

(0.545 mM) respectively (Codina et al., 1993). In some

Pseudomonas sp. DAF1, Pseudomonas sp. RWW2, Bacillus sp. DISK1 and Escherichia coli

cases, the toxicity threshold reported in this study

0.791
1.309
0.461
0.882
1.109
0.728
1.716
0.900
0.507
2.197
3.182
0.067
KI

corroborates other reports in the literature. Variations in

the toxicity threshold are attributable to a number of
factors including the genetics of the bacteria and the
9.588 x 1013
0.00001

-0.0147
0.0452

0.0013
0.0445
0.0245
0.0228
0.0494
0.0471
0.0500

0.0500

assay conditions.
a

The reciprocal of the inhibition coefficient

represents the affinity of the enzyme to the toxicants.
Small Ki indicates strong affinity between the enzyme
0.987
0.999
0.990
0.996
0.990
0.998
0.999
0.999
0.996
0.991
0.999
0.999
adj
R2

and the toxicant, and thus the more strongly the enzyme
is inhibited at a given concentration of toxicant.
-7
Equation 9

Therefore, smaller Ki means higher toxicity and smaller

10
0.3798
0.0093
0.3286
0.6265
0.2149
0.3263
0.0022

0.0125
0.0005
0.0008
0.1083
x

IC50. The Ki estimates were compared with IC50s

Ki

1.1382

obtained with each metal ion. The Pearson

product-moment correlation coefficient (r) is calculated
as 0.9802 for Zn 2+, 0.9834 for Cd2+ and 0.7122 for Co2+.
1.023
1.307
0.433
0.887
1.279
0.874
2.119
5.101
1.175
4.396
2.881
0.334
KI

Based on Ki, the order of sensitivity is Escherichia coli >

Bacillus sp. DISK1 > Pseudomonas sp. DAF1 >
Pseudomonas RWW2 for Zn2+, Bacillus sp. DISK1 >
Metal

Cd2+

Cd2+

2+

Cd2+
Co2+

2+

2+

Co2+
Zn2+

2+

Zn2+

Zn2+
Co

Cd
Co
Zn

Escherichia coli > Pseudomonas sp. DAF1 >

Pseudomonas sp. RWW2 for Cd2+ and Bacillus sp.
Bacteria

RWW2

DISK1

E. coli

DISK1 > Escherichia coli > Pseudomonas sp. RWW2 >

DAF1

Pseudomonas sp. DAF1 for Co2+. This sequence is

Journal of Research in Biology (2012) 2(4): 392-402 398

 Nweke and Okpokwasili, 2012

Table 2: Inhibition threshold concentrations of metals

Toxicity thresholds (mM)
Bacteria Metal
IC20 IC50 IC80
Zn2+ 0.113 0.018 0.397 0.046 1.392 0.104
Pseudomonas sp. DAF1 Cd2+ 0.010 0.002 0.028 0.002 0.081 0.004
Co2+ 0.004 0.000 0.081 0.016 1.608 0.688
Zn2+ 0.137 0.035 0.634 0.095 2.949 0.140
Pseudomonas sp. RWW2 Cd2+ 0.096 0.016 0.302 0.038 0.955 0.084
Co2+ 0.061 0.021 0.278 0.069 1.286 0.192
Zn2+ 0.032 0.001 0.055 0.002 0.092 0.007
Bacillus sp. DISK1 Cd2+ 0.033 0.001 0.043 0.001 0.057 0.001
Co2+ 0.009 0.000 0.028 0.001 0.084 0.010
Zn2+ 0.119 0.007 0.172 0.004 0.249 0.003
Escherichia coli Cd2+ 0.048 0.007 0.083 0.008 0.142 0.006
Co2+ 0.001 0.001 0.009 0.007 0.113 0.050

similar to that based on IC50. Ren and Frymier (2003)
reported similar agreement between Ki and IC50 obtained
from bioluminescence inhibition data. This indicates that
Ki can be used as a measure of toxicity. It can be seen
from equations 8 and 9 that Ki is actually a function of
KI
IC50 and is related as Ki = IC50 Similarly, the Ki
estimates obtained from equations 9, 11 and 12 were
compared. The Pearson product-moment correlation
coefficient (r) for equation 9 versus equation 11
2+
comparison were 0.9999 (Zn ), 0.9998 (Cd ) and
2+
0.9995 (Co ). In equation 9 versus equation 12
comparison, r was 0.9998 (Zn2+), 0.9997 (Cd2+) and
2+
0.9987 (Co ). The 2-way ANOVA results showed that
the dehydrogenase activity varied significantly (p < 0.05)
with bacterial type and the concentration of metal ions.
The toxicity threshold concentration a, represent
the maximum concentration of the metal ion required for
normal physiological activity of bacteria. Although the
2+ 2+
minimum concentrations of Zn , Cd and Co
would inhibit dehydrogenase activity in the bacterial
strains were not determined in this work, it would be the
concentration that caused 100% inhibition. Based on non
competitive inhibition of dehydrogenase
2+
(equation 12), toxicity threshold a of Zn was reported
as 0.145mM and 0.099 mM respectively
Pseudomonas sp. PLK5 and Escherichia sp. PLK1
399
isolated from river water (Nweke, 2009). While equation
12 predicts negative inhibition of dehydrogenase activity
for I < a, inhibition of dehydrogenase activity for I < a is
undefined with equation 11. However, equation 12 was
not used to predict the effect of metal ions at
concentrations below a. In some organisms, the actual
threshold concentrations were not detected. However, the
models returned negative values. This is attributed to
high percentage inhibition of dehydrogenase activity at
2+
low concentration (0.05 mM) of the respective metals
indicating that relatively lower concentrations of the
metal ions are required to inhibit dehydrogenase activity
in the organisms. Thus, it means that the threshold metal
ion concentration (a) would be below 0.05 mM. Below
a, stimulation of dehydrogenase activity is expected.
Such stimulation was observed in Pseudomonas sp.
RWW2 at 0.05 mM Cd2+ where dehydrogenase activity
was stimulated by 6.613 4.675%. The reason for this
2+
that stimulation is not known, as cadmium is not known for
any physiological function.
The results of this study have shown that zinc,
cadmium and cobalt are toxic to bacterial metabolic
activity activities. Degradation of organic matter is a
dehydrogenation process. Therefore, inhibition of
for dehydrogenase enzyme activity would result to the
inhibition of biodegradation activities. The
Journal of Research in Biology (2012) 2(4): 392-402
Nweke and Okpokwasili, 2012
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402
 Journal of Research in Biology
Stability Ball Exercises In Type 2 Diabetic Patients
Journal of Research in Biology
Authors:
Subramanian SS1 and
Venkatesan P2.
Institution:
1. Principal, Sree Balaji
College of Physiotherapy,
Chennai -100, India.
2. UGC Professor Emeritus
in Zoology, Loyola College,
Chennai -34, India.
Corresponding author:
Subramanian SS.
Web Address:
http://jresearchbiology.com/
documents/RA0242.pdf.
Journal of Research in Biology
An International
Scientific Research Journal
An International Scientific Research Journal
Original Research
ABSTRACT:
Number of people with diabetes in India is 40.9 million and is expected to get
rise to 69.9 million by 2025. Proper management can improve health of individuals
with diabetes and minimize many complications that may occur among diabetic
patients, along with due medications and regular physical exercises. Exercises using
Stability ball were quite effective in improving the glycemic control among Type 2
diabetic patients.
Keywords:
Type 2 Diabetes, Stability ball
Abbreviations:
Stability Ball Exercises (SBE)
Fasting Blood Sugar (FBS)
HbA1C - Glycocelated Haemoglobin
Note:
Stability ball is also called physio ball, swiss ball / gym ball, which is an air
inflated ball of 55cm size widely used as a rehabilitation tool in physiotherapy.
Article Citation:
Subramanian SS and Venkatesan P.
Stability Ball Exercises In Type 2 Diabetic Patients.
Journal of Research in Biology (2012) 2(4): 403-409
Dates:
Received: 14 May 2012 Accepted: 25 May 2012 Published: 14 Jun 2012
This article is governed by the Creative Commons Attribution License (http://creativecommons.org/
licenses/by/2.0), which gives permission for unrestricted use, non-commercial, distribution and
reproduction in all medium, provided the original work is properly cited.
403-409 | JRB | 2012 | Vol 2 | No 4
www.jresearchbiology.com
 Subramanian and Venkatesan, 2012

INTRODUCTION Balaji College of Physiotherapy, Chennai, India between

The prevalence of Diabetes is rapidly rising all May 2010 and July 2010.
over the globe at an alarming rate (Huizinga MM and 80 male Type 2 diabetic subjects between 30 - 60
Fothman, 2006). Last three decades, the status of years were randomly assigned to 12 weeks supervised
diabetes has changed from being considered as a mild control group (n=40) or moderate intensity resistance
disorder of the elderly to one of the major causes of exercises using stability ball (n=40). Fasting blood sugar,
morbidity and mortality affecting the youth and middle Post prandial sugar, Glycocelated haemoglobin and
aged people (Wild et al., 2004). India leads the world Waist circumference were measured before training
with the largest number with (40.9 million) diabetic (i.e., 0 week) and after 12 weeks of training.
patients (Sicree et al., 2006). The most disturbing trend Inclusion criteria were as follows:
is the shift in age of onset of diabetes to a younger age in Established Type 2 diabetes,
recent years, which could have long lasting adverse An inactive life style,
effect on nation's health and economy (Suresh et al., Not Insulin dependant,
2005). Male between 30 - 60 years.
Reduced physical activity, changes in dietary The eligible subjects underwent a medical
pattern, and sedentary occupational habits are the major screening and physical evaluation to exclude individuals
causes for concern(Misra, Pandey et al., 2001). The with subjective or objective evidence of Uncontrolled
American diabetes association (ADA) recommends that hypertension, Coronary artery disease, Advanced
individual with Type 2 diabetes perform at least 150 retinopathy, Neuropathy and severe orthopaedic
minutes of moderate intensity aerobic exercises or at conditions restricting physical activity.
least 90 minutes of vigorous aerobic exercises per week Graph 1 showing
Subjects Pre & Post
were assigned mean values
at random to oneofof the
(American Diabetes Association, 2002). Resistance two groups:control group &ball
- Stability Stability ball (n=40)
exercises exercises
or Control
exercise training is effective in improving glycaemic group (n=40).group
All onthe
Fasting bloodgave
subjects sugartheir written
control and can be used as an adjunct to standard care of
180
Type 2 diabetic patients (Carmen Castaneda 2002). 151 146
160 143
Vibration exercises are an effective, low time consuming
Fasting Blood Sugar in mg

140 124
tool to enhance glycaemic control in Type 2 diabetic
120
patients (Klans Banm 2007).
100
The objective of the study was to assess the
80
effects of 12 weeks of moderate intensity resistance
60
exercises using stability ball on glycaemic control on
40
male Type 2 diabetic patients was analysed, which is the
20
first of its kind study among Indian population.
0
Pre & Post mean values Pre & Post mean values
MATERIALS AND METHODS ofControl Group of SBE group
Subjects were recruited through diabetic camp Pre Test Mean value Post Test Mean value
organized during May 2010 through advertisements
Graph 1 showing pre & post mean values of control
given in regional news paper, The Hindu and Velachery group & stability ball exercises group on fasting blood
sugar
times respectively. The study was conducted at Sree
404 Journal of Research in Biology (2012) 2(4): 403-409
Subramanian and Venkatesan, 2012

informed consent to participate in the study.
Outcome measures
The subjects were tested on two occasions by
using same protocols. Baseline measurement was taken
before the intervention and after the study all the
measurements were taken again.
Venous blood sample of all subjects were taken
for analysis of Fasting blood sugar, Post prandial and
Glycocelated haemoglobin.
Anthropometric measures
Waist circumferences were measured in
centimetres around iliac crest before and after the study.
Intervention
Stability Ball Exercise (SBE) Group
Subjects allotted to this group have performed
systemic supervised resistance training in line with
(American Diabetic Association) ADA and ACSM
(American College of Sports Medicine) guidelines.
Subjects exercised for three times per week. Each session
comprises of 10 exercises for major muscle groups of
lower extremities including Lumbar spine extensors,
Abdominals, Gluteus Maximus, Quadriceps femoris,
Hamstrings, Gastrocnemius. For a period of 12 weeks
subjects have performed 3 sets of 5 repetitions of each
was designed in such a way that up to 4 weeks no
holding of each physical activity, from 4 - 8 weeks. 5
seconds hold of each exercises and 10 seconds hold of
each exercise during the period from 8 - 12 weeks.
Concept of technique
All the exercises performed were in the nature of
isometric contraction of major muscle groups and closed
kinematic chain exercises of both lower extremities.
Hence body weight of the subjects providing resistance
to each activity and the peak torque produced with every
physical activity done using stability ball.
Care points
Subjects were advised not to hold breath during
exercises. Two hypoglycaemic incidents had occurred
and due medical treatment was given. All the subjects
completed the training schedule of 12 weeks.
Control Group (CG)
Subjects underwent no specific training other
than their day to day routine physical activities. All the
subjects in Control Group and Stability Ball Exercise
Groups continued their prescribed medication and daily
Graph 2 showing Pre & Post mean values of
routine activities.
control group & Stability ball exercises
group on Post Prandial blood sugar
Post Prandial Blood Sugar

exercise per session. Progressive increase in intensity 200 201.47

189
Table 1: Baseline charecteristics of all the subjects 200 175.35
Parameters Group Group
Control Stability 150
in mg

Group Ball
(n-40) Exercise
Group 100
(n-40)
Age in years 30-40 7 11
50
41-50 18 13
51-60 15 16
Family Mother 12 11 0
History Father 7 7 Pre & Post mean Pre & Post mean
Both Parents 12 17 values ofControl values of SBE group
Nil 0 2
Unknown 9 3
Group
Cigarette Smokers 25 30 Pre Test Mean value Post Test Mean value
Smoking Non- 15 10
Smokers Graph 2 showing pre & post mean values of
Hypertension Hypertensive 17 17 control group & Stability ball exercises group on
Normotensive 23 23 post prandial blood sugar

Journal of Research in Biology (2012) 2(4): 403-409 405

 Graph 3 showing Pre & Post mean
Graph 4 showing Pre & Post mean
values of control group & Stability ball values of control group & Stability ball
exercises group on Waist exercises group onand
Subramanian HbA1C %
Venkatesan, 2012
Circumference in cm
120 10
94 95 92 89.77
100 9
Waist Circumference in cm

7.9 7.77
8 7.5
80

in%in %
7 6.36
60

Haemoglobin
6

GlycelatedHaemoglobin
40
5
20
4

Glycocelated
0
Pre & Post mean Pre & Post mean 3
values ofControl values of SBE
Group group 2
Pre Test Mean value Post Test Mean value
1
Graph 3 showing pre & post mean values of control
group & Stability ball exercises group on waist 0
Circumference in cm Pre & Post mean Pre & Post mean
values ofControl values of SBE group
The interactions of nine single nucleotide
Group
polymorphisms and cigarette smoking on blood pressure PreTest
Pre TestMean
MeanValue
value PostTest
Post Test Mean
Mean value
Value
levels were detected (Rui-Xing Yin et al., 2012) Graph 4 showing Pre & Post values of control group
An association of insertion / deletion & Stability ball exercises group on HbA1C %
polymorphism of alpha-adrenoceptor gene in essential Post prandial blood sugar was reduced following stability
hypertension with or without Type 2 Diabetes mellitus ball exercises by a mean value of 36 among the pre and
was proven (Vasudevan et al., 2008). post mean scores of Stability ball Exercise group, so is
Among stability ball exercise group, Fasting significant statistically at 5% probability level as P < .05.
blood sugar post mean value has decreased by 22 and Glycocelated haemoglobin mean values have
P < .05, Post prandial blood sugar post mean value decreased by 1.41% among pre and post mean values of
lowered by 36 and is significant at P <.05. Glycocelated Stability ball exercise group, so that is highly significant
Haemoglobin post mean value lowered by 1.41, and is at 0.1% probability level with P < .001. Waist
statistically significant at P < .001. Waist circumference circumference has decreased in the mean values of pre
post mean value lowered by 2.33 and with statistical and post mean scores of Stability Ball Exercise group by
significance of P < .001. 2.33, indicating of high statistical significance at 0.1%
Initial measurements and post training changes probability level with P < .001. Whereas among the
were analysed using paired t test. Statistical tests were control group subjects, Fasting Blood sugar, post
performed using SPSS software (Table 2). As displayed prandial blood sugar, Glycocelated haemoglobin and
in the above table Fasting blood sugar has reduced by a waist circumference level were statistically insignificant
mean value of 22 among the pre and post test mean among their pre and post test scores.
scores among Stability Ball Exercise group, hence is
statistically significant at 5% probability level as P < .05.
406 Journal of Research in Biology (2012) 2(4): 403-409
Subramanian and Venkatesan, 2012

Table 2: Results of paired t test among Stability Ball Exercises group:

Mean S.D Significance
Pre Test 146.25 45.56
Fasting Blood Sugar (mg) P < .05
Post Test 124.2 23.56
Pre Test 201.47 58.85
Post Prandial Blood Sugar (mg) P < .05
Post Test 175.35 43.58
Pre Test 7.77 1.21
HbA1C P < .001
Post Test 6.36 1.26
Pre Test 92 6.73
Waist Circumference P < .001
Post Test 89.77 6.28

DISCUSSION glucose levels (Baldi JC and Snawling N, 2003). A better

This study confirms that following Stability Ball
Exercises significant improvement in Glycocelated
Haemoglobin, Fasting Blood Sugar, Post Prandial Blood
Sugar and Waist circumference measures compared to
control group. Moderate intensity resistance training
results in a mean reduction of Glycocelated
Haemoglobin by 1% to 2% (Dustan et al., 1998). 0.5 to
1% reduction of Glycocelated Haemoglobin in response
to resistance exercises among women Type 2 Diabetes
(Cuff et al., 2003). In this study Glycocelated
Haemoglobin among Stability Ball used moderate
resistance exercises has decreased by 1.41%. 1%
decrement in glycocelated haemoglobin following
therapies to lower Glycocelated Haemoglobin can
reduce the risk of diabetic complications such as
myocardial infarction and microvascular disease
(Patel et al., 2008 & Stratton et al., 2006).
Leg exercises accelerate insulin absorption from
the leg, than arm exercises (Koiviste VA and Fligp
1978). As the Glycaemic control of this study with
reduction of Glycocelated Haemoglobin by 1.41 used
only exercises of lower extremity hence supports better
glycaemic control among Type 2 Diabetic subjects.
Glycaemic control improves with resistance training
(University of Calgary 2007). Resistance training
involving major muscle groups have been shown to
improve glycaemic control and reduced Fasting Blood
glucose control was observed due to improvement in
Insulin sensitivity and effects of glucose transporters due
to muscular hypertrophy and blood flow (Ploug and
Ralston 2002 & Rattigan et al., 2001).
Obesity is a most powerful determinant and a
risk factor for developing diabetes (WHO 2004).
Increase in waist circumference was demonstrated to
increased risk to complications in Type 2 diabetic
patients among Asian (Ramachandran, 1999). Among
Indian diabetic patients higher Body Mass Index and
Waist circumference were recorded (chandalia, 1999).
Waist circumference measurements may be a stronger
predictor than Body Mass Index for the identification of
metabolic and cardiovascular disease- associated risk
factors (Baik et al., 2000). In this study where waist
circumference has decreased by a mean value of 2.33cm,
indicating that SBE can be used to prevent many obesity
related diabetic complications.
CONCLUSION
Along with diabetic medications, dietary
restrictions, physical activities such as aerobic exercises,
vibration exercises, resisted exercises using dumbell,
bands. This study using stability ball to provide a new
form of resisted exercises in the comprehensive
management of diabetic type 2 patients can be
considered. Exercises using stability ball were effective

Journal of Research in Biology (2012) 2(4): 403-409 407


in glycaemic control of male, diabetic patients as well in
body weight reduction, which can be used in the
comprehensive diabetic care, which is time conserving
and cost effective.
Limitations and recommendations
With longer study period, more sample to be
studied and a combination of Aerobic and stability ball
exercises may provide further evidence.
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