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Journal of Research in Biology An International Scientific Research Journal

Original Research

Effect of age, sex and hemoglobin type on adaptive and blood biochemical
characteristics in Red Sokoto Goats
Journal of Research in Biology

Authors: ABSTRACT:
Akpa GN, Alphonsus C This study was conducted to evaluate the effect of haemoglobin (Hb) types,
and Usman N. sex and age on adaptive and blood biochemical characteristics of Red Sokoto goats.
Ninety four (94) goats were sampled from two locations: Dei-dei and Gwagwalada
grazing reserved, Abuja. Data were collected on adaptive characteristics {heart rate
(HR) and rectal temperature(RT) and adaptive coefficient (AC) was calculated from
the HR and RT} and blood biochemical characteristics{ haemoglobin (Hb) types,
Hb-concentration (Hb-conc), Potassium concentration (K-conc) and albumin
concentration (alb-conc)}. The effects of haemoglobin type, sex and age on the
adaptive and blood biochemical characteristics of the goats was analyzed by general
Institution: linear model (GLM) procedure of SAS. The results showed that the mean RT of the
Animal Science sampled goats was 38.9C with very minimal variations (CV=0.5). The mean HR of the
Department, Ahmadu Bello
goats was 76.1bpm, with min and max HR of 70 and 80bpm. The mean albumin,
University, Zaria, Nigeria.
Hb and K concentration were 38.4g/l, 8.9g/dl and 4.0Mmol/l, respectively. The
variation of Hb type with adaptive and blood biochemical characteristics was
significant (P<0.05) except Hb concentration. Higher HR was observed in goats with Hb
AA and AB. Age and sex had significant effect (P<0.05; P<0.01) on HR, AC and albumin
concentration of the goats. Although there was no trend in the variation of HR and AC
with age, but HR and AC were higher in the older goats than the younger, however the
albumin concentration significantly decreased with progressive increase in age of the
goats.
Corresponding author: Keywords:
Alphonsus C. Adaptive coefficient, heart rate, rectal temperature, blood biochemical
characteristics.

Email: Article Citation:


mcdyems@gmail.com Akpa GN, Alphonsus C and Usman N.
Effect of age, sex and hemoglobin type on adaptive and blood biochemical
characteristics in Red Sokoto Goats
Journal of Research in Biology (2013) 3(3): 870-875

Web Address: Dates:


http://jresearchbiology.com/ Received: 15 Dec 2011 Accepted: 01 Jan 2012 Published: 16 Apr 2013
documents/RA0167.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.

870-875 | JRB | 2013 | Vol 3 | No 3


Journal of Research in Biology
An International Scientific
Research Journal www.jresearchbiology.com
Akpa et al.,2013

INTRODUCTION related to farm animals. Comprehensive reviews have


In recent years, advances in the field of appeared under the authorship of Alderson, 1992,
biotechnology have opened up a completely new area at Derman and Noakes, 1994, Tambuwal et al., (2002),
molecular levels with the introduction of techniques such Otoikhian et al., 2009, chukwuka et al., 2010,
as routine electrophoresis employed for detection of Opara et al., 2010, Gurcan et al., 2010 and a lot of
polymorphism at protein and enzyme loci as well as others. Research results reported in this paper is intended
other serological and immunogenetic procedures for the to supplement data reviewed by authors listed.
measurement of variations (Salako et al., 2007). Data Eventually, accumulated data will permit specific
obtain from this type of study could be useful as genetic recommendation on breeding, feeding and management
markers for important economic characteristics and of farm animals.
could aid significantly in selection of superior animals This study therefore aimed at studying the effects
for breeding purposes. of Haemoglobin type, sex and age on adaptive and blood
Haemoglobin typing is very important as biochemical characteristics of Red Sokoto goats.
different Hb types may have selective advantage in
different geographical regions (Ndamukong, 1995). MATERIALS AND METHODS
Economic pressures of various kinds are forcing the Location
production of livestock into climatic environments that The study was conducted at the Federal Capital
are increasingly more remote from the considered ideal Territory Abuja, located within the Northern guinea
for optimal production and feed utilization. Savanna zone of Nigeria. It is laying between latitudes
Thermal stress, which is one of the major factors 8.25 and 9.0 N of the equator and Longitude 6.45 and
that affect the productivity of many farm animals can be 7.39 E of the Greenwich Meridian. (Presentation
reflected in an easily observable changes in pulse rate, Copyright@ falling Rain Genomics, 1996-2010).
respiration rate, and rectal temperature, although the Data collection
whole body reacts to thermal stress by an elaborate series Ninety four (94) goats were sampled from two
of chain reactions (Ahmed, 2004). The most obvious locations: Dei-dei and Gwagwalada grazing reserve,
index of thermal stress is body temperature response. Abuja. Data were collected on the adaptive and blood
Deviation from normal rectal temperature indicates that biochemical traits. The adaptive traits were Heart rate
the animal is under stress, that its homeothermic (HR), Rectal temperature(RT) and Adaptive coefficient
mechanisms are overtaxed Ahmed, 2004). (AC) while the blood biochemical characteristics were
Adaptive characteristics of animals serve as a haemoglobin (Hb) types, Hb-concentration (Hb-conc),
key to managing any livestock operation. Adaptive traits Potassium concentration (K-conc) and albumin
such as rectal temperature, heart rate, and flank concentration (alb-conc).
movement have been documented to have some
significant effect on genetic variations. Every normal METHODS OF MEASUREMENTS
animal has a range of individual adaptive traits in Adaptive Traits
relation to a specific physiological pattern. Heart Rate (HR)
Because study of environmental physiology Heart rate was taken by placing stethoscope on
involves so many variables and scientific discipline, femoral artery of the hind limb of the goat to count the
much is being published on this subject especially as number of beat per minute.
871 Journal of Research in Biology (2013) 3(3): 870-875
Akpa et al.,2013

Rectal Temperature (RT) the Rectal Temperature (RT) and Heart Rate (HR) as
This was taken using clinical thermometer which thus
was inserted into the rectum of the goat and left for Adaptive coefficient (AC) = (RT/38.33) + (HR/23.00)
45-50 seconds. It was then removed and the temperature The effects of haemoglobin types, sex and age on
level was read. The values read were recorded, and the the adaptive and blood biochemical characteristics of the
process was repeated for the other goats. goats were determined by general linear model (GLM)
Blood Biochemical Characteristics procedure of SAS, (2005).
Blood samples were taken from each of the
experimental goats through the jugular vein. 5 mls of the RESULTS AND DISCUSSION
blood was taken from each goat, from which 2 mls was Rectal Temperature (RT) is directly affected by
put into heparinized vacutainer tubes containing the surrounding and ambient temperature, and high
anticoagulant ethylene diamine tetra acetic acid (EDTA). ambient temperature has a negative effect on
The remaining 3 mls of the blood was put into sterile productivity of the animal. Chukwuka et al., (2010)
vacutainer tubes (without anticoagulant). The samples reported that negative effect of high ambient temperature
were labeled accordingly. The blood samples in the is direct in the form of stress suffered by the animal and
sterile vacutainer tube were centrifuged in order to have the diversion of energy from the purpose of production to
a clear layer of serum. This serum was pipetted into regulation of body temperature and indirectly by
another sterile bottle and store in a refrigerator. The affecting the availability of feed resources upon which
blood samples were taken to the Haematological production is dependent. In this study, the mean RT of
laboratory of Ahmadu Bello Teaching Hospital from the goats was 38.9C with minimum and maximum body
where the analysis of blood biochemical characteristics temperature of 38.1 and 39.4 C (Table 1). These values
was carried out. were within the reference range of previous study
A spectrometer with wavelength capability of of goats in thermal neutral condition (Otoikhian et al.,
600-650 nm (Zenway 5041 colorimeter) was used 2009) and this indicate that the goats used for this
to analyzed for the albumin concentration, while the research showed no clinical signs of stress during the
K concentration was analysed using Corning research period. The body temperature of the goats
flame photometer 410. Electrophoresis and exhibited minimal variations (CV=0.5%), thus implying
Cyanmethaemoglobin method was used to analyzed for that goats are homoeothermic animals, they can maintain
Hb-types and Hb-conc, respectively. near constant body temperature under wide range of
Data Analysis environmental conditions.
The adaptability of the goats were measured by The Heart Rate (HR) is the pulse that helps to
determining the adaptive coefficient from the values of know the beating rate of the heart which is measured

Table 1: Summary Statistics of the measured characteristics in Red Sokoto Goats


Characteristics N MeanSE CV(%) Min Max
Rectal Temperature (C) 94 38.90.02 0.5 38.1 39.4
Heart Rate (bpm) 94 76.10.39 3.8 70.0 81.0
Adaptive coefficient 94 4.30.02 5.0 4.1 4.6
Albumin concentration (g/l) 94 38.40.34 8.5 31.0 48.0
Hemoglobin concentration (g/dl) 94 8.90.16 17.1 4.0 12.7
Potassium concentration (Mmol/l) 94 4.00.06 14.7 3.0 5.8

Journal of Research in Biology (2013) 3(3): 870-875 872


Akpa et al.,2013

in beats per minute (bpm) using stethoscope amount of K-concentration is fairly high at intracellular
(Otoikhian et al., 2009). The mean HR of the goats used membranes (Gurcan et al., 2010). The values of
in this study was 76.1bpm, with the min and max HR of K-concentration reported by Opara et al., (2010) for
70 and 80bpm. This is slightly higher than the range of WAD bucks and does were 17.8 and 6.9mmol/l,
70-75bpm reported by Derman and Noaks (1994) in respectively. These values were higher than the mean
goats. The minor difference observed in the values of the value of 4.0mmol/l observed in this study; this is
HR may be explained by differences in geographical probably due to differences in breed and physiological
conditions, season or climate and physiological conditions of the sampled animals. Researchers had
conditions of the sample goats. identified the existence of different type of K in different
Rectal temperature and heart rate have been species of animals, and that in sheep for instance, there
shown to be good indicators of the thermal stress and are two types of K which is high and low K with the
may be used to assess thermal adversity of the low K type dominant over the high K type
environment (Al- Haidary, 2004). (Soysal et al., 2003). Also Gurcan et al., (2010) reported
The Adaptive Coefficient (AC) (which is the a range of 4.23 to 11.69mmol/l for low K type in
function of RT and HR) signifies the level of adaptability animals.
of the goats to the environments varied significantly The concentration of albumin in this study
(P<0.05) with Hb types, sex and age of the goats. The (38.4g/dl) was slightly higher than the 34.5g/dl reported
goats with Hb AA and AB had higher AC than those by Opara et al., (2010).
with BB and AC; likewise the bucks had higher AC than The variation of Hb type with adaptive and blood
the does. biochemical characteristics was significant (P<0.05)
Potassium is one of the intracellular elements except Hb-concentration (Table 2). The relationship
that regulate the intracellular density of the cell. The between Hb types and HR can be linked to the different
Table 2: Effect of hemoglobin type on adaptive and blood biochemical characteristics
Hemoglobin Type
Characteristics
AA BB AB AC SEM LOS
Rectal Temperature (C) 39.0a 39.0a 39.0a 38.9b 0.02 *
Heart Rate (bpm) 76.6a 75.3b 76.2a 75.1b 0.40 *
Adaptive coefficient 4.4a 4.3b 4.4a 4.3b 0.02 *
Albumin concentration (g/l) 37.6b 39.2a 38.6a 38.8a 0.34 *
Hemoglobin concentration (g/dl) 9.1 8.6 8.8 8.6 0.16 ns
Potassium concentration (Mmol/l) 3.8b 3.9a 4.0ab 4.4a 0.06 *
Number of observations 30 11 41 12 94
ab
: means within the same row with different superscripts differ significantly(P<0.05); ns:not significant;

Table 3: Effect of Sex on adaptive and blood biochemical characteristics


Sex
Characteristics
Buck Doe SEM LOS
Rectal Temperature (C) 39.0 39.0 0.03 ns
Heart Rate (bpm) 78.5a 75.0b 0.49 **
Adaptive coefficient 4.4a 4.3b 0.02 **
a
Albumin concentration (g/l) 39.8 37.7b 0.49 **
Hemoglobin concentration (g/dl) 9.1 8.7 0.26 ns
Potassium concentration (Mmol/l) 3.9 4.0 0.08 ns
Number of observations 30 64 94
ab
: means within the same row with different superscripts differ significantly(P<0.01); ns:not significant;
873 Journal of Research in Biology (2013) 3(3): 870-875
Akpa et al.,2013

Table 4: Effect of Age on adaptive and blood biochemical characteristics


Age of goat
Characteristics
12mon 18mon 24mon 30mon SEM LOS
Rectal Temperature (oC) 39.0 39.0 39.0 39.0 0.02 ns
Heart Rate (bpm) 77.1b 74.4c 74.4c 78.5a 0.37 *
Adaptive coefficient 4.4b 4.3c 4.3c 4.5a 0.02 *
Albumin concentration (g/l) 39.2a 37.4b 36.7c 36.5c 0.32 *
Hemoglobin concentration (g/dl) 9.1 8.7 8.3 8.5 0.16 ns
Potassium concentration (Mmol/l) 4.0 4.0 4.0 4.0 0.06 ns
Number of observations 55 25 12 2 94
ab
: means within the same row with different superscripts differ significantly(P<0.05); ns:not significant;

levels of oxygen carrying capacity of the different albumin concentration significantly decreased with
Hb types. In this study, higher HR was observed in goats progressive increase in age of the goats. The observed
with Hb AA and AB, and Hb A is known to be the significant influence of age on albumin concentration
haemoglobin allele with highest affinity for oxygen. This is at variance with the earlier studies of
is in line with the earlier report of Huisman et al., (1959) Piccione et al., (2009) and Opara et al., (2010) who
who relates the preponderance of Hb A to it greater reported non-significant effect of age on albumin
affinity to oxygen. This could also explain the high concentration of WAD goats.
adaptive coefficient observed on goats with Hb types AA
and AB since adaptive coefficient is a function of HR CONCLUSION
and RT. The mean body temperature (38.9C) of the
The variation of HR, AC and Albumin goats used was within the reference normal range for
concentration with sex was highly significant (P<0.01; goats in thermal neutral condition and this indicates that
Table 3). The RT of the buck and does were similar the goats showed no clinical signs of stress during the
however, the HR was higher in bucks (78.5bpm) than the research period.
does (75.0bpm) this is probably due to the high sexual
The albumin concentration, heart rate and
activity of the bucks. There was no significant (P>0.05)
adaptive coefficient of the goats had clear variation
difference between the bucks and does in Hb and
based on differences in haemoglobin type, sex and age of
K concentration. This is contrary to the study of
the animals.
opera et al., (2010) who reported significant differences
between WAD bucks and does in there Hb and
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Haematological parameters and blood chemistry of
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Otoikhian CSO, Orheruata MA, Imaseuen JA and Submit your articles online at www. jresearchbiology.com
Akporhuarho OP. 2009. Physiological response of local
Advantages
(West African Dwarf) and adapted Switzerland Easy online submission
(White Bornu) goat breed to varied climatic conditions in Complete Peer review
South-south Nigeria. African Journal of General Affordable Charges
Agriculture. 5(1):1-6. Quick processing
Extensive indexing
Piccione G, Casella S, Lutri L, Vazzana I, Ferrantelli You retain your copyright
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submit@jresearchbiology.com
Haematological, Haematochemical and electrophoretic www.jresearchbiology.com/Submit.php.
parameters in the Girgentana goats. Faculty of Vetrinary

875 Journal of Research in Biology (2013) 3(3): 870-875


Journal of Research in Biology An International Scientific Research Journal

Original Research

Eco-biology of Common Emigrant Catopsilia pomona Fabricius (Lepidoptera: Pieridae) with


special reference to its life table attributes in Tripura, India
Journal of Research in Biology

Authors: ABSTRACT:
Samit Roy Choudhury and Butterflies of the family Pieridae are common in tropical parts of the world.
Basant Kumar Agarwala* They are considered as major pollinators as well as pests of economically important
plants. Catopsilia pomona is a dominant pierid butterfly found in association with wild
plants of Tripura, northeast India. It is abundant throughout the year. Present study
was conducted to document the eco-biology of Catopsilia pomona with special
reference to its life table attributes in the state of Tripura. Survival rates of life cycle
Institution:
Ecology & Biodiversity stages in the semi-natural as well as in the field were the maximum during the wet
Laboratories, Department of and hot season. Mortality (k value) of different life cycle stages as a proportion of
Zoology, Tripura University, individuals dying during development varied from 0.16 to 0.46 in different seasons.
Suryamaninagar- 799022, Results suggested that abiotic factors and mortality factors of egg significantly
Tripura, India. influenced the survival rate of C. pomona population. This butterfly depends on three
species of Cassia plants, all shrubs, for their oviposition and larval development in the
environment of Tripura.

Corresponding author: Keywords:


Basant Kumar Agarwala Catopsilia pomona butterfly, Pieridae, eco-biology, life table, Tripura, north
east India.

Email: Article Citation:


bagarwala00@gmail.com Samit Roy Choudhury and Basant Kumar Agarwala.
Eco-biology of Common Emigrant Catopsilia pomona Fabricius (Lepidoptera: Pieridae)
with special reference to its life table attributes in Tripura, India.
Phone No: Journal of Research in Biology (2013) 3(3): 876-885
0091 381 237 9083/9123

Web Address: Dates:


http://jresearchbiology.com/ Received: 22 May 2012 Accepted: 28 May 2012 Published: 17 Apr 2013
documents/RA0245.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.

876-885 | JRB | 2013 | Vol 3 | No 3


Journal of Research in biology
An International Scientific
Research Journal www.jresearchbiology.com
Roy Choudhury and Agarwala, 2013

INTRODUCTION plants of north east India, including Tripura. However,


Host selection for survival, development and information on life table and host selections are available
reproduction in majority of insects often vary in space on other pierid species that feed and oviposit on crop
and time (van Nouhuys et al., 2003; Nylin et al., 2009) plants (Chew, 1995). C. pomona, a dominant pierid
which, in turn, depends on the availability (minimum butterfly, is found throughout the year in the state of
density per unit area) of closely related host plant species Tripura (Agarwala et al., 2010; Majumder et al., 2011;
(Thorsteinson, 1960), and trade off between host Roy Choudhury et al., 2011). It prefers green and moist
preference by females for oviposition and larval lands, pasture lands, farms, and edge of drains, moist
performance of insects (van Nouhuys et al., 2003). deciduous forests, hillocks, and semi-arid areas with high
However, adult butterflies and their caterpillars show abundance of grasses, small herbs and shrubs i.e.
preference for certain host plants for tender shoots, secondary type of vegetation (Atluri et al., 2004).
pollen and nectar as food source. Thus, butterfly Reported larval host plants of common emigrant
diversity of a particular habitat generally reflects the comprise of Cassia fistula L., C. sophera L.,
overall plant diversity of that habitat. Butterflies are C. occidentalis L., C. tora L., C. siamea (Lam.) Irwin et
essential component of any natural ecosystem. Their Barneby, Butea frondosa, and Bauhinia racemosa L.
value as indicators of biotope quality is being recognized (Kunte, 2000; Atluri et al., 2004). Among these plants
because of their sensitivity to minor changes in micro- C. fistula, C. tora, C. occidentalis, C. sophera, and
habitat, climatic conditions as well as seasonal changes B. racemosa are important as medicinal plants
(Kremen, 1992; Murugesan and Muthusamy, 2011). (Anonymous, 2004; Danish et al., 2011; Harshal et al.,
They are considered as ideal subject for ecological 2011; Singh and Dubey, 2012), and C. siamea is used in
studies of terrestrial landscapes (Thomas and Malorie, social forestry (Atluri et al., 2004, Borikar et al., 2009).
1985). Hence, it is very important to document the seasonal
North eastern region of India is blessed with occurrence and its host plant preference for oviposition
vegetation rich landscapes that support diverse butterfly and larval development of C. Pomona. With this view,
fauna and other insects (Alfred et al., 2002). The state of the present study was conducted to know the eco-biology
Tripura, being a part of this region, also contains large of Catopsilia pomona with special reference to its life
number of butterfly species which is evident from history attributes in the state of Tripura.
infrequent records of these taxa (Mandal et al., 2002; Study site
Agarwala et al., 2010; Majumder et al., 2011; Roy Present study was conducted in Trishna Wildlife
Choudhury et al., 2011). Butterflies of the family Sanctuary of south Tripura district (2326.137 N,
Pieridae are common in tropical parts of the world and 9128.184 E: 51-82 m asl), having an area of about
are considered as major pollinators of crop plants 194.7 sq. km. Study location is characterized by patches
(Borges et al., 2003), and a few of them are also of secondary moist deciduous forests and surrounded by
considered as pests of economically important plants swamp areas. Forest patches are rich in sal trees, garjan
(Anonymous, 2007; Capinera, 2008). Despite their trees, bamboo bushes, herbs, shrubs and climbers.
common occurrence, there is a lack of substantial study Trishna sanctuary is known by 230 tree species, 110
on the ecology, seasonal abundance, host preference and species of shrubs, 400 species of herbs, and 150 species
life history of the most common pierid species of climbers (Economic review of Tripura, 2008-2009).
Catopsilia pomona F. found in association with wild Among the known host plants of C. pomona, the study
877 Journal of Research in Biology (2013) 3(3): 876-885
Roy Choudhury and Agarwala, 2013

area contains three species of Cassia only viz. plastic tags. Thus, sixty plants from three species were
Cassia sophera, C. tora and C. occidentalis which are selected from transects. Ovipositing females were
considered to be the preferred hosts of larvae. Some part followed in the selected host plants for recording number
of the study area is used for rubber cultivation and paddy of eggs laid per female per leaf. Binoculars were used to
cultivation (Figure 1). The area has a tropical climate, observe the females from a distance (about 2 m) without
with cold weather from November to February. Average disturbing them. The same host plant was also observed
daily temperature varies from the minimum of 6.8C in for presence of larvae. All the females seen ovipositing
January to the maximum of 37.7C in June. The area on the selected host plants was recorded during the
receives, on an average, 3353.4 mm rainfall annually. transect walk. Two transects were walked in two
consecutive days in a week. Ten apical leaves were
MATERIALS AND METHODS observed within a selected plant for egg and larval counts
Field census of eggs, larvae and oviposition which were made between 8.00 AM to 12.00 noon local
preference of C. pomona time. When a female was found to either laying eggs or
Prior to the study a reconnaissance survey was seen perching near a host plant, halt was made for
made in the Trishna study area to locate the available approx. 8 to10 minutes, and then move to the subsequent
host plants distribution of C. pomona. Walk census for host plants along the transect. Different host plants
leaves of host plants containing eggs and larvae were selected by females for oviposition were recorded,
held at an interval of 7-days from March 2007 to photographed, collected and later identified by
February 2008. For this, two line transects (approx. 1 km comparing with the herbarium deposited in the gallery of
long and 5 m wide) were set up in the study area. Thirty Plant Taxonomy and Biodiversity Laboratories,
host plants, 10 plants each of C. sophera, C. tora and Department of Botany, Tripura University.
C. occidentalis, were randomly selected for the study
along the length of transects and were marked with

Figure 1. Geographical map of Trishna and landscape of the Study area.

Journal of Research in Biology (2013) 3(3): 876-885 878


Roy Choudhury and Agarwala, 2013

Larval host range and seasonal variation in Survival rate and K-factor analysis
development An age-specific life table was constructed
Leaves of the host plant species found to contain following the method of Stiling (2002). To prepare the
freshly laid eggs of C. pomona in field were brought to life table, records were made on the larval durations and
the field station (3 km from the study area), and survival rate at each developmental stage i.e. eggs to
transferred individually to 10 cm diameter paired Petri emergence of adults from pupae. For this purpose,
dishes lined with corrugated papers. These were fed with 409 eggs and 317 eggs of C. pomona were studied in
surplus quantity of tender leaves of respective host plants natural (in field) and in controlled conditions (ambient
from which they were actually collected. Twenty condition of field station), respectively. Meteorological
replicates were used for each host plant species. Food data of Trishna study area were collected from the
was changed every 24 hrs intervals. Petri dishes were records maintained by the Department of Agriculture,
cleaned at the time of food change. These were observed Govt. of Tripura at Arundhuti Nagar, Agartala.
twice in a day at 11 am and again at 5 pm to record the Data analysis
incubation period of eggs, developmental time of larvae, Field data on proportion of host plants used by
and pupae. Mortality in development, if any, was also C. pomona for laying of eggs and distribution of eggs per
recorded. This was simultaneously done on each host leaf of the different host species during a year were used
plant, once in five different seasons to record the to draw population curves. For this purpose, weekly data
seasonal variation, if any. Experiments were set up at the were pooled on monthly basis. Developmental time from
field station (Temp: 18C ~ 27C, RH: 45~75%, and egg to the eclosion of pupae on different host plants and
L: D: 16:8h) i.e. in the controlled environment. between different seasons was subjected to one-way
Larval development in field analysis of variation (ANOVA). Mean values of
Selected plants with freshly laid eggs and development time on different host plant species and
subsequent developmental stages were provided with between different seasons were compared by Tukeys
coloured tags and these were numbered for easy multiple comparison test. Differences in development
identification. Individual eggs, larvae and pupae were time recorded in field and in field station were compared
followed daily, and the disappearance of individuals or by Students t-test. A significance level of 0.05 was used
those that failed to develop in to the next stage at to reject the null hypothesis. Field data on distribution of
different life stages were recorded. Larvae were found to eggs on different host plant species were subjected to
be slightly sluggish and females laid solitary eggs, regression analysis to reveal the relationship between
usually one on each leaf. The study was repeated once in oviposition preference and host utilization. Based on the
different seasons. life table data, survival rate and K factor value that
closely mirrors the overall population mortality was

Table 1. Oviposition preference of C. pomona females on different host plants in the study area
Host plant No. of leaves No. of larvae Mean (+SEM) no ANOVA No. of eggs Mean (+SEM) ANOVA
observed counted of larvae/ leaf counted no of eggs/ leaf

C. sophera 4800 984 0.21 + 0.01 1237 0.26+0.02


F = 6.909 , F = 5.26,
C.occidentalis 4800 563 0.12 + 0. 02 df = 2,14397, 899 0.19+0.03 df = 2,14397,
P = 0.0001 P = 0.006
C. tora 4800 647 0.13 + 0.01 816 0.17+0.02

879 Journal of Research in Biology (2013) 3(3): 876-885


Roy Choudhury and Agarwala, 2013

Table 2. Development time (in days) of C. pomona on different host plant species
Month N Mean + SEM value (days)
C. sophera C. occidentalis C. tora
1 1
March 36 24.50 + 0.26 a 24.75 + 0.25 a 24.74 + 0.33 1 a
May 36 20.67 + 0.31 2 a 20.92 + 0.42 2 a 20.92 + 0.42 2 a
August 36 18.92 + 0.23 3 a 19.42 + 0.63 3 a 19.00 + 0.28 3 a
October 36 21.17 + 0.24 2 a 21.33 + 0.28 2 a 21.25 + 0.25 2 a
December 36 30.67 + 0.47 4 a 30.83 + 0.41 4 a 31.00 + 0.41 4 a

Dissimilar numbers following means in a column denote significant difference and similar letters accompanying
means show no difference between them by Tukeys multiple comparison range test at 5% significant level.

determined. At each life stage, number of deaths found with one or more eggs. Between the three host
(k value) was calculated as under: k = log Nt - log Nt+1, plant species, common emigrant females selected the
where Nt is the density of the population before it is highest proportion of C. sophera for oviposition during
subjected to the mortality and Nt+1 is the density hot and wet months, and the maximum was recorded in
afterward. Total generational mortality factor K is the month of August (Figure 2). In comparison,
determined as the sum of the individual mortality factors distribution pattern of eggs on C. occidentalis plants
k at egg, larval and pupal stage of the C. pomona species showed marked difference from the distribution of eggs
(Stilling, 2002). For interpretation of colleted data, the on C. sophera. Higher proportion of this host plant
year was divided in to five seasons: spring (March, species was recorded during dry and cooler months, and
April), summer (May, June), rain (July, September), the maximum was recorded in the month of January
autumn (October, November), and winter (December- (69.47%) (Figure. 2). In case of C. tora, the trend of egg
February). To determine the relationship between distribution was found to be nearly similar to that of
successful development (%) of C. pomona eggs and C. sophera but the proportion of host use was found to
climatic factors in the study area regression analysis was be much lower than C. sophera (Figure. 2). Occurrence
carried out. Origin 7 software (www.originlab.com) was of eggs showed that 4800 leaves each of C. sophera,
used for the analysis of data. C. occidentalis and C. tora that were surveyed during the
year, contained 1237, 899 and 816 eggs, respectively
RESULTS (mean + SEM: C. sophera: 0.26+0.02 eggs per leaf,
Egg abundance and oviposition preference C. occidentalis: 0.19+0. 03 eggs per leaf and C. tora:
Females of C. pomona laid solitary eggs at edges 0.17+0.02 eggs per leaf, ANOVA: F = 5.26, df = 2,
and on undersides of tender or young leaves (one egg/ 14397, P = 0.006) (Table 1).
leaf/female) of C. sophera, C. occidentalis and C. tora Larval host range
plants throughout the year (Table 1, Figure. 2). In the Larvae of C. pomona were found to feed on
2
year-round census of 10000 m (1000 m long x 5 m wide tender leaves of the three host plant species, viz.
x 2 transects @ 1 ha) which represents less than 0.5% of C. sophera, C. occidentalis and C. tora. Higher
the study area (19.47 ha), 52.54% to 85.07% of proportion of C. sophera plants were used as food and
C. sophera plants, 21.31% to 69.47% of C. occidentalis maximum was recorded in the hot and wet month of
plants and 23.88% to 56.52% of C. tora plants were August (26.70%). Incidence of larvae on C. occidentalis

Journal of Research in Biology (2013) 3(3): 876-885 880


Roy Choudhury and Agarwala, 2013

Proportion of host plants selected for oviposition 0.9 C. occidentalis


C. sophera
C. tora
0.8

0.7

0.6

0.5

0.4

0.3

0.2

Jan Feb Mar Apr May Jun July Aug Sep Oct Nov Dec
Months

Figure 2. Proportion of host plants of three Cassia Figure 3: Mean number of larvae of C. pomona
species recorded with eggs of C. pomona in different recorded on different host plants.
months of the year in the study area.

0.13 + 0.01 larva per leaf, ANOVA: F = 6.909,


df = 2,14397, P = 0.0001) (Table 1).
Developmental time and seasonal variation
Developmental time of different immature stages
(egg to pupae) of C. pomona was found to vary in
different seasons but did not show difference in any one
season between different host species (Figure 4).
Development time was recorded to be the longest at
lower temperature and lower relative humidity
corresponding to the month of December (controlled
condition: average temperature=18C, average relative
Figure 4. Development time (in days) of C. pomona on
different larval host plants in different months of a humidity=51.33%) and shortest at higher temperature
year. Similar alphabets accompanying bars denote no and higher relative humidity in August (average
significant difference between the mean values in that
month. temperature=27.91C, average relative humidity
=77.07%) (Table 2).
plants was recorded to be the highest in January Survival rate and K factor analysis
(20.61%) and lowest in August (1.64%), respectively. In Results showed that in field about 30% of the
case of C. tora host, the highest proportion was recorded eggs deposited by C. pomona developed in to pupae
in the month of June (17.24%) and the lowest in the during the months of July and August (average
month of January (5.33%) (Figure. 3). Occurrence of temperature 31.09C, average humidity 70%, mean
larvae showed that 4800 leaves each of C. sophera, rainfall 7.45 cm). Developmental success was limited to
C. occidentalis and C. tora that were surveyed during the 13.04% in the month of December (average temperature
year, contained 984, 563 and 647 larvae, respectively 19.330C, average humidity 51%, rainfall 0 cm).
(mean + SEM: C. sophera: 0.21 + 0.01 larva per leaf, Regression analysis of survival rate showed positive
C. occidentalis: 0.12 + 0. 02 larva per leaf and C. tora: correlations with average temperature (y =1.08 + 0.87x,
881 Journal of Research in Biology (2013) 3(3): 876-885
Roy Choudhury and Agarwala, 2013

Number of eggs that developed successfully in fields


(24.03+1.46; n=240) and in semi natural condition
(76.36+0.90; n=240) showed significant difference
(t =30.54, df =478, P =0.000).
K-value analysis showed maximum mortality in
development (0.46) in the month of December and
minimum (0.16) in the month of September. K - values
of the eggs recorded in different seasons were found to
be very high (0.21) and very low (0.09), respectively,
during these two months. Analyses showed that mortality
in the egg stage influenced the total K value the most
(Figures. 6a, b).

DISCUSSION
Natural populations of phytophagous insects
including butterflies frequently encounter wide choice of
host plants of differing suitability (Badeness et al., 2004;
Dennis et al., 2006). The dominant strategy among
herbivorous insects involves specialization on a set
of closely related plants that will maximize offspring
survival and fitness (Ward and Spalding, 1993; Gibbs et
al., 2006), and also to the phenological characteristics of
host plants. It is evident from the present study that
C. pomona butterflies utilize three species of Cassia for
oviposition and larval development in Trishna study
area. Among these host plants, maximum number of
C. pomona eggs were found in C. sophera with higher
proportion recorded during hot and wet months, and
lowest in dry and cooler months of the year. During dry
and cool months, females choose C. occidentalis in
higher proportion for oviposition followed by C. tora.
This might be due to the availability of more young
Figure 5. Regression analysis between successful leaves in C. occidentalis and C. tora compared to
development (%) of C. pomona eggs and climatic
C. sophera in dry and cooler months of the year. Results
factors: (a) average temperature (oC), (b) average
relative humidity (%), and (c) mean rainfall (cm). indicated that common emigrants preferred C. sophera
than the two other host plants but utilized three hosts
2
r =0.74) (Figure 5a), average relative humidity throughout the year depending on the host plant
2
(y = 3.87 + 0.33x, r = 0.52) (Figure 5b), and with mean phenology, and made the larval host range wider.
2
rainfall (y =20.07 + 1.64x, r = 0.64) (Figure 4c). Patterns of host use have several effects on butterfly

Journal of Research in Biology (2013) 3(3): 876-885 882


Roy Choudhury and Agarwala, 2013

relation to host plants in time and space (Begon et al.,


1996; Scheirs and Bryn, 2002).
Population of C. pomona showed strong
relationships with climatic factors. They took longer time
for development in the dry and cooler months when the
suitable habitat for oviposition and larval development
were minimum than in wet and hot months. But,
developmental time on the different host plants did not
differ during a particular season that suggested possible
qualitative similarity between host plants. However,
several studies showed that ovipositing females of
phytophagous butterflies typically show a preference for
host plants that are capable of supporting fast larval
growth (Thompson, 1988a, b, c; Janz et al., 1994).
Climatic factors are well known for their
significant influence on population dynamics of animal
communities (Leonard et al. 1998). Analysis of K-value
in this study has revealed that the average temperature,
the average relative humidity and the mean rainfall
showed strong positive relationships with survival rate of
C. pomona. In the present study no biotic factors such as
parasites, predators were noticed which can also
Figure 6. Key- factor analysis of development of influence the population dynamics of C. pomona
C. Pomona: (a) mortality in developmental stages butterfly.
expressed as k values, (b) regression fit of mortality in
egg stage (k1) to the total K value.
CONCLUSION
population dynamics (Hanski and Singer, 2001). Food Results revealed that C. pomona females
plant-insect herbivore association is based on resource occurred and laid eggs throughout the year on three host
size and optimal synchronization of their respective plant species of Cassia. It preferred C. sophera host over
life-cycles. If resource size in time and given space is C. occidentalis and C. tora for oviposition and larval
large, insects will show monophagism. In comparison, if development. Pattern of egg distribution i.e. oviposition
resource size is short and patchy, then insect herbivores was found to be linked with host plant phenology. Egg
are generally polyphagous or oligophagous (Price, 1997; mortality was the major influencing factor in
Dixon, 1998; Nylin et al., 2009). In this study, determination of survival rate. The k-value of egg
availability of taxonomically closely related Cassia mortality (k1) and total mortality factor (K) showed
plants in time and given area under study on which strong positive relationship.
C. pomona successfully completed their life history
attributes might widen their host range. This finding is in ACKNOWLEDGEMENT
conformity with the optimisation theory of species in Authors are thankful to the Head, Department of
883 Journal of Research in Biology (2013) 3(3): 876-885
Roy Choudhury and Agarwala, 2013

Zoology, Tripura University for the laboratory facilities. Properties. Journal of Natural Product Plant Resources.
1(1): 101-118.

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Journal of Research in Biology An International Scientific Research Journal

Original Research

Local peoples attitude towards conservation and development around


Pichavaram mangrove ecosystem, Tamil Nadu, India.
Journal of Research in Biology

Authors: ABSTRACT
Lakshmi Kodoth and
Ramamoorthy D. Studies in mangrove ecosystem are often focused on biological or ecological
criteria and interdependency between mangroves and people is normally neglected.
The situation is similar in Tamil Nadu; India which has a coastline of about 950 km.
One of the major mangrove forests in Tamil Nadu is situated in Pichavaram, Cuddalore
Institution:
district. The present study was carried out in the seventeen hamlets, which are
Department of Ecology &
Environmental Sciences, directly or indirectly dependent on the Pichavaram mangrove wetlands for their
Pondicherry University, livelihood and survival. These seventeen hamlets consist of over 2600 households
Puducherry. many of whom derive their principal income from fishing and related activities.
Individual surveys were carried out for 10% of the households in each of the selected
hamlets. Semi-structured questionnaires were used for surveys to study the attitude
and perception of the community on the conservation and importance of mangrove
wetlands and resources. The study was conducted to assess the awareness, attitudes
Corresponding author: and views of people dependent on the mangrove ecosystem towards conservation
Lakshmi Kodoth. issues and development options. It was observed that a large percentage of the
sampled population showed a positive inclination towards conservation of the
ecosystem and were well aware of their responsibility towards it.

Email: Keywords:
lakshmi.kodoth@gmail.com Mangrove ecosystem, Livelihood, Attitudes, Conservation, Development.

Web Address: Article Citation:


http://jresearchbiology.com/ Lakshmi Kodoth and Ramamoorthy D.
documents/RA0274.pdf. Local peoples attitude towards conservation and development around Pichavaram
mangrove ecosystem, Tamil Nadu, India.
Journal of Research in Biology (2013) 3(3): 906-910

Dates:
Received: 08 Aug 2012 Accepted: 26 Aug 2012 Published: 06 May 2013

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.

906-910 | JRB | 2013 | Vol 3 | No 3


Journal of Research in Biology
An International Scientific
Research Journal www.jresearchbiology.com
Kodoth and Ramamoorthy, 2013

INTRODUCTION The Pichavaram mangrove wetland is located in


The Mangrove ecosystem has been studied the northern extreme of Cauvery delta between the
extensively by scientists more in the ecological and Vellar and Coleroon estuaries (figure 1). Geographically,
biological sense. During the 1980s and early 1990s, more it is located between 7947E longitude and 1127N
attention was given to research involving the human latitude. The Pichavaram mangrove forests have an area
interactions with the forested wetlands (FAO, 1985; of about 1,350 ha, which are colonised by 13 true
Hamilton et al., 1989; FAO, 1994; Cormier-Salem, mangrove species. Rhizophora Sp and Avicennia Sp are
1999). Mangrove wetlands are a dominant feature of the the predominant mangrove species present in the
intertidal areas of the tropical and subtropical regions in Pichavaram mangrove forests. Pichavaram mangrove
between 25N and 25S latitudes. The mangrove wetland is also rich in its fishery resources (figure 2).
ecosystem provides a number of ecological services: Annually about 245 tons of fishery resources are
provision of plant and animal products (Macnae, 1974; harvested from this mangrove wetland, of which prawns
Rasolofo, 1997; Spaninks and Beukering, 1997), alone contribute 85% of the catch (Selvam et al., 2003).
sediment trapping and nutrient uptake and transformation Methods
(Furukawa et al., 1997; Hussain and Badola, 2008), they People belonging to 17 hamlets surrounding the
provide detritus food for the aquatic fauna, harbour
migratory and aquatic birds, serve as spawning ground
for fishes, mussels and prawns. They also act as a natural
shield against storms and tidal waves (Kathiresan and
Rajendran, 2005).
The coastal communities are largely dependent on
the mangrove forests for firewood, timber, honey, fodder
and for its fishery resources. Most coastal communities
in the tropics are significantly dependent on the harvest
of marine and coastal resources for sustaining their
livelihoods (Kunstadter et al., 1986). The majority of
people living near the mangrove areas derive their Figure 1 Glimpse of the Pichavaram mangrove forest

income predominantly from fishing and related activities.


Hence, the present study was carried out as it is essential
to understand peoples attitude and perception towards
the mangrove ecosystem as they derive their livelihood
from it; it helps us in formulating better policies and
enhances the developmental plan for the ecosystem.
Study Area
India has a coastline of 7,516 km of which Tamil
Nadu has about 950 km. Extensive mangrove wetlands
are located in two places namely, i) in Pichavaram,
Cuddalore district and ii) Muthupet in Thivarur and
Tanjore districts. Figure 2 Fishing in the mangrove backwaters
907 Journal of Research in Biology (2013) 3(3): 906-910
Kodoth and Ramamoorthy, 2013

Table 1: Attitude of people towards Pichavaram Mangrove Ecosystem and conservation (n= 324)
Questions Yes (%) No (%) Dont Know (%)
Are you aware that Pichavaram Mangrove Ecosystem is declared
91 9 -
as Protected area?
Do you feel any sense of responsibility for the protection of the
84 13.8 2.2
ecosystem?
Do you feel your rights have been violated after the declaration of
11.9 80.5 7.6
PA?
Do you face any problem because of PA? 15.8 78.6 5.6
Are you in favour of the implementation of an ecodevelopment
76.7 15.3 8
project?
Would you like to co-operate with the forest department with regard
67 23 10
to the ecodevelopment project?

Pichavaram mangroves wetland were selected for survey. RESULTS AND DISCUSSION
For each selected hamlet 10% of the households were Assess the awareness and views towards conservation
picked up randomly for the household survey. Using The results (Table 1) showed that majority of the
semi-structured questionnaires, information on the respondents i.e. 91% (n=324) were aware that
demography, land use, income and occupational pattern Pichavaram mangrove was as declared protected area
as well as local dependence on the mangrove resources and this awareness was gained largely because of,
were gathered (Badola and Hussain, 2003; Glaser,2003) . NGOs working in that area and the forest department.
Few open ended questions were also included to An overwhelming percentage (84%) of the local
determine the attitude and perception of villagers population felt responsible towards the protection of the
towards development and conservation issues. A total of mangrove ecosystem and another 76.7% are in favour of
324 households were surveyed. The responses we got eco-development projects in the area. Out of the 324
were mostly in terms of yes, no and we dont know. respondents, 67% of the people are willing to cooperate
with the forest department for the same. Only a small
percentage of people feel their rights being violated
Table 2: View of respondents towards
Eco-Development itiatives (n = 324) because of the protected area status if the ecosystem.
Views Frequency Percentage When questioned regarding their views on
Want through govt. eco-development initiatives and its implementation, a
75 23.1
initiative
Want through majority of the respondents (44.7%) were in favour of
145 44.7
Community initiative the community led initiatives. 32% felt that NGOs
Want through NGO should take lead in eco-development and the rest
104 32
initiative
23% felt that the government should take up

Table 3: View of local people towards various eco-developmental projects by itself ( Table 2).
management alternatives (n = 324) The importance of the mangrove forests to the
Management Alternatives Responses (%) local population was emphasized when a majority of
Forests should be cut and land used
0.6 people were against cutting down of the forests. A
for other purposes
Current situation of protecting the majority of the respondents (71%) felt that more
28.4
forests is good mangrove plantations need to be carried out, while
Increase in mangrove plantations
71 28.4% felt that the present conditions of the mangrove
needed
forests were good (Table 3).

Journal of Research in Biology (2013) 3(3): 906-910 908


Kodoth and Ramamoorthy, 2013

Table 4: View of local people towards various developmental options (n = 324)


Queries Yes (%) No (%) Dont know(%)
Are you in favour of developing eco tourism in the area 76 16 8
Are you in favour of shrimp farms 8 83 9
Has Shrimp farms been useful to you? (n=14) 47 46 7
The findings in this study are similar to that of highest ranking to contribution of mangroves towards
the study in Bitarkanika mangrove ecosystem in Orissa fishing. 63% gave agriculture as their second preference.
(Badola and Hussain, 2003) which shows that the Incase of ranking ecological functions performed by
villagers are well aware of their responsibility to the the Pichavaram mangrove ecosystem, 77.8% of the
ecosystem and willing to participate in the conservation responses favoured Tsunami/cyclone mitigation. 67.2%
efforts of both the government and NGOs. gave second preference to nutrient cycling (Table 6).
Developmental Options The results show that the respondents were aware
Recently, eco tourism has been promoted to a of both the direct and indirect benefits of the mangrove
large extent in Pichavaram mangrove forests. Majority of ecosystem. It is evident from the results that people value
the respondents (76%) were in favour of developing eco the uses or function which more beneficial to them in
tourism as it will improve job opportunities for the local their day today lives.
population. Shrimp farms are not favoured in the area as
83% of the responses were against setting up of such CONCLUSION
farms. This is primarily due to the fact that shrimp farms The results showed that in general people have a
in the area are the reason for increase in salinity of the positive attitude towards conservation and are aware of
canal water (Table 4). their responsibility in sustaining these mangrove forests.
Ecological functions and values identified by local The socio economic and market conditions influence the
community peoples attitude towards the resources. Eco
The respondents were given a list of ecological developmental plans were in favour with the local
functions to find out how much they were aware of the population since it will be helpful in formulating
functions and its direct or indirect importance in their sustainable policies for ecosystem. The promotion of eco
livelihoods. tourism in the area had a largely positive response hence
Table 5 shows ranking of use values, 76% gave it should be capitalised on to improve local economy.
Inclusion of the local people in decision making process
Table 5: Ranking of the use values in Percentage
(n=324) can lead to successful management of the Pichavaram
Use values Rank 1 (%) Rank 2 (%) Rank 3 (%) mangrove ecosystem.
Fishing 76 18 6
Agriculture 26 63 11
Tourism 35 56 9 REFERENCE
Badola R and Hussain SA. 2003. Valuation of the
Table 6: Percentage ranking of various functions
(n=324) Bhitarkanika mangrove ecosystem for ecological security
Ecological Rank 1 Rank 2 Rank 3 and sustainable resource use. Study report. Wildlife
functions (%) (%) (%)
Institute of India, Dehra Dun.
Fish 59.4 34.3 6.3
Aesthetic 38 59 3
Tsunami/cyclone
77.8 22.2 0
mitigation
Nutrient 32.2 67.2 0.6
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be clearedfor social scientists. Hydrobiologia. 413:
Rasolofo MV. 1997. Use of mangroves by traditional
135-142.
fishermen in Madagascar. Mangroves Salt Marshes.
FAO. 1985. Mangrove management in Thailand, 1:243-253.
Malaysia and Indonesia. FAO Environment Paper 4,
Selvam V, Ravichandran KK, Gnanappazham L and
Food and Agriculture Organization of the United
Navamuniyammal M. 2003. Assessment of
Nations, Rome.
community-based restoration of Pichavaram mangrove
FAO. 1994. Mangrove forest management guidelines. wetland using remote sensing data. Current Science.
FAO Forestry Paper 117, Food and Agriculture 85:6,794-798.
Organization of the United Nations, Rome.
Spaninks F and Beukering PV. 1997. Economic
Glaser M. 2003. Interrelations between mangrove Valuation of Mangrove Ecosystems: Potential and
ecosystem, local economy and social sustainability in Limitations. CREED Working 14.
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Furukawa K, Wolanski E and Mueller H. 1997.


Currents and sediment transport in mangrove forests.
Estuarine, Coastal and Shelf Science. 44:301-310.

Hamilton LS, Dixon JA and Miller GO. 1989.


Mangrove forests: an undervalued resource of the land
and of the sea. In: Borgese EM, Ginsburg N, Morgan JR.
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Hussain SA and Badola R. 2008. Valuing mangrove


ecosystem services: linking nutrient retention function of
mangrove forests to enhanced agroecosystem production.
Wetlands Ecology and Management. 16:441-450.

Kathiresan K and Rajendran N. 2005. Coastal Submit your articles online at www.jresearchbiology.com
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and Shelf Science. 65:601-606. Easy online submission
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Journal of Research in Biology (2013) 3(3): 906-910 910


Journal of Research in Biology An International Scientific Research Journal

Original Research

Phenol and Heavy Metal Tolerance Among


Petroleum Refinery Effluent Bacteria
Journal of Research in Biology

Authors: ABSTRACT:
Nwanyanwu CE,
Nweke CO, Orji JC, Bacterial isolates from petroleum refinery effluent were evaluated for growth
Opurum CC. in increasing doses of phenol and heavy metal ions. All the test organisms were able
to grow in mineral salt medium with phenol concentration of 15.0 mM ( 1412.0 mg/l)
except Pseudomonas sp. RBD3. Aeromonas sp. RBD4, Staphylococcus sp. RBD5 and
Pseudomonas sp. RBD10 showed the highest tolerance to 15.0 mM of phenol followed
Institution:
Department of by Corynebacterium sp. RBD7 while Escherichia coli RBD2 and Citrobacter sp. RBD8
Microbiology, Federal showed the least tolerance to 15.0 mM of phenol. The minimum inhibitory
University of Technology, concentrations (MICs) ranged from 1.0 mM for mercury and 4.5 mM for chromium,
P.M.B. 1526, Owerri, nickel, lead and copper. The bacterial strains were most susceptible to mercury
Nigeria. toxicity. Viable counts of the organism on mineral salt-phenol agar showed a typical
growth pattern for inhibitory substrate. The threshold concentration is 0.5 mM for
Bacillus sp. RBD1, Escherichia coli RBD2, Bacillus sp. RBD6, Citrobacter sp. RBD8,
Streptococcus sp. RBD9, Pseudomonas sp. RBD11 and Escherichia coli RBD12 and
1.0 mM for Pseudomonas sp. RBD3, Aeromonas sp. RBD4, Staphylococcus sp. RBD5,
Corresponding author:
Nwanyanwu CE. Corynebacterium sp. RBD7 and Corynebacterium sp. RBD10. The results suggest that
microorganisms isolated from petroleum refinery effluent are potentially useful for
detoxification of phenol impacted systems in the presence of heavy metals.

Email: Keywords:
cnwanyanwu2000@yahoo.com Phenol, heavy metals, refinery effluent bacteria.

Web Address: Article Citation:


http://jresearchbiology.com/ Nwanyanwu CE, Nweke CO, Orji JC, Opurum CC.
documents/RA0317.pdf. Phenol and Heavy Metal Tolerance Among Petroleum Refinery Effluent Bacteria.
Journal of Research in Biology (2013) 3(3): 922-931
Dates:
Received: 24 Dec 2012 Accepted: 09 Jan 2013 Published: 10 May 2013

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.

922-931 | JRB | 2013 | Vol 3 | No 3


Journal of Research in Biology
An International Scientific
Research Journal www.jresearchbiology.com
Nwanyanwu et al., 2013

INTRODUCTION Discharge of these metals into natural waters at


Petroleum refinery effluents are wastes liquids increased concentration in refining operations can have
that resulted from the refining of crude oil in petroleum severe toxicological effects on aquatic environment and
refinery. The effluents are composed of oil and grease humans. Heavy metals as well as phenol are known to
along with many other toxic organic and inorganic be harmful pollutants emanating from industrial
compounds (Diyauddeen et al., 2011). Among the toxic wastewaters that have negative effects on
components of these effluents are heavy metals. Heavy microorganisms. These metals are in the form of
metals include cobalt, chromium, nickel, iron, inorganic and metallo-organic compounds while phenol
manganese, zinc, etc. They usually form complexes with appears to be a soluble component of the industrial
different non metal donor atoms which account for their effluents (Nwanyanwu and Abu, 2010; Hernandez et al.,
participation in various microbial metabolisms in the 1998). These environmental pollutants which are
environment (Kamnev, 2003). Some of these heavy environmentally mobile tend to accumulate in organisms,
metals such as cobalt, chromium, nickel, iron and become persistent because of their chemical stability
manganese, zinc, etc. are required in trace amount by or poor biodegradability (Emoyan et al., 2005).
microorganisms at low concentration as nutrients, since Contamination of wastewater with high concentration of
they provide vital co-factors for metalloproteins and heavy metals caused a significant decrease in
enzymes and are known as essential metals while others the numbers of bacteria in biological system
such as cadmium, mercury, lead, etc have no (Otokunefor and Obiukwu, 2005). It is obvious that
physiological functions and are known as nonessential heavy metals are one of the toxic contaminants in
metals (Sevgi et al., 2010). At high concentration both wastewaters and causes disorder in biological wastewater
essential and nonessential heavy metals exert an treatment (Saidi, 2010). Microorganisms being
inhibitory action on microorganisms by impairing the ubiquitous in nature have been reported to be found in
essential functional groups as well as modifying the inhospitable habitats such as petroleum refinery
active conformation of biological molecules. This results effluents, coke effluents, etc (El-Sayed et al., 2003;
in reduction of microbial activity leading to increased lag Hidalgo et al., 2002) as the effluents are characterized by
phase as well as slow growth rate (Aleem et al., 2003). the presence of phenols, metal derivatives, surface active
It is expected that petroleum refinery effluents substances and other chemicals (Suleimanov,1995).
will contain some of these metals in reasonable quantity Bruins et al., (2000) in their work reported that
as well as aromatic compounds such as phenols. Organic organisms in such inhospitable environment must have
and inorganic mixed pollutants are known to be developed metal resistance systems in an attempt to
commonly present in industrial effluents and also other protect sensitive cellular components. On the other hand,
contaminated sites. In this case, apart from affecting the utilization of phenol and other pollutant is enhanced by
viability of the microbiota, the metal activity may have adaptation and production of appropriate enzymes by
synergistic effect on biodegradation processes of the organisms for the removal of the toxicants
aromatic compounds. Thus studies related to the (Nwanyanwu et al., 2012).
association of the bacterial tolerance properties to metals This study investigated the tolerance to heavy
and degradation of phenolic compounds may be relevant metals and phenol by bacterial population in petroleum
to applications in bioremediation processes (Silva et al., refinery effluent.
2007).
923 Journal of Research in Biology (2013) 3(3): 922-931
Nwanyanwu et al., 2013

MATERIALS AND METHODS allowed to cool at room temperature (282oC).


Sample collection Thereafter, 0.1 ml aliquot of cell suspensions were
Petroleum oil refinery effluent was collected seeded into the tubes and incubated at 30oC for 96 h. The
from Biological treatment plant unit (Rotary biodisk, final concentrations of phenol in the tubes ranged from
RBD) of Port Harcourt oil refinery complex and 0.1-100 mM. Controls included cells in mineral salt
transported to the laboratory for physicochemical medium without phenol and mineral salt medium
analysis which includes pH, total dissolved solids, supplemented with phenol but without cells.
biological oxygen demand (BOD), chemical oxygen Development of turbid culture depicted tolerance to
demand (COD), phosphate (PO4), nitrate (NO3), oil and phenol stress. Isolates that exhibited phenol tolerance
grease, phenol, electrical conductivity and heavy metals from 5.0 mM and above were used for further phenol and
content. The methods used for the analysis were as heavy metal toxicity assay.
shown elsewhere (Nwanyanwu et al., 2012).
Microbiological analysis
Table1: Physicochemical and microbiological
Microbiological counts were estimated by plating analyses of biological treatment unit of petroleum
0.1 ml of the 102 - 106 decimally diluted effluent samples refinery wastewater
in physiological saline on appropriate agar plates. Total Parameter/ unit Value
heterotrophic bacterial count was done on nutrient agar pH 8.18
plates while phenol-utilizing bacterial count was done on Elect. conduct (s/cm) 485
phenol-agar medium of Hill and Robinson (1975). The Oil and grease (mg/l) 15.0
inoculated plates were incubated for 24 h at 30oC for the
TDS (mg/l) 250
heterotrophic bacterial count and 72 h for phenol-
BOD (mg/l) 8.0
utilizing bacteria count.
COD (mg/l) 76.0
Isolation and identification of bacterial strains
Phenol (mg/l) 13.6
The discrete bacterial colonies that developed on
PO42- (mg/l) 0.14
phenol-agar medium were purified, characterized
-
NO3 (mg/l) 1.20
biochemically and identified as described by
Metal concentration
Nwanyanwu et al., (2012).
2+
Zn (mg/l) 0.02
Preparation of inoculum
Cu2+ (mg/l) <0.02
The organisms were grown in nutrient broth
2+
medium contained in Erlenmeyer flasks (100 ml) at Cr (mg/l) 0.05

282oC for 48 h. Thereafter, the cells were harvested and Pb3+ (mg/l) <0.01
2+
washed in sterile deionized distilled water. The cell Ni (mg/l) 0.02

suspensions were standardized by adjusting the turbidity Cd2+ (mg/l) <0.01


to an optical density of 0.1 at A540. Microbial load
Screening of isolates for phenol tolerance THBC (CFU/ml) 2.52 x 108
Into 5.0 ml mineral salt broth medium contained TPUBC (CFU/ml) 1.14 x 108
in 15.0 ml screw capped glass culture tubes were added % TPUBC (%) 45.24
aliquots of phenol stock solution (200 mM). The tubes THBC = Total Heterotrophic bacterial count;
o
were sterilized by autoclaving at 121 C for 15min and TPUBC = Total phenol-utilizing bacterial count

Journal of Research in Biology (2013) 3(3): 922-931 924


Nwanyanwu et al., 2013

Growth on phenol-mineral salt agar properties of the petroleum refinery effluent are shown in
The isolates were tested for their ability to grow Table 1. Phenol-utilizing bacteria represented 45.24% of
on mineral salt agar medium (MSM) amended with the microbial load of biodisk effluent. The high
increasing phenol concentrations. An aliquot (100 l) of population of phenol-utilizing bacteria obtained could be
decimally diluted standardized inoculum of each isolate related to natural selection and adaptation to phenol at
in physiological saline was spread plated onto surface the unit. The concentration of heavy metals in the
of MSM plates with 2.0-20 mM of phenol effluent present in the effluent may be as a result of
concentrations. Control included cells in MSM plates physicochemical treatment (oxidation and reduction,
o
without phenol. The culture was incubated at 30 C for chemical precipitation, etc) given to the raw wastewater
72 h (Kahru et al, 2002). The number of the colony that before been channeled into the biological treatment unit.
developed was enumerated as colony forming unit per ml The result of screen test for phenol tolerance is
(CFU/ml). shown in Table 2. With the exception of Pseudomonas
Minimum inhibitory concentration (MIC) sp. RBD3 that tolerated phenol up to 10 mM, all the
determination organisms are able to tolerate phenol stress up to
Stock solutions of Cd, Zn, Hg, Cu, Pb, Ni, Co 15.0 mM. The growth of the isolates in the medium with
and Cr as salts of CdCl2, ZnSO4, HgCl2, CuSO4, PbCl2, phenol concentrations above 10.0 mM may be attributed
Ni(NO3)2, CoCl2.6H2O and K2Cr2O7 were prepared in to previous exposure to phenolic raw wastewater influent
deionized distilled water. All the chemicals used were into the biological treatment unit (RBD). This is in line
analytical reagent grade. with the report of Santos et al., (2001) in which they
The minimum inhibitory concentrations (MIC) of related the growth of Trichosporom sp. in phenolic
eight heavy metal ions at which no growth was observed amended medium of 10.0 mM concentration to previous
were determined at pH 7.2 against each bacterial isolate phenolic wastewater shock load from stainless steel
using tube dilution method (Hassen et al., 1998) with industry. Moreso, the tolerance of the organisms to high
little modifications. Graded concentrations of each heavy concentration of phenol (15.0 mM) may be the ease with
metal ranging from 0.05 mM to 10.0 mM were prepared which the isolates open the phenol ring for its subsequent
in tryptone soy broth (TSB) contained in screw capped uptake as carbon and energy source (Ajaz et al., 2004).
culture tubes. The supplemented TSB-heavy metal Gurujeyalakshmi and Oriel (1989) in their work have
o
medium was sterilized by autoclaving at 121 C for reported that Bacillus stearothermophilus strain BR219
o
15 min. On cooling to room temperature (282 C), the could grow on phenol at levels up to 15 mM. In contrast,
tubes were seeded with 100 l of the bacterial growth inhibition of Bacillus, Pseudomonas and
suspension and incubated at 30oC for 72 h. Inoculated Citrobacter species at phenol concentration above
medium free of heavy metal ions and uninoculated 1.0 mM has been reported by many authors
medium with metal ions served as positive and negative (Obiukwu and Abu, 2011). Janke et al., (1981) reported
controls respectively. The MIC of the metal to the test inhibition of phenol hydroxylase activity in
isolates is the lowest concentration that totally inhibited Pseudomonas species at 0.25 mM phenol concentration.
growth of the organisms. Yang and Humphrey (1975) found that the growth
of Pseudomonas putida was strongly inhibited
RESULTS AND DISCUSSION above phenol concentration of 0.5 mM. Buswell and
The physicochemical and microbiological Twomey (1975) reported that growth of
925 Journal of Research in Biology (2013) 3(3): 922-931
Nwanyanwu et al., 2013

Table 2: Phenol tolerance of the test isolates in different concentrations of phenol

Growth in mineral salt broth with added phenol


Phenol concentration (mM)
Bacteria
0.1 0.2 0.5 1 2 5 10 15 20 50 100
Bacillus sp. RBD1 + + + + + + + + - - -
Escherichia coli RBD 2 + + + + + + + + - - -
Pseudomonas sp. RBD 3 + + + + + + + - - - -
Aeromonas sp. RBD 4 + + + + + + + + - - -
Staphylococcus sp. RBD 5 + + + + + + + + - - -
Bacillus sp. RBD 6 + + + + + + + + - - -
Corynebacterium sp. RBD7 + + + + + + + + - - -
Citrobacter sp. RBD8 + + + + + + + + - - -
Streptococcus sp. RBD9 + + + + + + + + - - -
Pseudomonas sp. RBD10 + + + + + + + + - - -
Corynebacterium sp. RBD11 + + + + + + + + - - -
Escherichia coli RBD12 + + + + + + + + - - -
+ = growth ; - = no growth

Bacillus stearothemophilus was inhibited at phenol of Pseudomonas sp. RBD3, Aeromonas sp. RBD4,
concentration above 5.0 mM. Staphylococcus sp. RBD5, Corynebacterium sp. RBD7
The effect of increasing doses of phenol and Corynebacterium sp. RBD10 with a total viable
(0.05 - 15.0 mM) on the population of the test organisms count of 8.4 x 106, 7.5 x 106, 7.2 x 106 and
are shown in Figure 1. Generally, the viable counts 8.4 x 106 CFU/ml respectively, were stimulated.
increased with the concentration of phenol until a certain Thereafter, the total viable counts progressively
concentration when the growth of the organisms was decreased as the phenol concentration increases. This
inhibited. The growth of the organisms on phenol growth pattern is typical of in an inhibitory substrate like
followed a substrate inhibition pattern. Increasing phenol phenol. The inhibition of bacterial growth by phenol is
concentration resulted in decrease in microbial growth well-documented. However, some bacteria are more
and eventually very minimal growth was detected at the tolerant to phenol than others. For instance, the growth
highest phenol concentration (15.0 mM) in all the test inhibition constant (Ki) for bacteria degrading phenol
organisms. The growth of Bacillus sp. RBD1, have been reported as 54.1mg/l (0.57 mM)
Escherichia coli RBD2, Bacillus sp. RBD6, Citrobacter (Monteiro et al., 2000), 129.79 mg/l (1.379 mM) (Kumar
sp. RBD8, Streptococcus sp. RBD9, Pseudomonas sp. et al., 2005), 2434.7 mg/l (25.87 mM) (Arutchelvan et
RBD11 and Escherichia coli RBD12 with a total viable al., 2006) and 7.818 mM (Wei et al., 2008). In this study,
6 6 6 6
count of 7.1 x 10 , 8.0 x 10 , 7.2 x 10 , 7.8 x10 , all the test organisms tolerated phenol up to 10.0 mM
6 6 6 6
7.5 x 10 , 8.8 x 10 , 7.4 x 10 and 7.4 x 10 CFU/ml ( 941 mg/l) and with the exception of Pseudomonas sp.
respectively were stimulated at phenol concentrations up RBD 3, all the bacterial strains tolerated 15 mM
to 0.5 mM ( 47.06 mg/l). Similarly, at phenol ( 1412 mg/l). This is in line with the report of Worden
concentration up to 1.0 mM ( 94.11 mg/l), the growth et al., (1991) that Bacillus stearothermophilus BR219

Journal of Research in Biology (2013) 3(3): 922-931 926


Nwanyanwu et al., 2013
Total Viable Count (x 106 CFU/ml)

10

8 Pseudomonas sp. RBD3

10
Citrobacter sp. RBD8
8

0
0 4 8 12 16

Phenol (mM)
Figure 1: Growth of bacteria on mineral salt agar medium supplemented with increasing doses of phenol.

tolerated phenol concentration of 15.0 mM. Similarly, phenol concentration (Hossein and Hill, 2006; Kotturi et
Corynebacterium species was reported to resist 15 mM al, 1991). Li and Humphrey (1989) as well as
phenol while Staphylococcus, Corynebacterium, Bacillus Gurujeyalakshmi and Oriel (1989) have reported
and Proteus were found to resist 10 mM of phenol microbial growth inhibition at relatively low
(Ajaz et al., 2004). However, many authors have concentrations of 2.0 mM and 0.25 mM respectively.
reported inhibition of microorganisms at such high
927 Journal of Research in Biology (2013) 3(3): 922-931
Nwanyanwuet al., 2013

Table 3: Minimal inhibitory concentrations of heavy metals


MIC of metal (mM)
Organism Cd Zn Hg Cu Pb Ni Co Cr
Bacillus sp. RBD1 3.5 2.0 1.5 4.0 4.5 3.5 2.0 4.0
Escherichia coli RBD2 3.5 2.5 1.0 4.0 3.0 4.0 2.5 3.5
Pseudomonas sp. RBD3 4.0 3.0 1.5 4.5 3.0 4.5 3.0 4.5
Aeromonas sp. RBD4 3.5 3.0 1.0 3.0 4.0 4.0 2.0 4.0
Staphylococcus sp. RBD5 4.0 2.0 1.0 3.5 3.0 4.0 3.0 4.0
Bacillus sp. RBD6 3.0 2.5 1.5 3.5 4.0 4.0 3.0 4.0
Corynebacterium sp. RBD7 3.0 2.0 1.5 3.0 3.5 3.5 2.5 3.5
Citrobacter sp. RBD8 3.5 1.5 1.0 2.5 3.0 3.0 2.0 2.5
Streptococcus sp. RBD9 4.0 2.0 1.0 3.5 2.5 4.0 3.0 2.5
Pseudomonas sp. RBD10 3.5 3.0 1.5 4.5 4.0 4.5 3.5 4.0
Corynebacterium sp. RBD11 2.5 2.0 1.0 4.0 4.0 3.0 4.0 4.5
Escherichia coli RBD12 3.0 1.5 1.0 3.0 3.5 3.5 2.5 3.0

The tolerance levels of refinery wastewater reported for cadmium, chromium, lead, cobalt, mercury
phenol-utilizing bacteria to heavy metals expressed as and copper respectively (Nweke et al., 2006a). These
minimal inhibitory concentrations (MIC) are shown in reported MICs in most cases corroborates the values
Table 3. The test isolates in this study showed similar observed in this study. The MIC in growth inhibition
trend of susceptibilities to heavy metal ions based on assay is analogous to the concentration of metal ion
minimal inhibitory assay. The high MIC values obtained that exhibited 100 % inhibition in dehydrogenase
in the study may be as a result of long term exposure of activity assay. Thus, the MIC of zinc against river
the organisms to metal ions in the refinery effluent. water planktonic bacteria have been reported as
Highest MIC values were exhibited in Chromium, 1.558 0.037 mM, 1.283 0.068 mM,
Copper and Nickel while the least MIC was shown in 2.469 0.045 mM and 1.328 0.094 mM for
mercury among the isolates with a maximum value of Escherichia, Proteus, Micrococcus and Pseudomonas
>3.0 mM and minimum value of <2.0 mM. species respectively (Nweke et al., 2006b). Likewise, the
Pseudomonas sp. RBD3 showed maximum MICs value concentration of zinc that gave 100% inhibition of
range of 1.5 - 4.5 mM whilst Escherichia coli RBD12 dehydrogenase activity in sediment Bacillus and
showed minimum MICs value range of 1.0 - 3.5 mM in Arthrobacter species are 1.442 0.062 mM and
all the metals tested. The MICs are higher than that 1.199 0.042 mM respectively (Nweke et al., 2007).
reported by El-Deeb (2009) for some phenol-degrading Also, Hassen et al., (1998) have reported MIC values of
bacteria. However, the MIC values are similar to the 0.1, 0.8, 1.5, 1.6 and 1.8 mM for Mercury, Cobalt, Zinc
values reported elsewhere (Nieto et al., 1989, and Cadmium, Copper and Chromium respectively on
Nweke et al., 2006a, Akinbowale et al., 2007). The MIC Pseudomonas aeruginosa, Citrobacter freundii,
of metal ranging from 0.5 - 2.5 mM, 1.25 - 2.5 mM, Staphylococcus aureus, Streptococcus sp. and
5.0 - 12.0 mM, 1.0 - 1.25 mM, 0.25 - 1.0 mM and Bacillus thurieniensis. Hassen et al., (1998) in their work
1.25 - 5.0 mM against hydrocarbon-utilizing bacteria was reported 3.0 mM chromium as the MIC for

Journal of Research in Biology (2013) 3(3): 922-931 928


Nwanyanwu et al., 2013

Pseudomonas aeruginosa S8 and Citrobacter freundii Akinbowale OL, Haihong P, Peter G, Barton MD.
S24. The variation in the tolerance of heavy metal could 2007. Antibiotic and heavy metal resistance in motile
be attributed to the bacterial strain involved, assay aeromonads and pseudomonads from rainbow trout
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Aleem A, Isar J, Malik A. 2003. Impact of long-term
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931 Journal of Research in Biology (2013) 3(3): 922-931


Journal of Research in Biology An International Scientific Research Journal

Original Research

Biodegradation of phenol at low and high doses by bacterial strains indigenous to


Okrika River in the Niger Delta of Nigeria
Journal of Research in Biology

Authors: ABSTRACT:
Nwanyanwu CE 1*
Abu GO2. Assessments on biodegradation at low and high doses of phenol by bacterial
strains indigenous to Okrika River in Niger Delta of Nigeria were carried out. Growth at
low dose of 0.01 g/l phenol showed that highest and lowest cell density values of
OD540nm of 0.15 and 0.09 in Pseudomonas sp. SD1 and Citrobacter sp. RW1 while at
1.0 g/l phenol concentration the highest cell density values of OD 540nm of 0.28 was
Institution:
1.Department of observed in Staphylococcus sp. RW2. The highest specific growth rate of 0.019 h-1 at
Microbiology, Federal 500 mg/l of phenol was obtained for Pseudomonas sp. SD1 while Citrobacter sp. RW1
university of Technology, had the lowest specific growth rate of 0.014 h-1 at 500 mg/l of phenol. The specific
P.M.B.1526, Owerri, phenol degradation rate ranges from 55.35 to 130.98 mg/(L.h.OD). The order
Nigeria. of specific phenol consumption rate at 1000 mg/l by the organisms is: Bacillus sp.
SD3>Pseudomonas sp. SD1>Citrobacter sp. RW1>Staphylococcus sp.
2.Department of RW2. Citrobacter sp. RW1 exhibited highest growth yield in 250 mg/l of phenol with
Microbiology, University of the growth yield of 6.24 (x 10-4 A540 unit.l/mg). The results showed that the test
Port Harcourt, P.M.B. 5323, organisms might be the most suitable bacterial strains for removal of phenols at low
Port Harcourt, Nigeria. and high doses in phenolic polluted media.

Keywords:
Corresponding author:
Nwanyanwu CE. Biodegradation, phenol, bacteria, Okrika River.

Email: Article Citation:


cnwanyanwu2000@yahoo.com Nwanyanwu CE and Abu GO.
Biodegradation of phenol at low and high doses by bacterial strains indigenous to
Okrika River in the Niger Delta of Nigeria.
Journal of Research in Biology (2013) 3(3): 911-921
Web Address:
http://jresearchbiology.com/ Dates:
documents/RA0318.pdf.
Received: 26 Dec 2012 Accepted: 17 Jan 2013 Published: 06 May 2013

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.

911-921| JRB | 2013 | Vol 3 | No 3


Journal of Research in biology
An International Scientific
Research Journal www.jresearchbiology.com
Nwanyanwu and Abu, 2013

INTRODUCTION (2000) and Polymenakou and Stephanou (2005).


Contamination of aquatic environment brought Microorganisms indigenous to aquatic environment are
about by the discharge of wastewater resulting from crucial for the biodegradation of organic matter and the
anthropogenic activities clearly continues to be a major cycling of nutrients, while these microorganisms are
environmental issue. Effluents are very important susceptible to toxic pollutants from industrial effluent
sources of chemicals entering aquatic ecosystems. They discharges, especially petroleum refinery. Therefore,
may contain hundreds, or even thousands of chemicals, perturbations of aquatic microbial communities could
but only a few of them are responsible for effluent have consequences for the higher trophic levels and for
toxicity (Tisler et al., 1999). High strength wastewaters the overall aquatic environment.
have been reported to be associated with chemical The composition of effluents from petroleum
processing industries. Wastewaters generated from these refineries varies according to their origin, storage and
processing industries such as petrochemical, oil treatments as these wastewaters are enriched with
refineries, coke-processing plants, etc contain a large different pollutants. Phenol and its derivatives along with
number of organic and inorganic pollutants at high other organic and inorganic compounds is one of the
concentrations that exhibit adverse effect on the most common contaminants present in refinery effluents
environments when released (Papadimitriou et al., 2009). (Jena et al., 2005) which renders refinery effluents its
The presence of high level of these contaminants formed toxic nature. Phenols as constituents of industrial
the major pollutant in the water body as a result of effluents may remain in water body for much longer
continuous discharge of effluents by industries into the period if it is continually or consistently released into the
ecosystem. In water these pollutants of the discharged aquatic environments from sources thereby increasing its
effluent sorbs onto particulate materials and if not elevation in the environment. The toxic nature of phenol
degraded eventually end up in sediments. As an ultimate and its derivatives to microbial cells is well documented
respiratory of most xenobiotic contaminants that enter (Kahru et al., 2002; Keweloh et al., 1990). Owing to
water bodies, sediments act as both carrier and sources of toxic nature of phenol, its contact with microorganisms
contaminants in aquatic environment (Akan et al., 2010). always results in the decrease of microbial enzyme
Thus, the contaminated sediments may represent a activity (Nwanyanwu and Abu, 2011) as well as leading
continual threat of recontamination of the aquatic to death of organisms at higher concentration.
environment as the adsorbed pollutants if not degraded, A large number of microbial genera possess the
in turn lead to the exposure of aquatic life to organic capability to degrade organic pollutants. Among the
pollutants such as phenol (Mort and Dean-Ross, 1994). bacterial genera implicated in the degradation of phenol
On the other hand, the release of contaminants from include Pseudomonas, Bacillus, Corynebacterium
sediments could increase the amount of toxic compounds species etc. The ability of organisms to degradation
in the waters making them more available to organisms phenol and other toxicants is related to adaptation of the
and affecting their life cycles, reproduction, metabolism microorganisms to the compound of concern and
and physiology. Microorganisms being ubiquitous in adaptation is associated with synthesis of new enzymes
nature exploit many carbon and energy sources in its capable of transformation of the toxicant to harmless
niche for growth. Several species of micro-organisms substances (Jaromir and Wirgiliusz, 2007). The resultant
inhabiting hostile ecological niche have been reported by effect of biodegradation of phenol and other organic
Colwell and Walker (1977), Atlas (1981), Heinaru et al. compounds is growth as the organic pollutants are used
912 Journal of Research in Biology (2013) 3(3): 911-921
Nwanyanwu and Abu, 2013

as the source of carbon and energy. 0.4 at 540 nm and used as inocula.
This research assessed the growth and utilization Assay for isolates growth in very low concentrations
of phenol at low and high doses by bacterial strains of phenol
indigenous to Okrika River in the Niger Delta of Nigeria. The ability of the isolates to grow and utilize
phenol at low concentrations (0-1.0 g/l) was assessed in
MATERIALS AND METHODS sterile Bushnell Haas (BH) mineral salt broth medium.
Chemical reagents The assay was carried out as described by
All chemical reagents used in the study were of Nwanyanwu et al., (2012) with little modification. The
analytical grade and were obtained from sigma chemical medium without agar was used instead for the assay.
company, St Louis Missouri, USA, BDH chemicals, After inoculation of the flasks, growth profile of the
Poole, England and HACH chemical company. organisms was monitored by the optical density
Sample collection and analysis (OD540nm) on daily basis.
The Okrika River is a small tidal river that Growth and biodegradation of phenol at high
empties into Bonny estuary in Niger Delta of Nigeria. concentration
The River is highly polluted as a result of effluent Degradation of phenol at high concentration by
discharges from Port Harcourt petroleum refinery the organisms was carried out in sterile BH medium
industry sited along its bank (IAIA09 Conference contained Erlenmeyer flasks. The flasks were
Proceeding, 2009). Sediment and water samples were supplemented with aliquot of sterile phenol (2000 mg/l)
collected from the river as described by Nweke et al., to bring the final phenol concentrations in the flasks to
(2007) and the samples analyzed within few hours of 250, 500, 750 and 1000 mg/l. The flasks after inoculation
collection. The results of the physicochemical analysis of with the test organisms were incubated at 30oC in an
the samples are as shown in Table 1. incubator. At predetermined time, samples were
Isolation and identification of bacterial strains withdrawn to determine cell growth and phenol
The bacterial strains used in this work were concentration. Controls, one without phenol and another
isolated from the samples by spreading one tenth of without cells in BH medium were set up. At
decimally diluted sediment suspension and water predetermined time, samples were removed and used to
samples on mineral salt agar-phenol (2.5 mM) medium measure for cell growth (optical density, OD540nm) and
and the isolated organisms identified as described
Table 1: Physicochemical characteristics
elsewhere (Nwanyanwu et al., 2012). The isolates were of Okrika River
designated according to their sources (RW for River
Sample source
water, SD for sediment) and were then maintained on Parameter/unit Water Sediment
nutrient agar slants. pH 8.90 6.90
Elect. conduc. (scm-1) 364 615
Preparation of inoculum
Oil and grease (mg/l) 16.0 103.0
The bacterial strains used for the assay were BOD (mg/l) 8.16 -
grown in 100 ml of sterile nutrient broth media for 48 h. COD (mg/l) 84.0 -
PO4 (mg/l) 0.15 0.90
The turbid culture medium were harvested, washed SO4 (mg/l) 118 117
and suspended in deionized distilled water then Phenol (mg/l) 6.1 15.5
Zn (mg/l) 0.03 3.48
followed by standardization of the suspensions Cu (mg/l) <0.01 0.06
spectrophotometrically to an optical density of Pb (mg/l) <0.01 <0.01

Journal of Research in Biology (2013) 3(3): 911-921 913


Nwanyanwu and Abu, 2013

Table 2: Yield factor (Y) of biomass after growth at dX


different initial phenol concentrations Y 3

dS
-4
Yield factor, Y(x 10 A540 Where dX is the change in cell biomass related to
unitsa. l/mg)
Bacteria Phenol concentration (mg/l) the change in substrate concentration dS. X was replaced
with the OD at 540 nm.
250 500 750 1000
Citrobacter sp. RW1 6.24 4.46 2.69 3.11
Staphylococcus sp.RW2 4.96 3.80 3.28 3.00 RESULTS AND DISCUSSION
Pseudomonas sp. SD1 4.96 3.80 3.28 3.00
Bacillus sp. SD3 3.28 4.46 2.69 3.11 The phenol content of Okrika River water and
a
A540 units = optical density at 540 nm sediment were 6.1 and 15.5 mg/l while oil and grease of
the River water and sediment were 16.0 and 103.0 mg/l
phenol residue (4-amino antipyrine) in cell free samples.
respectively (Table 1). This level of oil and grease as
Analytical methods
well as phenol in the River water and sediment were
Cell growth was determined
much higher than the previously reported levels of
spectrophotometrically while phenol was analyzed by
10.56 and 15.23 mg/l (oil and grease) and 5.13 and
photometric method using 4-aminoantipyrine as the
16.0 mg/l (phenol) (Otokunefor and Obiukwu, 2005).
colouring agent and measuring the absorbance at 500 nm
This indicated that these compounds have accumulated
(Folsom et al., 1990).
in Okrika River over time and pose the major pollutants
Data Analysis
of the river.
Specific growth rate
Figure 1 shows the growth of the test organisms
The specific growth rate () for each
in low concentration of phenol amended mineral salt
concentration of phenol was calculated from the slope of
medium. All the organisms showed progressive growth
linear logarithmic plots of optical density against time as
in low phenol concentration medium. Highest growth of
expressed in equation 1 (Gokulakrishnan and Gummadi,
the organisms was observed in phenol concentration of
2006):
1.0 g/l followed by 0.1 g/l. The least growth was
In X 2 X 1 observed in 0.01 g/l. Among the test organisms,
1
t 2 t1 Staphylococcus sp. RW2 showed the highest growth in
Specific degradation rate 0.1 and 1.0 g/l of phenol with optical density (OD)
The specific degradation rate (Qs) was values of 0.23 and 0.28 respectively while Citrobacter
determined through the relationship of equation 2 (Loh sp. RW1 showed the least growth in all the low
and Wang, 1998): concentrations (0.01, 0.1 and 1.0 g/l) of phenol
d Ph / dt amended medium with OD values of 0.09, 0.11 and 0.13
Qs 2
X respectively. Growth of microorganisms especially
Where: [Ph] denotes phenol concentration bacterial species at phenol concentration as low as
(mg/l), t denotes incubation time (h) and X denotes cell microgram per litre have been reported by many authors.
concentration (optical density, OD540 nm). Chesney et al., (1985) have reported growth of water
Yield factor microorganism in water sample supplemented with 0.001
Yield factor (Y) of the biomass was calculated to 1.0 g/ml of phenol. Also Goldstein et al., (1985)
using equation 3 (Bajaj et al., 2009): have reported the growth of Pseudomonas sp. in a

914 Journal of Research in Biology (2013) 3(3): 911-921


Nwanyanwu and Abu, 2013

Absorbance (A540 nm)

Time (h)

Figure 1: Growth profile of the bacteria in mineral salt medium fortified


with phenol concentrations

medium amended with 1.0 and 10.0 g/l concentration of concentrations of 500, 750 and 1000 mg/l was degraded
2, 4-dichlorophenol. Pahm and Alexander (1993) found completely within 96, 132 and 156 h by Pseudomonas
that Pseudomonas sp. K, Flavobacterium sp. M4, sp. SD1 while same concentrations of phenol was
Flavobacterium sp. M1 and Pseudomonas sp. SP3 grown degraded completely within 108, 144 and 180 h by other
in p-nitrophenol (PNP) of concentration of 0.1 g/l test organisms. Time-dependent degradation of organic
reached a total viable count of 105 and 106 cells/ml. compounds has been reported to be linked with
Figures 2 and 3 showed typical profiles of cell concentration of the organic compound as observed by
growth and biodegradation of phenol at high many authors (Colwell and Walker, 1977; Kotresha and
concentrations by bacterial strains of Okrika River Vidyasagar, 2008). This may be due to changes in the
ranging from 250 to 1000 mg/l. The lag phase of the transport mechanism of the substrate across the cell
organisms in phenol fortified medium was short. The membrane in response to high phenol concentration
short in lag phase period depends on the pre-exposure of hence diminished capacity to catabolize phenol. This is
the organism. Phenol was completely utilized by the in line with the reports of Gilbert and Brown (1978),
isolates within 180 h of incubation. Phenol Keweloh et al., (1990), Collins and Daugulis (1997) and

Journal of Research in Biology (2013) 3(3): 911-921 915


Nwanyanwu and Abu, 2013

Phenol (mg/l)
Absorbance (A540 nm)

Time (h)

Figure 2: Biodegradation and cell growth profile of planktonic bacteria of Okrika River in high
phenol concentrations

Nwanyanwu and Abu (2011) who observed the toxic the medium progressively decreased. This may be due to
effect of phenol at the membrane level, thereby high phenol concentration made available more carbon to
disrupting the activity of enzymes in phenol-utilizing the organism for growth. Pseudomonas sp. SD1
bacteria. Also, Joseph and Joseph (1999) and Ye and degraded 1000 mg/l of phenol in 160 h with a cell
Shen (2004) reported that phenol toxicity depends on the biomass (OD540nm) of 0.363.
sensitivity as well as source of organism. The dependence of specific growth rate on
The growth profiles of the pure cultures expressed phenol concentration is shown in Figure 4. From this
as optical density and phenol residues at different initial plot, the specific growth rate increased with increase in
concentrations are shown in figures 2 and 3. The cells the initial phenol concentration upto 250 mg/l and then a
gradually increase in number as the phenol residues of progress decrease started with increase in phenol
916 Journal of Research in Biology (2013) 3(3): 911-921
Nwanyanwu and Abu, 2013
Phenol (mg/l)
Absorbance (A540 nm)

Time (h)
Figure 3: Biodegradation and cell growth profile of sediment bacteria of Okrika River in high
phenol concentrations

concentration. In the present study, at 500 mg/l of phenol concentration and then started decreasing to a constant
concentration, the specific growth rate of Pseudomonas (0.057 h-1) at 600 and 700 mg/l of phenol. This trend
sp. SD1 is increased (highest =0.017 h -1). For suggested that the phenol is an inhibitory substrate. Thus
concentration higher than 500 mg/l, the specific growth the parameter has been found to be a strong function of
rate of Pseudomonas sp. SD1 decreases and became initial phenol concentration. At 250 and 500 mg/l, the
-1
almost constant at 750 mg/l ( = 0.011 h ) and highest specific growth rate values of 0.026 and 0.017 h -1
1000 mg/l ( = 0.011 h -1) of phenol. This is quite similar were observed in Citrobacter sp. RW1 and Pseudomonas
to the result obtained by Dey and Mukherjee (2010) who sp. SD1 respectively while the lowest specific growth
-1
observed increase in specific growth rate (0.093 h ) of rate of 0.016 and 0.014 h -1 at the same concentration of
mixed microbial culture up to 300 mg/l of initial phenol phenol was observed in Pseudomonas sp. SD1 and

Journal of Research in Biology (2013) 3(3): 911-921 917


Nwanyanwu and Abu, 2013

Specific degradation rate (mg/(L.h.OD))


Specific growth rate (h-1)

Phenol, So (mg/l)
Phenol, So (mg/l)
Figure 5: Specific degradation rate at different
Figure 4: Specific growth rate of the organisms initial phenol concentrations by the bacterial strains
at different initial phenol concentrations

Citrobacter sp. RW1 respectively. However, the concentration decreases. This is in line with the work of
growth rates of the test organisms are similar Cho et al., (2000) who observed an increase in specific
to that of Pseudomonas aeruginosa and degradation rate as phenol concentration increases in
Pseudomonas pseudomallei degrading phenol in saline their assessment of influence of phenol on
solutions (Afzal et al., 2007). biodegradation of p-nitrophenol by freely suspended and
The specific rate of phenol degradation of the immobilized Nocardioides sp. NSP41. Agarry and
organisms is depicted in figure 5. The specific Solomon (2008) also made similar reports in their work
degradation rate (Qs), was estimated by correlating on kinetics of batch microbial degradation of phenols by
phenol concentration versus culture time using indigenous Pseudomonas fluorescence.
regression technique in Microsoft Excel to obtain the Table 2 shows the growth yield of the test
equation of best fit of the degradation curve. The organisms expressed as absorbance, A at 540nm unit litre
correlation were differentiated with respect to time and of cells produced per mg of phenol substrate utilized.
then divided by the cell mass (Loh and Wang, 1998). The growth yield varied among the test organisms
The specific degradation (consumption) rate of a ranging from 2. 69 to 6.24 (x 10-4A540 units. l/mg). High
compound was suggested to be a measure of microbe growth yield were obtained at low concentration of
performance. The highest specific consumption rate of toxicant (phenol) while low values of growth yield were
phenol was observed in Bacillus sp. SD3 with specific obtained at high phenol concentration. At 250 mg/l
degradation rate value of 130.98 mg/(L.h.OD) at highest and lowest growth yield were observed in
1000 mg/l while Staphylococcus sp. RW2 showed the Citrobacter sp. RW1 and Bacilllus sp. SD3 with cell
least specific consumption rate of phenol with a specific yield coefficients of 6.24 and 3.28 (x 10-4A540 units.l/mg)
degradation rate value of 99.83 mg/(L.h.OD) at the same respectively. The higher value of Y observed in
concentration. The organisms in this work showed a Citrobacter sp. RW1 indicate that phenol was degraded
robust decrease in specific degradation rate as the phenol very efficiently by the organism. All the growth yields
918 Journal of Research in Biology (2013) 3(3): 911-921
Nwanyanwu and Abu, 2013

reported here were lower than those reported by other Mineralizing Microorganisms in Fresh Water. Appl.
authors. Yield coefficients of 0.14 and 0.16 have been Environ. Microbiol. 49: 15 18.
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Cho YG, Rhee SK, Lee ST. 2000. Influence of phenol
reported by Yoong et al., (1997) are 0.16 and 0.27.
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As Citrobacter sp. RW1, Staphylococcus sp. RW2,
and immobilized Nocardioides sp. NSP41.
Pseudomonas sp. SD1and Bacillus sp. SD3 shown high
Biodegradation 11(1): 21 28.
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Journal of Research in Biology An International Scientific Research Journal

Original Research

Evaluation of the Impact of Oil and Gas Pollutants on the Chemical Composition of
Abelmoschus esculentus Moench and Pterocarpus mildbraedii Harms.
Journal of Research in Biology

Authors: ABSTRACT:
Ujowundu CO1, The phytochemical, proximate, mineral and vitamin contents of
Nwaogu LA1, Igwe KO1, Abelmoschus esculentus Moench and Pterocarpus mildbraedii Harms were
Ujowundu FN1, investigated. Plant samples were harvested from Polluted Environment (PE) at
Belonwu DC2. Izombe in Oguta Local Government Area- an oil drilling and gas flaring environment.
The results obtained were compared to identical vegetables harvested from Eziobodo
in Owerri West Local Government Area, designated as Unpolluted Environment (UPE).
Our result showed that A. esculentus and P. mildbraedii have excellent nutritional
value, which can confer biochemical and physiological advantage to humans. The
quantitative proximate composition showed that the carbohydrate and ash contents
Institution: of samples harvested from PE differed significantly (P<0.05) from samples obtained
1. Department of from unpolluted environment. The protein, crude fibre, moisture and total fat
Biochemistry, Federal contents of samples from PE differed non significantly (P<0.05) when compared with
University Technology samples obtained from UPE. The phytochemical contents of A. esculentus and
Owerri, Nigeria. P. mildbraedii were significantly higher in samples from UPE than in samples from PE.
2. Departmentof The mineral and vitamin contents were also determined. The concentration of
Biochemistry, University of nutritionally important macro and micro elements indicates that the two vegetable
Portharcourt, Nigeria. samples studied are rich sources of minerals and, therefore, can be used to improve
the diet of both humans and livestock. This study also showed that environmental
pollutants emanating from the activities of oil and gas industries can impact negatively
on some important chemical and nutritive compositions of edible vegetables.

Corresponding author: Keywords:


Ujowundu CO. Oil and gas, Pollution, Phytochemicals, Vitamins, Oha, Okra.

Email: Article Citation:


ujowundu@yahoo.com Ujowundu CO, Nwaogu LA, Igwe KO, Ujowundu FN, Belonwu DC.
Evaluation of the Impact of Oil and Gas Pollutants on the Chemical Composition of
Abelmoschus esculentus Moench and Pterocarpus mildbraedii Harms.
Journal of Research in Biology (2013) 3(3): 861-869

Web Address: Dates:


http://jresearchbiology.com/ Received: 16 Jan 2013 Accepted: 09 Feb 2013 Published: 11 Apr 2013
documents/RA0327.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.

861-869 | JRB | 2013 | Vol 3 | No 3


Journal of Research in Biology
An International Scientific
Research Journal www.jresearchbiology.com
Ujowundu et al., 2013

INTRODUCTION pollution.
Nigeria, a major producer of crude oil, benefits The most glaring sight in gas production flow
as well as suffers from the positive and negative effects station is the ten-meter-high flame that burns
of crude oil drilling and gas flaring (Adeniye et al., continuously from vertical pipes at many facilities owned
1983). Gas flaring is the unscientific burning of excess by oil companies. These vertical pipes are fed with gas
hydrocarbons gathered in an oil/gas production flow given off during production. Gas flaring, for about four
station. Gas flaring is a major source of pollution in decades has contributed to the high pollution level, and
Nigeria's Niger Delta because it is the preferred means of the ecosystem of Izombe may have been impacted
disposing waste gas associated with oil exploitation in negatively. A good example of such negative effect is
that region by many multinational oil companies that high soil acidity that creates chemical and biological
operate its fields. Gas flaring releases carbon monoxide, conditions which may be harmful to the soil and plants
oxides of sulphur and nitrogen, hydrocarbons, soot and (Nwaugo et al., 2006). One of these conditions is the
heavy metals (Coker, 2007; Ikoro, 2003). These reduction in the capacity of plants to absorb cations
pollutants actually interfere with growth and survival of (Wild et al., 2005). The higher acidic nature of soil is
living organisms in such environment especially in attributable to high concentrations of sulphur dioxide and
plants. It pollutes seedlings and fruits of plants which in particulates from gases flared into the atmosphere which
turn have a devastating effect on humans who consume is washed back to the soil as acid rain.
them. Such effects include respiratory or cardiovascular This study has two objectives, first to contribute
diseases. This study used Abelmoschus esculentus to the knowledge of nutritional and antinutritional
Moench and leaves of Pterocarpus mildbraedii Harms, composition of A. esculentus Moench and P. mildbraedii
two commonly consumed indigenous vegetables to Harms. Secondly, to evaluate the effect of environmental
evaluate the biochemical effects of these environmental pollution resulting from crude oil exploration and
pollutants. exploitation and other industrial processes within the
Abelmoschus esculentus Moench (local name, area of study. The arable nature and vast land mass of
Okra) and leaves of Pterocarpus mildbraedii Harms Izombe, confers it the status of food basket of Imo State,
(local name, Oha) are vegetables commonly consumed Nigeria.
as a source of food and medication for their high content
of nutrients and phytochemicals and mainly used for MATERIALS AND METHODS
soup preparation. Consumption of vegetables provide Collection and preparation of plant samples
taste, palatability, increases appetite and provides fiber Samples of A. esculentus Moench and
for digestion and to prevent constipation. They play key P. mildbraedii Harms were obtained at Izombe, in Oguta
roles in neutralizing acids produced during digestion of Local Government Area and at Eziobodo, in Owerri
proteins and fatty foods and also provide valuable West Local Government Area both in Imo state, Nigeria
roughages which helps in movement of food in the and identified by a plant taxonomist in the Federal
intestine. Some of these vegetables possess the ability to University of Technology, Owerri (FUTO). Izombe is a
reduce or reverse so many disease conditions and rainforest ecosystem, which hosts multinational
disorders such as those which require a reduced intake of industries specialized in crude oil exploration and
glucose (diabetes) (Mcdowell, 2001: Ogbonnia et al., exploitation. Flaring of gases constitutes the major
2008). These vegetables can be deeply affected by method of waste gas disposal at these oil fields. Situate
862 Journal of Research in Biology (2013) 3(3): 861-869
Ujowundu et al., 2013

to these oil fields are communities of indigenous 1998). Saponin, alkaloid and flavonoid were done by
inhabitants whose occupation are subsistence and semi- method described by Harborne, (1973). The
commercial farming. Eziobodo is also within the spectrophotometric method as described by Griffiths and
rainforest region of Nigeria, occupied by indigenous and Thomas (1981) was used for determining phytate
FUTO students population. It has no known industrial content. Determinations were done in triplicates and
activities, except few automobiles that convey results were expressed as averages of percent values on
inhabitants in and out of the village. Samples were sorted dry weight basis.
by removing extraneous materials, spoilt and unhealthy Evaluation of vitamins content
ones. After washing, okro samples were carefully sliced. Retinol, ascorbic acid and -tocopherol contents
The samples were oven dried, macerated, sieved and in the samples were determined using the method of
properly stored. Association of Vitamin Chemist as described by Kirk
Evaluation of proximate composition and Sawyer (1998).
The method described by James (1995) and Evaluation of mineral content
Onwuka (2005) were used to determine crude fiber. Fat Some mineral contents were determined by
content was determined by the method of Min and Boft atomic absorption spectrophotometer (James, 1995). The
(2003). Moisture content was determined by the method dry samples were burnt to ashes to remove all organic
of AOAC (1990). The samples total protein content was materials leaving inorganic ash. The resulting ash was
determined by microkjeldhal method described by dissolved in 10 ml of 2 M HCl solution and diluted to
James (1995). Protein concentration was obtained by 100 ml with distilled water in a volumetric flask. The
determining total nitrogen and multiplied by the mixture was filtered and the resulting extract was used
factor- 6.25. Carbohydrate contents was calculated using for the specific evaluation of copper, zinc and iron.
the arithmetical difference method described by Pearson Sodium, potassium, calcium and magnesium were
(1976) and James (1995). determined with the aid of Jaway digital flame
Evaluation of phytochemical content photometer. Phosphorus was determined as phosphate by
Tannin content of samples were determined by the vanadomolybdate colorimetric method (Pearson,
Folin-Denis colorimetric method (Kirk and Sawyer, 1976)

Table 1: Proximate composition (%) of Abelmoschus esculentus Moench


Environment of Crude
Carbo- hydrates Ash Crude Fibre Moisture Total Fat
sampling Protein
Polluted 32.472.22a 14.280.30a 13.780.40a 22.131.40a 10.520.89a 6.820.90a
Unpolluted 37.941.78b 12.670.07a 7.990.16b 21.830.23a 12.711.71a 6.850.03a
Values (mean + SD of triplicate determinations) with different superscripts per column are significantly (P<0.05) different.

Table 2: Proximate Composition (%) of Pterocarpus mildbraedii Harms


Environment of Crude
Carbo- hydrates Ash Crude Fibre Moisture Total Fat
sampling Protein
Polluted 29.381.24c 8.061.43c 19.400.57c 17.650.79c 22.371.18c 3.140.33c
Unpolluted 34.820.30d 9.670.07c 12.050.18d 16.580.21c 23.670.29c 3.210.06c
Values (mean + SD of triplicate determinations) with different superscripts per column are significantly (P<0.05) different.

Journal of Research in Biology (2013) 3(3): 861-869 863


Ujowundu et al., 2013

Statistical analysis PE were higher than that of A. esculentus from PE. The
Da t a obt a i n ed wer e expr ess ed as highest concentrations of phytochemicals were observed
means standard deviation. Statistical Package for the in flavonoids (0.540.02%) and tannin (1.830.01%)
Social Sciences (SPSS) was used for the Analysis of from A. esculentus and P. mildbraedii respectively from
Variance (ANOVA) for the test of significant difference PE. However, alkaloids and tannins contents were
between means (P<0.05). highest in A. esculentus and P. mildbraedii respectively
from UPE.
RESULTS Vitamin contents were presented in tables 5 and
The proximate contents of A. esculentus and 6. Vitamin A concentration in A. esculentus and
P. mildbraedii are presented in Tables 1 and 2 P. mildbraedii were 627.590.47 mg/100g and
respectively. Results obtained from unpolluted 375.480.18 mg/100g respectively, indicating the
environment (UPE) showed that A. esculentus have highest vitamin content in the samples. Also, vitamin C
higher content of carbohydrate, protein, fibre and total and B5 contents in samples from UPE were significantly
fat compared to P. mildbraedii. However, higher ash higher (P<0.05) when compared to samples from PE.
and moisture content were observed in P. mildbraedii Similarly P. mildbraedii have significantly higher value
when compared to A. esculentus from UPE. of vitamin B5 (189.332.31 mg/100g) compared to
Carbohydrate content in samples obtained from polluted A. esculentus from UPE. In A. esculentus (table 5), all
environment (PE) were significantly (P<0.05) lower than the vitamins determined were significantly (P<0.05)
the UPE. But the ash contents were significantly higher lower except vitamin B5, vitamin B9 and vitamin E in
in samples from PE. The protein, crude fiber, moisture samples from PE. Also, samples of P. mildbraedii from
and total fat contents of the samples showed no PE when compared with samples from UPE showed
significant difference. significantly lower content in all the vitamins (table 6)
Results of phytochemical analysis are presented except in vitamin B2, vitamin B3 and vitamin E.
in tables 3 and 4. The concentrations of phytochemicals The mineral contents are shown in tables 7 and 8.
were significantly higher (P<0.05) in samples from PE. The concentrations of copper, iron, zinc and lead in
Also, the phytochemical contents of P. mildbraedii from A. esculentus from PE were significantly higher (P<0.05)
Table 3: Phytochemical Composition (%) of Abelmoschus esculentus Moench
Environment of
Saponins Tannins Phytates Alkaloids Phenols Flavonoids
sampling
Polluted 0.330.03a 0.370.01a 0.230.01a 0.430.01a 0.260.04a 0.540.02a
Unpolluted 0.120.05b 0.200.01b 0.090.01b 0.270.09b 0.110.00b 0.240.02b
Values (mean + SD of triplicate determinations) with different superscripts per column are significantly (P<0.05) different.

Table 4: Phytochemical Composition (%) of Pterocarpus mildbraedii Harms Vegetables


Environment
Saponins Tannins Phytates Alkaloids Phenols Flavonoids
of sampling
Polluted 0.410.02c 1.830.00c 0.370.01c 0.570.01c 0.440.02c 0.630.01c
Unpolluted 0.230.08d 1.560.14d 0.150.01d 0.280.12d 0.350.00d 0.450.08d

Values (mean SD of triplicate determinations) with different superscripts per column are significantly
(P<0.05) different.

864 Journal of Research in Biology (2013) 3(3): 861-869


Table 5: Vitamin Content (mg/100g) of Abelmoschus esculentus Moench
Environment
Vitamin A Vitamin B1 Vitamin B2 Vitamin B3 Vitamin B5 Vitamin B6 Vitamin B9 Vitamin C Vitamin E
of sampling
Polluted 592.7819.69a 0.020.02a 0.050.01a 0.830.10a 21.002.52a 0.580.02a 0.670.11a 68.412.31a 1.520.02a
Unpolluted 627.590.47b 0.080.01b 0.080.01b 1.120.01b 23.332.31a 0.810.10b 0.730.08a 78.031.02b 1.880.17a
Values (mean + SD of triplicate determinations) with different superscripts per column are significantly (P<0.05) different.
Ujowundu et al., 2013

Table 6: Vitamin Content (mg/100g) of Pterocarpus mildbraedii Harms Vegetables


Environment
Vitamin A Vitamin B1 Vitamin B2 Vitamin B3 Vitamin B5 Vitamin B6 Vitamin B9 Vitamin C Vitamin E
of sampling
Polluted 302.577.01c 0.070.02c 0.040.01c 0.650.09c 175.226.97c 0.250.08c 0.530.17c 85.291.79c 1.970.18c
Unpolluted 375.480.18d 0.120.00d 0.060.01c 0.570.02c 189.332.31d 0.590.05d 0.890.02d 99.152.69d 2.270.17c

Journal of Research in Biology (2013) 3(3): 861-869


Values (mean + SD of triplicate determinations) with different superscripts per column are significantly (P<0.05) different.

Table 7: Mineral Content (mg/100g) of Abelmoschus esculentus Moench.


Environment
Magnesium Calcium Phosphorus Sodium Potassium Copper Iron Zinc Lead
of sampling
Polluted 22.171.39a 70.232.02a 57.350.59a 6.340.46a 107.634.45a 0.420.15a 0.950.26a 0.480.14a 0.650.07a
Unpolluted 39.601.39b 82.832.32b 68.280.46a 7.470.23a 130.406.55b 0.060.01b 0.740.25b 0.320.07b 0.350.10b

Values (mean + SD of triplicate determinations) with different superscripts per column are significantly (P<0.05) different.

Table 8: Mineral Content (mg/100g) of Pterocarpus mildbraedii Harms Vegetables.


Environment
Magnesium Calcium Phosphorus Sodium Potassium Copper Iron Zinc Lead
of sampling
Polluted 48.674.38c 64.383.95c 409.890.43c 17.161.71c 250.734.29c 0.340.13c 0.770.18c 0.340.06c 0.500.22c

Unpolluted 52.802.40c 77.482.32d 413.8912.48c 21.130.12d 284.272.44d 0.040.00d 0.580.09d 0.160.02d 0.190.13d

865
Values (mean + SD of triplicate determinations) with different superscripts per column are significantly (P<0.05) different.
Ujowundu et al., 2013

when compared to samples from UPE. Results presented those from UPE but were not significantly different,
in table 8 showed that P. mildbraedii from UPE are which indicates that they were not adversely affected by
excellent source of phosphorus (413.8912.48), the pollution.
potassium (284.272.44), calcium (77.482.32) and The phytochemical results indicate that
magnesium (52.802.40). Also, the concentration of A. esculentus and P. mildbraedii are good sources of
minerals in P. mildbraedii from PE and UPE were these beneficial chemicals. They have antioxidative,
significantly different in all except in magnesium and hypocholesterolemic, chemoprotective and antibacterial
phosphorus. properties (Price et al., 1987; Enechi and Odonwodo,
2003; Okwu, 2004). Both vegetables are rich in
DISCUSSION alkaloids, flavonoids and tannins which indicates that
Gas flaring and other oil and gas activities for they have diuretic, antispasmodic, anti-inflammatory and
about four decades have contributed to pollution in analgesic effects (Owoyele et al., 2002; Nobre-Junior,
Oguta, which have impacted on the ecosystem. Soots 2007 ; Alisi et al., 2011). Comparatively, P. mildbraedii
were seen on vegetation within the communities around had higher content of the phytochemicals studied. Also,
the flaring site. Plants growing in such environment have significantly higher amount of phytochemicals were
over the years taken in varying doses of pollutants which observed in vegetables obtained from PE. The increase
invariably may affect the nutritional and chemical can be linked to their role in oxidative stress in plants.
contents. Phytochemicals are secondary metabolite of plants,
Our result showed that A. esculentus had better known to exhibit diverse pharmacological and
nutritional value than P. mildbraedii with respect to biochemical effects on living organisms. It has been
protein and carbohydrate contents. Also, A. esculentus reported that certain phytochemicals play important role
and P. mildbraedii showed higher values in proximate in antioxidant defense systems of vegetative plants
contents (except in protein) than A. hybridus as reported (Ugochukwu and Babady, 2003). Pollution by gas flaring
by Nwaogu et al., (2006). Carbohydrates provide energy is taught to generate free radicals in surrounding
to cells in the body, particularly to the brain, a environment. Thus, it is expected that plants may
carbohydrate dependent organ in the body. (Nelson and increase synthesis of antioxidant defense compounds.
Cox, 2005). These vegetables can supplement the daily These vegetables showed significantly high
energy intake of humans (Bingham, 1998; Effiong et al., amount of vitamins especially vitamins A, B1, B2, B5, B6
2009). The crude fibre content, indicates that the and C in samples from UPE when compared
vegetables are good sources of fibre, thus making them to samples from PE. These vitamins are involved in
veritable source of roughage. The concentrations of intermediary metabolism of both plants and animals
carbohydrate were significantly reduced while ash acting as part or whole coenzyme to some specific
contents were increased in plants from polluted enzyme system and playing important role in both
environment when compared to plants from unpolluted enzyme and non enzyme oxidative stress defense
environment (UPE). The reduced carbohydrate can be systems. The high concentrations of vitamins A and C
attributed to the effect of air pollutants as reported by will contribute significantly to the daily requirements in
Farzana (2005), in which he affirmed that it reduces view of the reports of Murray (1998). Vitamin C
photosynthesis in chloroplasts. The contents of protein, maintains blood vessel flexibility and improves
crude fiber and fat in samples from PE were lower than circulation in the arteries of smokers. The most important
866 Journal of Research in Biology (2013) 3(3): 861-869
Ujowundu et al., 2013

benefit of vitamins A and C is their involvement in free phosphorus are important and indispensable for the
radical scavenging processes (Trumbo et al., 2004; synthesis of strong bones and teeth, kidney function and
Nwaogu et al., 2011). These chemically active radicals cell growth (Uddoh, 1988; Brody, 1994). Phosphorus
are byproducts of many normal biochemical processes. and magnesium are also important in the regulation of
Their numbers are increased by environmental assaults acid-alkaline balance in the body (Fallon, 2001).
such as chemicals and toxins. The lower concentrations The mineral contents, like Mg, Ca, P, S and K in
of these vitamins in samples from PE suggest an inability vegetables from PE have significantly (P<0.05) reduced
of the plants to synthesize these vitamins in sufficiently value compared to vegetables obtained from UPE. The
large amount for their metabolic functions. Oxidative release of pollutants such as oxides of sulphur and
stress caused by gas flaring in Oguta community can nitrogen, hydrocarbons and other volatile organic
interfere with the synthetic mechanisms of the plants in carbons can create chemical and biological conditions
the environment (Farzana, 2005). which may be harmful to plants and soil microorganisms.
Som e of the mineral contents of One of such conditions is the reduction in the capacity of
A. esculentus Moench and P. mildbraedii Harms are plants to absorb cations (Wild et al., 2000). Crops grown
comparable or higher than that reported for in soil with low mineral contents exhibit various forms of
Amaranthus hybridus (Nwaogu et al., 2006) mineral deficiency. In plants, potassium is an essential
Mucuna utilis (Ujowundu et al., 2010), nutrient and has an important role in the synthesis of
Commelina nudiflora and Boerhavia diffusa (Ujowundu amino acids and proteins (Malik, 1982). Ca and Mg play
et al., 2008). The values obtained for the minerals significant role in photosynthesis, carbohydrate and
indicates that the samples are good sources of mineral nucleic acids metabolism (Russel, 1973). The reduced
and are of great nutritional importance. In animals, content of these minerals will definitely affect these
potassium and sodium are important electrolytes. important plant processes. Lead is yet to record any
Potassium is a major intracellular cation. Sodium is physiological role in the biological system and are
involved in the regulation of acid-base equilibrium, known to be extremely toxic even at the slightest
protection against dehydration and maintenance of concentration. Their presence in the samples calls for
osmotic pressure in living system. It plays a role in the serious concern
normal irritability of muscles and cell permeability This study has shown that A. esculentus Moench
(Schwart, 1975). Copper (Cu) is essential for and P. mildbraedii Harms are good sources of nutrients
haemoglobin synthesis, normal bone formation and the and their consumption should be encouraged. Improved
maintenance of myelin within the nervous system information on these plants will contribute to the
(Passmore and Eastwood, 1986). In animals, the awareness of their nutritive value, especially in this time
manifestations of copper deficiency include; anaemia, of increased food insecurity. Also, gas flaring showed
hypo-pigmentation, defective wool keritinization, negative effects on these plants, which could affect
abnormal bone formation with spontaneous reproductive animals that consume them. Similarly, the adverse health
and heart failure (Williams, 1982). In humans, it has consequences on the inhabitants around the gas flare
been established that occurrence of Cu absorption site are of great concern. Communities around such
disorder in after partial gastetomycin leads to severe environment should be enlightened on the inherent
malnutrition just as when protein is severely deficient in dangers. Oil and gas industries should be compelled to
the diet; as in kwashiorkor (Davies, 1972). Calcium and upgrade their waste disposal technologies, with emphasis

Journal of Research in Biology (2013) 3(3): 861-869 867


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Ugochukwu NH and Babady NE. 2003. Antioxidant Submit your articles online at www.jresearchbiology.com
effects on Gongonemia catifolium in hepatocytes of rat
Advantages
model of non insulin dependent diabetes mellitus. Easy online submission
Filoterapia 173(7-8):612-618. Complete Peer review
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Journal of Research in Biology An International Scientific Research Journal

Original Research

An ornithological survey in the vicinity of Agartala city of Tripura state,


north-eastern India
Journal of Research in Biology

Authors: ABSTRACT:
Partha Pratim Bhattacharjee,
Rahul Lodh, Dipten Laskar, North-east India is a part of Indo-Burma hotspot and among the richest bird
Joydeb Majumder and
zones in India. Tripura lies in the border of Indo-Burma global biodiversity hotspot
Basant Kumar Agarwala.
area but is poorly covered by ornithological works. Avifauna of Tripura state is known
by 277 species but there is lack of information about their distribution, particularly in
and around Agartala city, which is the capital of Tripura state and is a tourist
Institution: destination along the border of Bangladesh for its natural landscapes, inland water
Ecology & Biodiversity species, and strong presence of green flora. With a view to enhance its value for
Laboratories, Department of tourist attraction and naturalists, a study was conducted to record the species of birds
Zoology, Tripura University, that occur in and around the City. In the present study 73 bird species were recorded
Suryamaninagar-799 022, from Agartala city and its adjacent areas belonging to 41 families and 14 orders.
Tripura, India.

Corresponding author: Keywords:


Basant Kumar Agarwala. Avifauna, biodiversity hotspot, Agartala, Tripura, north-east India .

Email: Article Citation:


bagarwala00@gmail.com Partha Pratim Bhattacharjee, Rahul Lodh, Dipten Laskar, Joydeb Majumder and
Basant Kumar Agarwala.
An ornithological survey in the vicinity of Agartala city of Tripura state, north-eastern
India.
Journal of Research in Biology (2013) 3(3): 852-860
Web Address:
http://jresearchbiology.com/ Dates:
documents/RA0328.pdf. Received: 28 Jan 2013 Accepted: 15 Feb 2013 Published: 10 Apr 2013

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.

852-860 | JRB | 2013 | Vol 3 | No 3


Journal of Research in Biology
An International Scientific
Research Journal www.jresearchbiology.com
Bhattacharjee et al., 2013

INTRODUCTION little is known regarding the bird species found in the


Avifauna contributes most significantly to the vicinity of Agartala city, situated by international
diversity of terrestrial vertebrates, which have a special boundary of Bangladesh.
role in conservation of biodiversity of a particular area STUDY SITES
(Daniels, 1994). Birds are very good indicator Agartala city is situated in the western region of
of environmental changes as they respond in the minute Tripura state with the latitude of 2345' North and
change in habitat structure and composition longitude of 9145' East and an average elevation of
(Robert et al., 2001). Indian subcontinent harbour nearly 20.36 m above sea level. It is the capital town of Tripura
1300 species of birds, which is more than 13% of total with a mix of urban and semi urban complex and a rich
bird species of the world (Grimmet et al., 2004), and green cover. Forests and farms adjoin the town on three
more than 60% of Indian birds are found in north-east sides, and therefore, it is also called Green City. The
India (Choudhury, 2010). North-east India is one of the total city area is 62.02 km2 and is delimited on the west
most significant biodiversity hotspots of the world and side by international boundary with Bangladesh.
among the richest bird zones in India because of Climatic condition is of tropical monsoon type with an
convergence of the Indo-Malayan, Indo-Chinese and average annual rainfall of 220 cm. Average minimum
Indian biogeographical realms. As a result, it is unique in and maximum temperature recorded in the region are
providing an abundance of habitats that harbour diverse 6.8C in January and 37.70C in June, respectively.
biota with a high degree of endemism (Chatterjee et al., Present study was carried out in eleven
2006; Narwade et al., 2011). Tripura (2256- 2432 N different sites (viz., College Tilla lake area, Golbazar,
2
and 9110- 9221 E, with an area of 10,490 km ) is a Pratapgarh, Dashamighat, Arundhutinagar, Shanmura,
small state of north-east India bounded by Bangladesh Bhubanban, Barjala, Jagannath Bari lake area, G B Bazar
on three sides and with Assam and Mizoram on the other and Nandannagar) (Table 1, Figure 1) covering different
side. It lies in the border of Indo-Burma global sides of Agartala city and its adjacent areas.
biodiversity hotspot area (Myers et al., 2000) but
very poorly covered by ornithological works METHODOLOGY
(Choudhury, 2010). Although avifaunal checklist for The study sites were visited fortnightly
Tripura state listed 277 species (Choudhury, 2010) but throughout the study period from 2009-2011. Data on

Table 1: Geo-coordinate details of the study sites

Sl. No Sites Coordinates Altitude (m)

1. College Tilla lake area 2349'35.45" N; 9117'42.28" E 17


2. Golbazar 2349'38.30" N ; 9116'57.15" E 16
3. Pratapgarh 2349'08.98" N ; 9117'17.10" E 16
4. Dashamighat 2349'46.34" N ; 9115'51.45" E 16
5. Arundhutinagar 2349'01.44" N ; 9116'21.68" E 31
6. Shanmura 2350'51.98" N ; 9116'07.20" E 15
7. Bhubanban 2351'56.50" N; 9115'73.70" E 21
8. Barjala 2352'05.05" N ; 9116'32.13" E 23
9. Jagannath Bari lake area 2350'05.43" N; 9116'53.70" E 14
10. G B Bazar 2351'33.74" N ; 9117'33.97" E 27
11. Nandannagar 2351'43.68" N ; 9117'57.00" E 28
853 Journal of Research in Biology (2013) 3(3): 852-860
Bhattacharjee et al., 2013

Figure 1. Showing the study sites in and around Agartala City.

present bird species were collected by direct observations (2002) recorded 259 species of birds, belonging to
with the help of binoculars (VISTA LE 8 X 40). Almost 56 families and 16 orders. Recently Choudhury (2010)
all the species mentioned in the checklist were recorded 277 species of birds, in the annotated checklist
photographed. For this purpose, digital cameras of Canon from Tripura, but avifaunal diversity of Agartala city is
Power shot SX 200 IS (12 X Digital zoom), Cannon SRL not yet available. Out of 14 orders. Passeriformes was
EOS 50D and Panasonic Lumix DMC FZ 40 were used. found dominant with 22 families followed by
In lake areas birds were observed from the bank, Coraciiformes with 3 families and Pelecaniformes and
peripheral areas, and urban areas were surveyed on foot Piciformes with 2 families each. Dominance of
regularly. Farm and forested areas in the vicinity of the Passeriformes was also recorded by Choudhury (2010)
city were surveyed to record the assemblages of different and Majumdar et al., (2002) from the state and from
bird species. Most of visits were made in morning and Nagpur district of central India (Chinchkhede and Kedar,
afternoon time when birds are most active. Identification 2012). The resident birds such as Pond heron, Cattle
of birds was based on the field guides produced by Ali egret, Lapwing, Blue rock pigeon, Spotted dove,
and Ripley (1995), Ali (1996 and 2002) and Grimmett et Parakeets, Asian koel, Kingfisher, Bee eater, Lineated
al., (2003). barbet, Woodpecker, Bush lark, Bulbul, Shrike, Robin,
Status of the birds was classified as C-Common, Tailorbird, Cinereous tit, Sunbird, Sparrow, Starling,
MC-Most common, NC-Not common, S-Singleton, Myna, Oriole, Black drongo and Crow etc were found
W- Winter visitor. regularly throughout the study period. Little Cormorant,
Asian Openbill-Stork, Black headed Ibis, Lesser
RESULTS AND DISCUSSION Whistling Duck, Crested Serpent Eagle, Red Junglefowl,
In the present study 73 bird species were Red Collared Dove, Yellow-footed Green Pigeon, Brown
recorded from Agartala city and its adjacent areas Fish Owl, Asian Palm Swift, Indian Roller, Coppersmith
belonging to 41 families and 14 orders (Table 2, Plate 1 Barbet, Orange headed Thrush, Blue Rock Thrush,
and 2). There is no authentic information about the White-rumped Shama, Scarlet-backed Flower pecker,
avifauna of Tripura except that by Blyth (1845, 1846), Tricoloured Munia etc were found less common in this
Ali and Ripley (1968-74) and International Waterfowl study. Common Sandpiper and Black headed Ibis were
and Wetlands Research Bureau on Asian Waterfowl observed during the winter season only in the paddy
Census, 1989 (Scott and Rose 1989). Majumdar et al., fields of peripheral areas of the city. Common Hoopoe

Journal of Research in Biology (2013) 3(3): 852-860 854


Bhattacharjee et al., 2013

Table 2: List of birds in and around Agartala city during 2009-2011


Sl. Status
Common name Scientific name
No. IUCN Abundance
Cormorants [Phalacrocoracidae]
1. Little Cormorant Phalacrocorax niger Vieillot, 1817 LC NC
Herons & Egrets [Ardeidae]
2. Indian Pond Heron Ardeola grayii (Sykes, 1832) LC MC
3. Cattle Egret Bubulcus ibis (Linnaeus, 1758) LC MC
4. Median Egret Mesophoyx intermedia (Wagler, 1827) LC C
Storks [Ciconiidae]
5. Asian Openbill-Stork Anastomus oscitans Boddaert, 1783 LC NC
6. Black headed Ibis Threskiornis melanocephalus (Latham, 1790) NT W, NC
Ducks [Anatidae]
7. Lesser Whistling Duck Dendrocygna javanica (Horsfield, 1821) LC NC
Hawks & Eagles [Accipitridae]
8. Crested Serpent Eagle Spilornis cheela Latham, 1790 LC NC
9. Black Kite Milvus migrans (Boddaert, 1783) LC C
Pheasants [Phasianidae]
10. Red Junglefowl Gallus gallus (Linnaeus, 1758) LC NC
Rails & Coots [Rallidae]
11. White-breasted Waterhen Amaurornis phoenicurus Pennant, 1769 LC C
Lapwings [Charadriidae]
12. Red-wattled Lapwing Vanellus indicus (Boddaert, 1783) LC MC
Sandpipers [Scolopacidae]
13. Common Sandpiper Actitis hypoleucos (Linnaeus, 1758) LC W, C
Pigeons & Doves [Columbidae]
14. Blue Rock Pigeon Columba livia Gmelin, 1789 LC MC
15. Spotted Dove Streptopelia chinensis (Scopoli, 1768) LC MC
16. Red Collared Dove Streptopelia tranquebarica (Hermann, 1804) LC NC
Orange-breasted Green
17. Treron bicinctus (Jerdon, 1840) LC C
Pigeon
18. Yellow-footed Green Pigeon Treron phoenicoptera (Latham, 1790) LC NC
Parakeets [Psittacidae]
19. Rose-ringed Parakeet Psittacula krameri (Scopoli, 1769) LC C
20. Red-breasted Parakeet Psittacula alexandri (Linnaeus, 1758) LC C
Cuckoos & Coucals [Cuculidae]
21. Asian Koel Eudynamys scolopaceus (Linnaeus, 1758) LC MC
22. Greater Coucal Centropus sinensis (Stephens, 1815) LC C
Owls [Strigidae]
23. Collared Scops Owl Otus lettia Hodgson, 1836 LC S
24. Spotted Owlet Athene brama (Temminck, 1821) LC C
25. Brown Fish Owl Bubo zeylonensis (Gmelin, 1788) LC NC
855 Journal of Research in Biology (2013) 3(3): 852-860
Bhattacharjee et al., 2013

Swifts [Apodidae]
26. Asian Palm Swift Cypsiurus balasiensis Gray, 1829 LC NC
27. House Swift Apus affinis (J E Gray, 1830) LC C
Kingfishers [Alcedinidae]
28. Common Kingfisher Alcedo atthis (Linnaeus, 1758) LC MC
29. Stork-billed Kingfisher Halcyon capensis (Linnaeus, 1766) LC C
30. White-throated Kingfisher Halcyon smyrnensis (Linnaeus, 1758) LC MC
Bee-eaters [Meropidae]
31. Little Green Bee-eater Merops orientalis Latham, 1802 LC MC
Rollers [Coraciidae]
32. Indian Roller Coracias benghalensis (Linnaeus, 1758) LC NC
Hoopoe [Upupidae]
33. Common Hoopoe Upupa epops Linnaeus, 1758 LC S
Barbets [Capitonidae]
34. Lineated Barbet Megalaima lineata (Vieillot, 1816) LC MC
35. Coppersmith Barbet Megalaima haemacephala Muller, 1776 LC NC
Woodpeckers [Picidae]
36. Rufous Woodpecker Celeus brachyurus (Vieillot, 1818) LC C
37. Greater Flameback Chrysocolaptes lucidus (Scopoli, 1786) LC C
38. Fulvous-breasted Woodpecker Dendrocopos macei (Vieillot, 1818) LC C
Larks [Alaudidae]
39. Singing bush lark Mirafra cantillans Blyth, 1844 LC C
Pipits & Wagtails [Motacillidae]
40. Paddy field Pipit Anthus rufulus Vieillot, 1818 LC C
41. White Wagtail Motacilla alba Linnaeus, 1758 LC W,C
Bulbuls [Pycnonotidae]
42. Red-whiskered Bulbul Pycnonotus jocosus (Linnaeus, 1758) LC C
43. Red-vented Bulbul Pycnonotus cafer (Linnaeus, 1766) LC MC
Loras [Irenidae]
44. Common Lora Aegithina tiphia (Linnaeus, 1758) LC C
Shrikes [Laniidae]
45. Brown Shrike Lanius cristatus Linnaeus, 1758 LC W, MC
46. Grey-backed Shrike Lanius tephronotus (Vigors, 1831) LC W, C
Thrushes [Turdidae]
47. Orange headed Thrush Zoothera citrina (Latham, 1790) LC NC
Flycatchers [Muscicapidae]
48. Blue Rock Thrush Monticola solitarius (Linnaeus, 1758) LC NC
49. White-rumped Shama Copsychus malabaricus (Scopoli, 1786) LC NC
50. Oriental Magpie Robin Copsychus saularis (Linnaeus, 1758) LC MC

Journal of Research in Biology (2013) 3(3): 852-860 856


Bhattacharjee et al., 2013
Babblers [Timaliidae]
51. Rufous-necked Laughing- Garrulax ruficollis (Jardine & Selby, 1838) LC C
thrush

Warblers [Sylviidae]
52. Common Tailorbird Orthotomus sutorius (Pennant, 1769) LC MC
Flycatchers [Stenostiridae]
53. Grey-headed Canary- Culicicapa ceylonensis (Swainson, 1820) LC W, C
flycatcher

Tits [Paridae]
54. Cinereous Tit Parus cinereus Vieillot, 1818 LC MC
Flowerpeckers [Dicaeidae]
55. Scarlet-backed Flowerpecker Dicaeum cruentatum (Linnaeus, 1758) LC NC
Sunbirds [Nectariniidae]
56. Ruby-cheeked Sunbird Anthreptes singalensis (Gmelin, 1788) LC C
57. Purple-rumped Sunbird Nectarinia zeylonica (Linnaeus, 1766) LC C
58. Purple Sunbird Cinnyris asiaticus Latham, 1790 LC MC
White-eyes [Zosteropidae]
59. Oriental White-eye Zosterops palpebrosus (Temminck, 1824) LC C
Munias [Estrildidae]
60. Scaly-breasted Munia Lonchura punctulata (Linnaeus, 1758) LC C
61. Tricoloured Munia Lonchura malacca (Linnaeus, 1766) LC NC
Sparrows [Passerinae]
62. House Sparrow Passer domesticus (Linnaeus, 1758) LC MC
Weavers [Ploceidae]
63. Baya Weaver Ploceus philippinus (Linnaeus, 1766) LC C
Starlings & Mynas [Sturnidae]
64. Chestnut-tailed Starling Sturnus malabaricus (Gmelin, 1789) LC C
65. Asian Pied Starling Gracupica contra (Linnaeus, 1758) LC MC
66. Common Myna Acridotheres tristis (Linnaeus, 1766) LC MC
67. Jungle Myna Acridotheres fuscus (Wagler, 1827) LC MC
Orioles [Oriolidae]
68. Black-hooded Oriole Oriolus xanthornus (Linnaeus, 1758) LC MC
Drongos [Dicruridae]
69. Black Drongo Dicrurus macrocercus (Vieillot, 1817) LC MC
70. Greater racket-tailed Drongo Dicrurus paradiseus Linnaeus, 1766 LC NC
Crows & Treepie [Corvidae]
71. Rufous Treepie Dendrocitta vagabunda (Latham, 1790) LC C
72. House Crow Corvus splendens Vieillot, 1817 LC MC
73. Jungle Crow Corvus macrorhynchos Wagler, 1827 LC MC
Abbreviations:
Status: LC = least concern; NT = near threatened; C = common; MC = most common; NC = not common;
S = singleton; W = winter visitor.
857 Journal of Research in Biology (2013) 3(3): 852-860
Bhattacharjee et al., 2013

Plate 1. A-Lineated barbet, B-Cattle egret, C-Red-wattled Lapwing, D-Common Hoope, E-Little cormorant,
F-Stripe-breasted woodpecker, G-Rufous woodpecker, H-White throated kingfisher, I-Yellow footed green
pegion, J-Asian open bill stork, K-Chestnut-tailed starling, L-Collared scops owl.

Plate 2: M-Asian Koel, N-Crested serpent eagle, O-Common Tailorbird, P-Cinereous Tit, Q-Emerald dove,
R-Little green bee-eater, S-Grey-baked shrike, T-Indian pond heron, U-Red collared dove, V-White-rumped
shama, W-Singing bush lark, X-Black headed ibis.
Journal of Research in Biology (2013) 3(3): 852-860 858
Bhattacharjee et al., 2013

and Collared Scops Owl were sighted only once in the Ali S and Ripley SD. 1995. A pictorial guide to the
two years study. Brown Shrike, Grey-backed Shrike, birds of Indian Subcontinent. Bombay Natural History
Grey-headed Canary-flycatcher were observed in the Society, Mumbai.
winter season only (Table 2), which corroborates with
BirdLife International 2009: IUCN 2011. IUCN Red
the findings of Choudhury (2010) and Majumdar et al.,
List of Threatened Species. Version 2011.2.
(2002).
<www.iucnredlist.org>. Downloaded on 18 June 2012.

CONCLUSION Blyth E. 1845. Notices and descriptions of various new


The present avifaunal survey of Agartala city and or little known species of birds, Journal of the Asiatic
its adjacent areas revealed 73 bird species which is very Society of Bengal, 14: 546-602.
important as it is the first ornithological record of the city
Blyth E. 1846. Notices and descriptions of various new
and will give a baseline data for future study. Rich bird
or little known species of birds, Journal of the Asiatic
diversity is influenced by the topographical location of
Society of Bengal, 15: 1-54.
the city and adjacent areas of Bangladesh.
Expansion of the city by construction activities, Chatterjee S, Saikia A, Dutta P, Ghosh D, Pangging
reducing forest and farm areas with population pressure, G and Goswami AK. 2006. Biodiversity significance of
filling of pond and lake areas, dumping of wastes and north east India: WWF-India. New Delhi. pp-71.
garbage in the low lands, use of chemical pesticides in
Chinchkhede KH and Kedar GT. 2012. Avifaunal
agricultural fields and hunting of birds are the major
diversity of Koradi lake in Nagpur district of central
threats to the avifaunal diversity here which needs proper
India. Journal of Research in Biology, 2: 70-76.
conservation management practices.
Choudhury A. 2010. Recent ornithological records from
ACKNOWLEDGEMENT Tripura, north-eastern India, with an annotated checklist.
Authors are thankful to Mr. Dipankar Kishore Indian BIRDS 6(3): 66-74.
Sinha for his constant services, tireless field assistance
Daniels RJR. 1994. A landscape approach to
and in capturing photographs during the study.
conservation of birds. Journal of Bioscience 19(4): 503-
509.
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Journal of Research in Biology (2013) 3(3): 852-860 860


Journal of Research in Biology An International Scientific Research Journal

Original Research

Anti-inflammatory activity of lycopene isolated from Chlorella marina on


carrageenan-induced rat paw edema
Journal of Research in Biology

Authors: ABSTRACT:
Renju GL and
Muraleedhara Kurup G. Even though role of lycopene (all-trans) in controlling inflammation was
reported, lycopene (cis and all-trans 40:60) isolated from green algae Chlorella marina
was not reported so far. In this present study inflammation was induced in male
Sprague dawley rats and edema was produced acutely by injecting 0.1 ml of
carrageenan into the plantar region of the right hind paw of the rats subcutaneously.
Institution: Intra peritoneal administration of algal lycopene (AL) at the dose of 10 mg/kg b.wt
Department of Biochemistry, showed maximum (83%) inhibition on paw edema. The anti- inflammatory effect was
University of Kerala, significantly (P< 0.05) higher in rats fed with algal lycopene when compared to the
Trivandrum, India. standard drug voveran (71%) and all- trans tomato lycopene (TL) (63%). Carrageenan
induced rats showed elevated levels of cyclooxygenase (COX) and lipoxygenase (LOX)
activities in monocytes. Myeloperoxidase (MPO) in serum, C- reactive protein (CRP)
and ceruloplasmin activity in plasma was also high in carrageenan induced rats when
compared to normal. Lycopene from Chlorella marina showed significant effect in
Corresponding author: reducing the above parameters to that of the standard drug while tomato lycopene
Muraleedhara Kurup G. showed less effect when compared to algal lycopene. Therefore algal lycopene from
Chlorella marina would be recommended for the treatment of anti-inflammatory
disorders.

Email: Keywords:
gmkbio@gmail.com. Microalgae, Chlorella marina, lycopene, anti-inflammation.

Phone: Article Citation:


+919447251408. Renju GL and Muraleedhara Kurup G.
Anti-inflammatory activity of lycopene isolated from Chlorella marina on
carrageenan-induced rat paw edema.
Fax: Journal of Research in Biology (2013) 3(3): 886-894
91-471 2308078.
Dates:
Received: 02 Feb 2013 Accepted: 22 Feb 2013 Published: 23 Apr 2013
Web Address:
http://jresearchbiology.com/
documents/RA0329.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.

886-894 | JRB | 2013 | Vol 3 | No 3


Journal of Research in Biology
An International Scientific
Research Journal www.jresearchbiology.com
Renju and Kurup., 2013

INTRODUCTION was that algal lycopene contain cis-configuration


Inflammation is a response which protects and (5-cis, 9-cis, 13- cis and 15-cis). Recently it has been
heals the host tissue after infection or injury. (Nathan, reported that the cis form of lycopene is more
2002). However, it is frequent that the inflammatory biologically active than the trans form (Stahl and Sies,
response to several insults erroneously leads to the 1996).
damaging of normal tissues. Prostaglandin-E2 is
generated from arachidonic acid by the enzyme MATERIALS AND METHODS
cyclooxygenase (COX) at sites of inflammation in Chemicals
substantial amounts and can mediate many of the Lycopene, carrageenan, linoleic acid,
pathologic features of inflammation (Serhan and Levy, Histopaque, arachidonic acid other fine chemicals were
2003). One of the early cellular events in inflammation is purchased from Sigma, St. Louis, MO, USA. Diclofenac
the margination of leukocytes, primarily neutrophils and sodium (Voveran) was obtained from Novartis, India.
this can be measured by myeloperoxidase activity Salt and vitamin mixtures were purchased from Merck,
(Goulet et al., 1994). Germany. All other chemicals and reagents were
Currently, non steroidal anti-inflammatory drugs purchased from Sisco Research Laboratory Pvt.Ltd
(NSAIDs) were used for inflammatory diseases. Even (SRL), India, and were of analytical grade.
though this drugs transiently suppresses inflammation, Algal source
but their long term use cause ulceration in the Marine algae Chlorella marina Butcher was
gastrointestinal tract and renal morbidity (James and collected from the Vizhinjam coast of Kerala, located at
Hawkey, 2003). However research focused on finding Latitude 08 22 North Longitude. 76 59 East on the
newer drugs with pharmacological actions without side south west coast of India and was cultured under
effects. laboratory conditions. The microalgae were identified by
Several antioxidants have been reported to the botanist (Dr. G. Valsaladevi, Department of botany)
have anti-inflammatory and anti-arthritic activities and a voucher specimen (No. KUBH 5812) has been
(Maxwell et al., 2006). In the present study a culturable deposited in the Department of Botany, University of
marine edible algae Chlorella marina was selected to Kerala, India.
evaluate the anti-inflammatory activity of lycopene. Culture medium
Generally tomatoes are the source of lycopene, but it has Walnes medium (1970) was used as a basal
many disadvantages (Shi and Le mague, 2000). The medium for the cultivation of Chlorella marina. 5 g /L
content of lycopene in tomato is very less and the glucose was added to the basal medium. Flasks were
configuration of lycopene is all-trans. Even though incubated at 25C with continuous illumination. The pH
lycopene from algae has been reported (Ishikawa and was adjusted to 7.5. Nicotine (10 M/ L) was sterilized
Abe, 2004), no attempt has been made so far for the by autoclaving and was added to 5 days old cultures for
commercialization of algal lycopene. It can be seen that the production of lycopene.
marine sources especially algae are the least exploited Biomass harvest
for their bioactive molecules (Pinky and Goswai, 2012). Chlorella marina cells were grown in suspension
Work in our laboratory has shown that the lycopene cultures up to 30 to 40 days. The cells were harvested at
content in algae is comparatively high, when compared stationary phase by withdrawing the cultures in 50 ml
to tomato lycopene. The most interesting observation polypropylene tubes and centrifuged at 5000 rpm for
887 Journal of Research in Biology (2013) 3(3): 886-894
Renju and Kurup., 2013

10 minutes. Removed the medium and the pellets were concentrations of different fractions. All samples were
freeze dried, weighed and stored under nitrogen at -20C. injected in duplicate.
Isolation of lycopene from Chlorella marina (AL) and Experimental animals
analysis Male Sprague Dawley rats with the average body
Harvested biomass (5g dry weight) was weight of 150- 200 g of the same breed were selected for
suspended with 5 ml of 80% cold acetone and kept the study. These animals were housed in the department
overnight under 4C for better and easy recovery of animal house and provided standard pellet diet and water
carotenoids. The mixtures were vortexed for 2 minutes ad libitum and maintained with temperature at 25 1C,
and centrifuged at 5000 rpm for 20 minutes. After humidity (55-60%) and photoperiod (12:12 h) light and
repeated extractions (4 times), the supernatants were dark cycle. Experimental procedures conducted on rats
pooled and the colorless cell pellets were discarded. The were approved by the Animal Experiment Committee
extracts were dried over anhydrous sodium sulphate and (218/CPCSEA) for animal care of Kerala University
reduced to a minimum volume by evaporating the according to Government of Indian law on animal use
solvents using N2 stream. The crude extracts were kept and care.
for further separation of carotenoids in amber colored Induction of acute inflammation-Carrageenan
containers under nitrogen at -20C. All operations were induced rat paw edema
done at subdued light under nitrogen atmosphere. The Carrageenan-induced rat paw edema assay was
absorbance in the solvent phase was quantified by conducted according to the procedure as described by
spectrophotometric method at 470 nm as described by Winter et al., (1962). Five groups of six rats were
Lichtenthaler (1987). treated as AL and TL with doses 10 mg/kg and reference
Isolation of all-trans lycopene from tomato (TL) drug Voveran, a Diclofenac sodium preparation
Tomatoes obtained from the local market, (20 mg/kg) were given orally and intraperitoneally (i.p),
Trivandrum, India were used. The all-trans lycopene 1 h before the injection of carrageenan. Control rats were
from tomato was extracted and evaluated according to given 0.1 ml 1% carrageenan. Inflammation was
the procedure of Fish et al., (2002). induced by 0.1 ml, 1% carrageenan suspension in
Determination of lycopene by HPLC 0.9% NaCl solution was injected into the right hind paw
Lycopene extracted from algal cells and tomatoes after 1 hour. The volume of the right paw was measured
were determined by HPLC method at 450 nm as by paw edema meter before and after injection in
described by Shaish et al., (1992). HPLC analysis of the third and fifth hour. The paw edema and inhibition
lycopenes were performed using a silia chrom column was calculated by the equation: Activity= 100 - (100
(250 x 4mm + 5 x 4, NCLIOSIL 100-5-C18 5.0m), average drug treated/average for control).
K 1001 type pump and the UV detector type of K 2600, Treatment Protocol and Experimental Design in
Germany. Elution was performed isocratically with Acute Inflammation
methanol: acetonitrile (9:1) v/v at a flow rate of Edema was induced on rat right hind paw by
-1
1 ml min . A UV detector with a wavelength of 450 nm aponeurosis injection of 0.1ml of 1% carrageenan in
was employed. Lycopene (95%) obtained from Sigma 0.9% saline. The experimental groups consisted of 30
chemicals were used as standard. The retention time was rats were divided in to five groups.
recorded and peak areas of standards and tests were Group I: control (received saline only),
noted on each run and used for calculation of Group II: carrageenan alone

Journal of Research in Biology (2013) 3(3): 886-894 888


Renju and Kurup., 2013

Group III: carrageenan + algal lycopene (AL groups, carrageenan-induced paw edema. Algal lycopene showed
10 mg/kg i.p) maximum edema inhibition compared to all-trans tomato
Group IV: carrageenan + tomato lycopene (TL groups, lycopene and drug. AL exhibited 70% and 83% edema
10 mg/kg i.p) inhibition at third/fifth hours, respectively. This effect
Group V: carrageenan + Voveran (VOV groups, was comparable to the reference drug Voveran which
20 mg/kg i.p.) exerted 54% and 71% edema inhibition at third and fifth
At the end of third hour, the animals were hour, respectively. TL showed 51% and 63% edema
sacrificed by euthanasia. Blood was removed to ice cold inhibition at third and fifth hour after carrageenan
containers for various biochemical analyses. induction (Figure. 1).
Activity of Cyclooxygenase (COX) and COX activity in PBMC was significantly
Lipooxygenase (LOX) in Peripheral Blood (p<0.05) increased in carrageenan treated rats when
Mononuclear Cells (PBMC) compared to control rats (Figure. 2). Treatment with AL
Mononuclear cells were isolated the procedure showed significant (p<0.05) decrease in COX activity
described by Radhika et al., (2007). Cox activity was when compared to carrageenan induced rats.
measured by the method of Shimizu et al., (1984). Prostaglandin is formed by the interaction of two distinct
15-LOX activity was determined by the method of but related enzymes, COX-1 and COX-2 and plays an
Axelrod et al., (1981). important role in promoting the signs and symptoms
Biochemical analysis of inflammation (Otterness and Bliven, 1985;
Serum myeloperoxidase (MPO) activity was Ibegbulem et al., 2012). The activity of COX in PBMC
measured by Mullane et al., (1985). CRP in plasma was was decreased (p<0.05) in AL treated group when
determined by using Immunoturbidometric kit (Diasys compared to TL and voveran treated group. Reduction of
Diagnostics, Germany). Ceruloplasmin was estimated by paw swelling and decreased activity of COX showed the
the method of Ravin (1961). Protein was determined by immunological protection rendered by the algal
the methods of Lowry et al., (1951). lycopene. These results showed the anti-inflammatory
Statistical analysis potential of the AL.
The Statistical package for social sciences The activity of 5-LOX and 15-LOX in PBMC
(SPSS/PC+), version 11.5 (SPSS Inc; Chicago. IL, USA) was significantly (p<0.05) increased in carrageenan
was used to analyze the results for statistical significance induced rats when compared to normal rats
using one-way ANOVA followed by Duncans test. (Figure.3 and 4). Algal lycopene treatment significantly
P value < 0.05 was considered as significant. reduced (p<0.05) in 5-LOX and 15-LOX activity, when
compared to CII rats. The effect was significantly higher
RESULTS AND DISCUSSION (p<0.05) than TL and drug treated groups.
Sub plantar injection of carrageenan into the foot Lipoxygenases are a family of key enzymes in the
of rats caused a time-dependent increase in paw volume. biosynthesis of leukotrienes that are postulated to play an
The localized inflammatory response as evidenced important role in the pathophysiology of several
visually by the edema reached a maximum intensity at inflammatory diseases (Henderson, 1994; Yamamoto,
third hour after carrageenan induction and this maximal 1992). In the normal situation, cellular leukotriene
effect was seen until the fifth hour. Administration of AL production is suppressed by selenium dependent
and TL has showed significant effects in decreasing peroxidases (Werz et al., 1997). On receiving
889 Journal of Research in Biology (2013) 3(3): 886-894
Renju and Kurup., 2013

inflammatory stimuli, leukotriene production is elicited Table 1 also shows the variations in serum CRP
through the arachidonic acid cascade, causing micro and ceruloplasmin level in the test animals compared to
vascular injury, vasoconstriction and production of control. Serum CRP and ceruloplasmin levels were
pro-inflammatory cytokines (Peskar, 1991). Studies have significantly increased (p<0.05) in carrageenan induced
shown that LOX and leukotrienes have a profound role rats when compared to normal rats. Supplementation
in carrageenan-induced inflammation (Henderson, 1994; with AL significantly decreased (p<0.05) the serum
Gamache et al., 1986). In the carrageenan-induced CRP and ceruloplasmin levels when compared to
inflammation model, AL significantly reduced carrageenan induced rats. The levels of CRP and
carrageenan-induced 5-LOX and 15-LOX activities in ceruloplasmin were decreased significantly (p<0.05),
mononuclear cells, indicating decreased leukotriene when compared to TL and Voveran treated groups.
production and hence a protective effect. C-reactive protein is an acute phase protein that has been
MPO activity in serum was significantly identified as an important biomarker for various
increased (p<0.05) in carrageenan induced rats when inflammatory, degenerative, and neoplastic diseases.
compared to normal group (Table 1). Treatment with AL Elevated levels of CRP have been found in the blood
showed significant decrease (p<0.05) in MPO activity during virtually all diseases associated with active
when compared to carrageenan induced rats. The MPO inflammation or tissue destruction, particularly in
activity was significantly decreased when compared to patients with rheumatoid arthritis (Pepys and Hirschfield,
TL and drug treated groups. The activity of MPO is a 2003; Kushner, 1991). In our study the increased levels
marker of neutrophil infiltration (Bradley, 1982), and
was found to be significantly increased in the paw tissue
of carrageenan-induced rats. AL significantly decreased
(p<0.05) the elevated MPO activity, an indicator of
neutrophil in inflamed paws, suggesting that inhibition of
neutrophil infiltration might be another mechanism by
which AL achieves its anti-inflammatory effect.

Figure 2: Effect of algal lycopene on activity of COX in


PBMC of normal and experimental rats COX activity
is expressed as an optical density increase
(OD increase) per mg protein per minute. Val-
ues are expressed as mean SEM of six rats in each
group.
a
Statistical difference of Control group with CII
group when p < 0.05.
b
Statistical difference of CII group with group AL,
TL and VOV when p < 0.05.
c
Statistical difference of VOV group with group AL
and group TL when p < 0.05.
Figure 1: Effect of algal lycopene on carrageenan- d
Statistical difference of TL group with
induced paw edema in normal and experimental rats. AL when p <0.05.

Journal of Research in Biology (2013) 3(3): 886-894 890


Renju and Kurup., 2013

of CRP level was found to be significantly decreased in compared to TL and standard drug Voveran might be
algal lycopene treatment when compared to TL and having a protective response against free radical
Voveran treatments. mediated lipidperoxidation.
The serum protein, ceruloplasmin is a powerful Lycopene from edible marine microalgae
free radical scavenger that oxidizes iron from the ferrous C. marina showed higher anti-inflammatory activity than
to ferric state. Ceruloplasmin levels increase under all-trans tomato lycopene and standard drug Voveran.
conditions leading to the generation of oxygen products These effects might be due to the presence of two
such as the superoxide radical and hydrogen peroxides isomeric form of lycopene (cis and all-trans) in the
(Revnic, 1995). Serum ceruloplasmin level was microalgae. Reports available indicate that the
significantly increased in carrageenan induced rats when cis-lycopene has a high antioxidant potential when
compared to normal rats. Treatment with AL showed compared to all-trans lycopene (Stahl and Sies 1992;
significant decrease in the concentration of Clinton et al., 1996). Algal lycopene isolated from
ceruloplasmin. The increased levels of ceruloplasmin in C. marina could reduce cell influx, oedema formation
carrageenan induced rats could be decreased
significantly on treatment with algal lycopene when

Figure 3: Effect of algal lycopene on activity of


5- LOX in PBMC of normal and experimental Figure4: Effect of algal lycopene on activity of
rats 15- LOX in PBMC of normal and experimental
5-LOX activity is expressed as an optical density rats
increase (OD increase) per mg protein per min- 15-LOX activity is expressed as an optical den-
ute. Values are expressed as mean SEM of six sity increase (OD increase) per mg protein per
rats in each group. minute. Values are expressed as mean SEM of
a
Statistical difference of Control group with CII six rats in each group.
a
group when p < 0.05. Statistical difference of Control group with
b
Statistical difference of CII group with group CII group when p < 0.05.
b
AL, TL and VOV when p < 0.05. Statistical difference of CII group with group
c
Statistical difference of VOV group with group AL, TL and VOV when p < 0.05.
c
AL and group TL when p < 0.05. Statistical difference of VOV group with
d
Statistical difference of TL group with AL group AL and group TL when p < 0.05.
d
when p <0.05. Statistical difference of TL group with AL
when p <0.05.

891 Journal of Research in Biology (2013) 3(3): 886-894


Renju and Kurup., 2013

Table 1: Levels of CRP, Ceruloplasmin in plasma and Table 4: Statistical table of CRP in one way ANOVA
MPO in serum of experimental animals. followed by Duncans test

Groups MPO CRP Ceruloplasmin Ceruloplas- Sum of Mean


(m/min/mg) (mg/ml) (mg/dl) min Squares df Square F
Between
.196 4 .049 59.148
Control 5.85 0.37 22.0 1.24 0.10 0.006 Groups
Within
.021 25 .001
CII 20.52 1.11a 97.71 3.80a 0.34 0.014a Groups
Total .217 29
AL 7.45 0.37bc 45.94 2.01bc 0.19 0.007bc Where df is degrees of freedom, F is F- ratio.

TL 13.75 0.48bcd 80.64 3.18bcd 0.28 0.016bcd and release of mediators associated with inflammatory
condition, and therefore has the potential to be used as
VOV 9.74 0.39b 56.89 2.42b 0.22 0.010b
an anti-inflammatory agent. Further studies are in
Values are expressed as mean SEM of six rats in progress to evaluate the molecular mechanism of its
each group. anti-inflammatory activity.
a Statistical difference of Control group with CII
group when p < 0.05.
b Statistical difference of CII group with group AL, ACKNOWLEDGEMENT
TL and VOV when p < 0.05.
c Statistical difference of VOV group with group AL We express gratitude to Dr. Anantha Lekshmi,
and group TL when p < 0.05. Veterinary Doctor, Department of Biochemistry,
d Statistical difference of TL group with AL when
p <0.05. University of Kerala, Kariavattom, India for helping us
with the animal experiments.

Table 2: Statistical table of Myeloperoxidase in one


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and Ammon H. 1997. A test system for leukotriene Affordable Charges
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Journal of Research in Biology (2013) 3(3): 886-894 894


Journal of Research in Biology An International Scientific Research Journal

Original Research

Identification of Animal Pasteurellosis by PCR Assay


Journal of Research in Biology

Authors: ABSTRACT:
Venkatesan PS,
Deecaraman M and Diagnosis of pasteurellosis has become difficult, as there are five different
Vijayalakshmi M. capsular types and 16 somatic types. Molecular techniques like PCR are adapted
nowadays for rapid and accurate diagnosis in early stage of the disease and also it
provides useful information for epidemiological studies. The present study was
conducted to study the efficiency of polymerase chain reaction (PCR) in the
identification of P. multocida isolates and evaluation of different PCR methods viz.,
Institution:
Department of IBT, (i) PCR using genomic DNA (ii) PCR using culture lysate and (iii) PCR by colony touch
Dr. M.G.R. Educational & method. In the present study P. multocida specific PCR was performed by using
Research Institute, KMT1SP6 and KMT1T7 oligos. These oligos amplified the genomic DNA from
Department of IBT, P. multocida isolates only. All the three methods produced PCR amplified product at
Maduravoyal, 460 bp and colony touch method was found to be the best method.
Chennai - 600095.

Corresponding author: Keywords:


Venkatesan PS. Culture lysate, genomic DNA, Pasteurella multocida, PCR .

Email: Article Citation:


venkyvet74@gmail.com Venkatesan PS, Deecaraman M and Vijayalakshmi.
Identification of animal Pasteurellosis by PCR assay.
Journal of Research in Biology (2013) 3(3): 895-899

Web Address: Dates:


http://jresearchbiology.com/ Received: 04 Feb 2013 Accepted: 05 Mar 2013 Published: 30 Apr 2013
documents/RA0332.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.

895-899 | JRB | 2013 | Vol 3 | No 3


Journal of Research in Biology
An International Scientific
Research Journal www.jresearchbiology.com
Venkatesan et al., 2013

INTRODUCTION incubated at 37C with 5-10 % CO2 for 24-48 h. Plates


The various forms of pasteurellosis caused by were examined for colonies, the suspected colonies were
Pasteurella multocida are the major health problem for subjected to grams staining, and biochemical test as
livestock population worldwide. Diagnosis of per standard techniques. Standard vaccine strain of
pasteurellosis has become difficult, as there are five P. multocida P52 (B:2) was taken as reference strain.
different capsular types and 16 somatic types. Molecular Pathogenicity test in mice were carried out for all the
techniques like PCR are adapted nowadays for rapid and fifteen isolates and PCR was performed for all the
accurate diagnosis in early stage of the disease and also it isolates.
provides useful information for epidemiological studies. Isolation and Purification of Genomic DNA
Pasteurellosis has high impact on economic status of A 900 l cell suspension of each sample were
Indian farmers. The overall incidence rate of resuspended in 100 l of 10x Tris-EDTA (TE) buffer
haemorrhagic septicaemia (HS) was reported as (pH 8.3) with 10 mg of lysozyme and were incubated at
6.4 per lakh population during 1974-86, resulting in 37C for 1.5 h. Bacterial cultures were treated with 10 l
losses exceeding ten million rupees annually of proteinase K (10 mg/ml) and incubated at 50C
(Dutta et al., 1990; singh et al., 1996). for 1 h. The nucleic acid was extracted with
Isolation and identification of P. multocida from phenol-chloroform-isoamyl alcohol followed by ethanol
specimens like fresh tissues or heart blood followed by precipitation as per the method of Sambrook et al.,
the performance of various biochemical and serological (1989) and Sachithanandam et al., (2011).
methods have been used to study P. multocida. These PCR Using Culture Lysate
include catalase, indole, oxidase and sugar fermentation One Milliliter of 18 h broth culture or take few
tests. Due to time consuming procedure and limitations freshly grown pure colonies from blood agar plate and
of these methods, molecular techniques like polymerase suspend in 500 l sterile distilled water and centrifuge at
chain reaction (PCR) were adapted nowadays. PCR has 4000 g for 1 minute and collect the pellet. The pellet was
advantages over the conventional techniques in rapidity, washed with sterile distilled water, resuspended in 100 l
sensitivity and specificity to identify the P. multocida. sterile distilled water and boiled for 10 min. The samples
The present study was conducted to assess the efficiency were centrifuged to sediment cell debris and 10 l of the
of PCR in the identification of P. multocida from poultry supernatant was used in the PCR reaction.
and ruminants and to evaluate the different methods in PCR Using Colony Touch Method
PCR assay viz. PCR using genomic DNA, PCR using A single pure colony grown on agar plates was
culture lysate and PCR by colony touch method. used to perform PCR. A pipette tip was lightly touched
onto a colony and then suspend in PCR amplification
MATERIALS AND METHODS mixture.
Isolation and Identification of P. multocida PCR Technique
Fifty two samples were collected from various The species-specific primers KMT1SP6 and
geographical areas of Tamil Nadu, India. Specimens KMT1T7 designed by Townsend et al., (1998) were used
such as heart blood swab, liver, spleen and long bones in this study to amplify the gene sequences in
collected from various animals, were streaked directly P. multocida.
onto 5% sheep blood agar and Pasteurella multocida Primers 1 KMT1SP6 5-GCT GTA AAC GAA CTC
selective agar as reported earlier (Moore et al., 1974) and GCC AC- 3
896 Journal of Research in Biology (2013) 3(3): 895-899
Venkatesan et al., 2013

Primers 2 KMT1T7 5- ATC CGC TAT TTA CCC AGT Finland).


GG-3 The biochemical tests were carried out as per the
PCR mixture was prepared using PCR kit standard procedure followed in Arun kumar et al., (2012)
obtained from FINNZYME, Finland. The 50 l of
reaction mixture was prepared with 10 l template DNA, RESULTS
10ng of each primers, 200 M concentration of each Out of total collection of 52 suspected samples,
dNTPs, 10x PCR buffer and 1 unit Taq DNA procured from cattle sheep, goat and poultry, 15 samples
polymerase. PCR amplification was carried out in an were confirmed as P. multocida based on biochemical
automated thermal cycler (Perkin Elmer Gene AMP PCR tests (Table 1) and PCR. All the P. multocida isolates
system 2400) with the following thermal programme. were pathogenic to mice and dies within 24 h. PCR was
Initial denaturising at 95C for 4 min followed by performed for all the 15 isolates by 3 methods viz.,
30 cycles of denaturising at 95C for 1 min., annealing at colony touch method, culture lysate and with genomic
55C for 1 min., extension at 72C for 1 min. and final DNA. P52 strain of P. multocida, obtained from the
extension at 72C for 9 min, were carried out. After Institute of veterinary preventive medicine (IVPM)
amplification, PCR products were checked in Ranipet, Tamil Nadu, taken as a positive control
1.5% agarose gel electrophoresis along with the standard and the following bacteria Escherichia coli,
molecular weight marker (Lambda DNA Hind III digest Clostridium chauvoei, Salmonella enteritidis,
and X 174 DNA Hae III digest; FINNZYME, Salmonella typhimurium, Bacillus anthracis,
Table 1. Biochemical Profiles for the Identification of Pasteurella multocida Isolates

Tests Name of the Isolates


D1P D2P FP GP HP KP LP NP OP AS CS TS YS BG MC
Hemolysis on Blood
- - - - - - - - - - - - - - -
agar
Growth on MacConkey
- - - - - - - - - - - - - - -
agar
Motility - - - - - - - - - - - - - - -
Gelatin Liquefaction - - - - - - - - - - - - - - -
Methyl Red Test - - - - - - - - - - - - - - -
H2S (Hydrogen
+ + - - + + + - + + + + + + -
sulphide)
Catalase + + + + + + + + + + + + + + +
Oxidase + + + + + + + + + + + + + + +
Nitrate Reduction + + + + + + + + + + + + + + +
Indole + + + + + + + + + + + + + + +
Lysine Decarboxylase - - - - - - - - - - - - - - -
Ornithine
- + + + + + - + + + + + + + +
Decarboxylase
Urease - - - - - - - - - - - - - - -
Pyrase - - - - - - - - - - - - - - -
Esculin Hydrolysis - - - - - - - - - - - - - - -
VT (Voges Proskaeur - - - - - - - - - - - - - - -
Phenylalanine - - - - - - - - - - - - - - -
-Galactosidase
- - - - - - - - - - - - - - -
(ONPG)
-Glucuronidase + + + + + + + + + + + + + + +
-Galactosidase + + + + + + + + + + + + + + +
-Xylosidase - - - - - - - - - - - - - - -
N-acetyl
- - + + + + - + - - + + + + +
-D-glucosaminedase
+ : Positive, - : Negative,

Journal of Research in Biology (2013) 3(3): 895-899 897


Venkatesan et al., 2013

D1P D 2P FP GP HP PC NC M KP LP NP OP AS PC NC M

460bp

CS TS YS BG MC PC NC M

460bp

Figure 1: Pasteurella multocida specific PCR (PM-PCR) assay


These figures illustrate fragments specifically amplified by PCR in all the P. multocida isolates by means of the
primers KMT1SP6 and KMT1T7. Variation in the intensity of the amplified product was observed, due to variation in
DNA concentration of each sample.
D1P, D2P, FP, GP, HP, KP, LP, NP, OP, AS, CS, TS, YS, BG, and MC are the names of P. multocida isolates.

Staphylococcus aureus and Klebsiella spp. used as reference strain P52, but no amplified product was
negative controls. The expected amplification size of noticed among the negative controls. It is concluded that
460 bp was obtained in all the 15 isolates. PCR the primers were highly specific to P. multocida isolated
amplification was noticed at approximately 460 bp by all from various sources. The above result agrees with the
the three methods and in all the 15 isolates as like that of previous reports of earlier workers (Townsend et al.,
positive control (figure 1). No amplification product was 1998; Hunt et al., 2000; Miflin and Blackall, 2000; OIE
observed in negative controls (figure 1). Molecular manual, 2000; Dutta et al., 2001). In this study the
weight of PCR product was estimated based on the amplified product of approximately 460 bp was observed
standard molecular weight marker. using three different methods viz. colony touch method,
culture lysate method and purified genomic DNA
DISCUSSION method (figure 1). The intensity of the amplified PCR
The 15 isolates of P.multocida collected from product varies (figure 1), due to the variation in DNA
different places and sources of origin produced concentrations. Townsend et al., (1998) reported that
approximately 460 bp amplified product as that of PCR using colony touch method produced amplification
898 Journal of Research in Biology (2013) 3(3): 895-899
Venkatesan et al., 2013
product approximately at 460 bp and the intensity of the occurrence of haemorrhagic septicaemia in India. Indian
amplified product varied due to inconsistency of the Veterinary Journal 67(10): 893-899.
DNA concentration. Dabo et al., (2000) reported that the Dutta TK, singh VP and Kumar AA. 2001. Rapid and
boiled cell extract method has the advantages of Specific diagnosis of animal pasteurellosis by using PCR
simplicity and rapidity in the identification of assay. Indian Journal of Comparative Microbiology,
Immunology and Infectious Diseases 22(1):43-46
P. multocida isolates. Since the PCR amplified product
of 460 bp was noticed in all samples of poultry and Hunt ML, Alder B and Townsend KM. 2000. The
molecular biology of Pasteurella multocida. Veterinary
ruminants, using oligos KMT1SP6 and KMT1T7, the
Microbiology 72(1-2):3-5.
oligos are considered as specific to P. multocida
affecting all species of poultry and ruminants. Manual of Standards for diagnostics tests and
vaccines. 2000. Office International Des Epizootics
Considering the cost and time involved in the preparation
Manual, France. 446-456.
and purification of genomic DNA, the colony touch
Sachithanandam V, Mohan PM, Dhivya P,
method has advantages of simplicity and rapidity for
Muruganandam N, Baskaran R, Chaaithanya IK and
epidemiological surveys involving large number of
Vijayachari P. 2011. DNA barcoding, phylogenetic
P. multocida isolates. PCR using colony touch method relationships and speciation of Genus: Plectropomus in
would be an adaptable easy to perform method in Andaman coast. Journal of research in Biology. 1( 3):
regional laboratories for rapid diagnosis of HS and FC 179-183.
from field cases without the need to obtain pure culture Sambrook J, Fritisch EF and Mamiatis T. 1989.
and extensive biochemical and serological tests. Molecular cloning: a laboratory manual, Cold spring
harbor press, Plainview, N.Y. 2nd ed. 3.

ACKNOWLEDGEMENT Singh VP, Kumar AA, srivastava SK and Rathore


The authors thank the Head, department of BS. 1996. Significance of Haemorrhagic Septicaemia in
Asia: India. International workshop on diagnosis and
microbiology, Madras Veterinary College, Chennai, for
control of Haemorrhagic Septicaemia. Bali, Indonasia.
providing the facilities to carry out this work.
May 28-30.

Townsend KM, Frost AJ, Lee CW, Papadimitrion


REFERENCE
JM and Dawkins HJS. 1998. Development of PCR
Arun Kumar JM, Lakshmi A, Sangeetha Rani V and assays for species and type specific identification of
Sailaja B. 2012. Isolation and characterization of feather Pasteurella mutlocida isolates. Journal of Clinical
degrading bacteria from poultry waste. Journal of Microbiolgy 36(4):1096- 1100.
Research in Biology. 2(7): 676-682.

Blackall PJ and Miflin JK. 2000. Identification and Submit your articles online at www.jresearchbiology.com
typing of Pasteuruella multocida; A Review. Avian
Advantages
Pathology 29(4):271-287. Easy online submission
Complete Peer review
Dabo SM, Confer A and Lu YS. 2000. Single primer
Affordable Charges
polymerase chain reaction fingerprinting for Quick processing
Pasteurella multocida isolates from laboratory rabbits. Extensive indexing
American Journal of Veterinary Research 61(3):305-309. You retain your copyright

Dutta J, Rathore BS, Mullick SG, Singh R and submit@jresearchbiology.com

Sharma GC. 1990. Epidemiological studies on www.jresearchbiology.com/Submit.php.

Journal of Research in Biology (2013) 3(3): 895-899 899


Journal of Research in Biology An International Scientific Research Journal

Original Research

Effect of Chromolaena odorata leaf extract on haematological profiles in


Salmonellae typhi infested Wistar rats
Journal of Research in Biology

Authors: ABSTRACT:
Nwankpa P1, Ezekwe AS1, Haematological indices provide an insight about the internal environment of
Ibegbulem CO3 and a given organism. In this present study, the possible anti-haemototxic effect of
Egwurugwu JN2. Chromolaena odorata on Salmonellae typhi induced haematotoxicity in rats were
investigated. The experimental animals were divided into three groups. Group A
received only food and water (control). Group B and C received in addition to food and
water, single dose of stock Salmonellae typhi at a dose of 106cfu/ml. The animals in
group B and C were allowed to be infected with Salmonellae typhi for 7 days and
Institution:
1. Department of Medical confirmed by widal test, after which group C was treated with 750mg/kg body weight/
Biochemistry Imo State day ethanolic extract of Chromolaena odorata for 10 days. The result showed a
University, Owerri, Nigeria significant (p < 0.05) decrease in Red Blood Cells (RBC) count, packed cell volume
(PCV), haemoglobin (Hb), mean corpuscular haemoglobin (MCH), Mean Corpuscular
2. Department of haemoglobin Concentration (MCHC), neutrophil and increase in platelet, total White
Physiology, Imo State Blood Cell (WBC) and lymphocytes in animals infected with Salmonellae typhi when
University, Owerri, Nigeria compared to the control non-infected group. Treatment of animals in group C with
ethanolic extract of Chromolaena odorata showed a significant (P < 0.05) increase in
3. Department of mean values of RBC count, PCV, Hb, MCH, MCV, MCHC and decrease in platelets, WBC
Biochemistry, Federal and lymphocytes when compared to the group infested with Salmonellae typhi only.
University of Technology The results above suggest the anti-haematotoxic potential of ethanolic extract of
Owerri, Nigeria. Chromolaena odorata in Salmonellae typhi infested rats.

Corresponding author: Keywords:


Promise Nwankpa Salmonellae typhi, Chromolaena odorata, Blood cells, Anti-haematotoxic,
Rats.

Article Citation:
Web Address:
http://jresearchbiology.com/ Nwankpa P, Ezekwe AS, Ibegbulem CO and Egwurugwu JN.
documents/RA0337.pdf. Effect of Chromolaena odorata leaf extract on haematological profiles in
Salmonellae typhi infested Wistar rats.
Journal of Research in Biology (2013) 3(3): 932-939

Dates:
Received: 15 Feb 2013 Accepted: 05 Mar 2013 Published: 11 May 2013

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.

932-939 | JRB | 2013 | Vol 3 | No 3


Journal of Research in Biology
An International
Scientific Research Journal www.jresearchbiology.com
Nwankpa et al., 2013

INTRODUCTION of animals may be influenced adversely by diabetic


Enteric fever, also called typhoid fever caused by condition (Edet et al., 2011), phenylhydrazine (Sanni
the bacterium Salmonellae typhi, is an acute life et al., 2005), some anti-retroviral drugs (Kayode et al.,
threatening febrile ailment (Kotton, 2007). Typhoid fever 2011) and aqueous leaf extract of Ocimum gratissimum
is distributed worldwide and prevalent throughout the (Obianime et al., 2011).
tropics where it is the commonest cause of fever Chromolaena odorata (known as siam weed,
(Wilcocks and Manson-Bahr, 1972). Literature reports independent weed, killer weed) is a perennial shrub
have shown that two million cases of typhoid and 200 which grow in rainforest, grassland and arid bushvelds
thousand related deaths occur worldwide each year (Timbilla and Braimah, 2002). The leaves of the plant
(Steinberg et al., 2004). One challenge of development in has been reported to be widely used as herbal remedy for
developing countries, is the provision of portable water the treatment of various ailments. Available reports have
for the populace as poor sanitary condition and hygiene shown a decotion of the leaf extract effective in the
has been reported to increase the prevalence of treatment of malaria and cough (Suksamran et al., 2004).
Salmonellae typhi infection with reduced incidence in Akah (1990) has reported the haemostatic and
developed countries (Kotton, 2007). Available reports anti-inflammatory property of the leaf extract while
indicate that typhoid infection is the leading cause of Thang et al., (2005) has shown the stimulation of
morbidity and mortality in a developing country like granular tissue and re-epithelization of the epithelial
Nigeria where water carriage method of sewage disposal tissue during wound healing. Recently Nwankpa et al.,
is inefficient (Crump et al., 2004). Salmonellae typhi (2012) reported the antioxidative effect of ethanolic leaf
infection causes gastroenteritis which symptoms include extract of Chromolaena odorata in rats. Other medicinal
nausea, vomiting and diarrhea (Parry et al., 2002). The uses including anti-hypertensive, anti-diarrhoeal and
affected organs include spleen, liver and other tissues diuretic has been reported (Iwu, 1993).
which habor the bacterium before entering the blood In rural communities in Nigeria, the use of
(Jones and Falkow, 1996). During metabolism, bacterial Chromolaena odorata for treating Salmonellae typhi
cells, release chemical toxins which interactions damage infection is common but the effect of the plant on
the tissue of the host organism. This tends to disrupt the haematological indices in typhoid fever is not known.
blood components or blood forming tissues. This study was therefore designed to assess the effect of
Blood is one of the specialized body fluid Chromolaena odorata on haematological profiles in
responsible for the transportation of nutrients, oxygen, Salmonellae typhi infested rats.
hormones and other metabolites to the bodys cell and
metabolic waste products away from those cells to sites MATERIALS AND METHODS
of elimination. It is known to be the most important body Plant Material: The Chromolaena odorata leaves were
fluid that regulates various vital functions of the body collected from a natural habitat in Owerri and
such as excretion, respiration, circulation, osmotic and authenticated by professor S.C. Okeke, a taxonomist at
temperature balance etc. Mammalian circulation of blood the department of Plant Science and Biotechnology, Imo
transports specific nutrients, gases, metabolic products State University Owerri, Nigeria. The voucher specimen
and hormones between different tissues and organs was kept in the university herbarium for references.
(Baynes and Dominiczak, 2005). Literature reports Preparation of Extract: Large quantities of fresh leaves
indicated that haematological profiles of different species of Chromolaena odorata, washed free of sand and
933 Journal of Research in Biology (2013) 3(3): 932-939
Nwankpa et al., 2013

debris, were dried under shade at room temperature at with Salmonellae typhi and serve to monitor successful
27C for 3 weeks. Electric blender was used to induction of typhoid.
homogenize the dried leaves to a powder form. A 700g Group B: The rats in this group served as control. They
of the powder macerated in 1.1 litres of 80% (v/v) were fed with rat chow and had free access to water.
ethanol were allowed to stand for 24 hours. A chess clot Single dose of Salmonellae typhi at106cfu/ml was orally
was used to filter the mixture and the filtrate administered to rats in this group but were not treated
concentrated in vacuo at 37-40C to 10% its original with the plant extract.
volume using a rotary evaporator. The concentrate was Group C: The rats in this group were fed with
evaporated in a water bath at 40C to a solid residue, the rat chow and had access to water. Single dose
extract. The extract was dissolved in 100ml of 10% of Salmonellae tysphi at 106cfu/ml were orally
ethanol to an approximate concentration used for the administered to the rats in this group. After 7 days
experiment. of infection, 750 mg/kg ethanolic leaf extract of
Salmonellae typhi: The stock Salmonellae typhi was Chromolaena odorata were orally administered to the
procured from Federal College of Veterinary and animals daily for 10 days.
Medical Laboratory Technology of the National Collection and preparation of blood samples for
Veterinary Research Institute Vom, Jos, Plateau State, analysis
Nigeria. Nutrient agar plate, cesteine lactose electrolyte At the end of the treatment, the animals were
deficient plate (DCA) was used to sub-culture the micro- fasted for 24 hours, re-weighed and sacrificed under
organism which was incubated at 37C for 24 hours and chloroform anesthesia. By cardiac puncture, blood
examined for growth. The stock sample used for the sample was collected from each animal with a sterile
experiment was prepared as culture slants using syringe and needle, in EDTA anti coagulated bottle. The
McCartney bottle and nutrient agar. Salmonellae typhi anti-coagulated blood samples were used for
from the sub-cultured medium was aseptically incubated haematological analyses which were carried out within
for 18 hours at 37C. 24 hours of sample collection.
Animals: Albino Wistar rats of both sexes weighing Haematological analysis
between 150-200g were obtained from the animal house Full blood counts such as packed cell volume
of Faculty of Medicine, Imo State University Owerri, (PCV), Haemoglobin (Hb), Red Blood Cell (RBC), Total
Nigeria. They were maintained at room temperature and White Blood Cells (TWBC), Platelet count, differential
acclimatized for 12 days to daily handling. They were white blood cell (like lymphocytes, monocytes,
fed ad-libitum with commercial rat chow (Product of eosinophils, neutrophils) and red cell indices including
Pfizer Nigeria Ltd) and had free access to water. Mean Corpuscular Haemoglobin (MCH), Mean
Induction of typhoid: Each rat was orally administered Corpuscular Volume (MCV), Mean Cell Haemoglobin
6
with 1ml of Salmonellae typhi at a dose of 10 cfu/ml to Concentration (MCHC) were estimated using the
induce typhoid (Kirby, 1960). Sysmex Automated Haematology Analyzer KX-2IN,
Experimental design: Twenty - four albino Wistar rats Sysmex Corporation, Kobe, Japan.
were used for the study. They were randomly assigned Statistical analysis
into 3 groups. Each group has 8 rats. Data generated were statistically analysed by
Group A: The rats in this group were fed with rat chow one-way analysis of variance (ANOVA) of the SPSS
and had free access to water. They were not administered statistical programme of Microsoft Excel. Values were

Journal of Research in Biology (2013) 3(3): 932-939 934


Nwankpa et al., 2013

declared significantly different at p<0.05. Salmonellae typhi infested group (Table 2). However the
results of this study showed no significant (P > 0.05)
RESULTS AND DISCUSSION difference in RBC, Hb, PCV, MCV, MCH, MCHC,
Table 1 and 2 shows the effect of platelets, WBC, and lymphocytes in Salmonellae typhi
Salmonellae typhi infection and subsequent treatment infested rats treated with Chromolaena odorata
with ethanolic leaf extract of Chromolaena odorata on compared to the non-infested rats (Table 1 and 2).
haematological parameters in rats. The results showed a Haematological indices provide relevant
significant (P < 0.05) decrease in Red Blood Cells (RBC) information regarding the internal milieu of an organism.
count, haemoglobin (Hb), Packed Cell Volume (PCV), Nutritional, environmental and microbial infection are
Mean Corpuscular Haemoglobin (MCH), Mean among several other factors which have been reported to
Corpuscular Volume (MCV), Mean Corpuscular have adverse effects on the haematological profiles of
Haemoglobin Concentration (MCHC) and percentage most organisms. Vitamin B12 and folic acid deficiency
nuetrophil levels in Salmonellae typhi infested rats (Jee et al., 2005, Murray et al., 2007) and exposure to
compared to the non-infested group (Table 1 and 2). environmental pollutants such as carbondisulphide,
On the contrary, the total White Blood Cell (WBC), insecticide, hexane, gasoline vapour, nitrocellulose
platelets and lymphocyte levels in rats infested with thinner has been reported (Dhembara and Pandhe, 2000;
Salmonellae typhi showed a significant (P < 0.05) Uboh et al., 2007; 2009; 2012 and Savithri et al., 2010).
increase compared to the non-infested group (Table 2). Bacterial infection in living cells release toxins which
Treatment of the rats in group C with ethanolic leaf metabolism results to increase in release of free radical
extract of Chromolaena odorata showed a significant species with attendant damage to the cells (Stipanuk,
(P < 0.05) increase in RBC count, Hb, PCV, MCH, 2000). In this study, Salmonellae typhi infection
MCV, MCHC and percentage neutrophil levels significantly decreases the level of RBC, PCV, Hb,
compared to the Salmonellae typhi infested non-treated MCH, MCV, MCHC, neutrophils and increases the level
group (Table 1 and 2) while treatment of rats in group C of WBC and lymphocytes. The observation made in this
with ethanolic leaf extract of Chromolaena odorata study agrees with the report of Wilcocks and Manson-
showed a significant (P < 0.05) decrease in platelets, Bahr (1972) in Salmonellae typhi infection and Kumar
WBC and lymphocyte levels compared to the non-treated and Kuttan (2005) on cyclophosphamide induced

Table 1: Effect of Chromolaena odorata on mean values of red blood cells, packed cell volume, hemoglobin and
red cell indices in both experimental and control groups.
RBC Hb PCV MCV MCH MCHC
Group Treatment
X1012/L (g/dL) (%) (fL) (pg) (g/dL)
Negative
A 3.69 0.21 14.43 0.65 44.33 2.13 63.12 1.60 17.19 1.12 31.27 1.20
control/water
Salmonellae
B typhi (Positive 1.62 0.03a 10.09 0.71a 33.26 2.14a 54.85 1.55a 12.52 1.30a 24.12 1.23a
control)
Salmonellae
typhi +
C 3.49 0.05bc 14.15 0.79bc 43.40 2.34bc 61.95 1.32bc 16.55 1.02bc 30.12 1.33bc
Chromolaena
odorata
Mean SD (n = 8)
a
Significantly different compared with negative control (P < 0.05).
b
Significantly different compared with Salmonellae typhi (positive control) (P < 0.05).
c
No significant difference compared with negative control (P > 0.05).
935 Journal of Research in Biology (2013) 3(3): 932-939
Nwankpa et al., 2013

Table 2: Effect of CO on mean values of platelets, total white blood cells and differential cell counts in both
experimental and control groups
Platelets TWBC Lymphocytes Neutrophils Eosinophils Monocytes
Group Treatment
X103L-1 X103L-1 (%) (%) (%) (%)
Negative control/
A 855.18 2.11 16.24 0.78 70.11 2.01 20.19 1.15 1.98 0.6 2.51 0.11
water
Salmonellae typhi
B 880.13 1.5a 25.85 1.16a 82.14 2.11a 11.56 0.87a 3.20 1.10 2.90 0.55
(Positive control)
Salmonellae typhi
C + Chromolaena 858.82 1.46bc 17.14 1.21bc 72.18 1.88bc 19.26 1.11bc 2.10 0.80 2.6 0.52
odorata
Mean SD (n = 8)
a
Significantly different compared with negative control (P < 0.05).
b
Significantly different compared with Salmonellae typhi (positive control) (P < 0.05).
c
No significant difference compared with negative control (P > 0.05).

toxicity. The haematotoxic effect of Salmonellae typhi with Salmonellae typhi.


infection may be explained by the interaction of the Ethanolic extract of Chromolaena odorata
bacteria or its toxins with the blood forming tissues/ significantly increased the level of RBC, Hb, PCV,
organs which may inhibit the rate at which some specific MCV, MCH and MCHC thereby reducing and
or generalized haemopoeitic committed stem cells are ameliorating the anaemic condition induced by
synthesized by the tissues. Some reports have shown that Salmonellae typhi infection. The observed increase in
hexane, cyclophosphamide and benzene induced RBC, Hb, and PCV may be explained by the role of
haematotoxic effect is associated with the interaction of Chromolaena odorata extract in reversing bone marrow
their metabolites with the haematopoeitic tissues and depression with attendant improvement in erythrocyte
cause depression in their haematopoeitic activities membrane stability through the antioxidant potential of
(Synder and Hedli, 1996; Kumar and Kuttan, 2005). the plant extract, thus reducing haemolysis (Krause and
Increase in total white blood cells and lymphocytes as Mahan, 1984; Naaz et al., 2007, Nwankpa et al., 2012).
well as decrease in neutrophils seen in this study is The improvement on the haematopoetic activities of the
consistent with the reports on effect of insecticides and tissues and/or maintenance of red blood cell membrane
pesticides such as fenvalerate, lindane, aldrin among integrity relieves the anaemic condition observed in
others, on total white blood cells and the differential Salmonellae typhi infection.
counts in experimental animals (Synder and Hedli, 1996; Consequently, increase in RBC count on
Kumar et al., 1996; Savithri et al., 2010). This may be administration of Chromolaena odorata leaf extract
explained by increased lymphopoeisis and/or enhanced translates to an increase in MCV while increase in Hb
release of lymphocytes from lymph myeloid tissue (Das translates, to an increase in MCH and MCHC.
and Mukherjee, 2003). This response may be a direct Furthermore, inhibition of microbial growth by the plant
stimulatory effect of toxic substance on lymphoid tissue/ extract has been reported. Okigbo and Ajalie (2005) and
pollutant induces tissue damage and disturbance of the Alisi et al., (2011) showed that Chromolaena odorata
non-specific immune system leading to increase in leaf extract possess antibacterial activity which inhibit
production of leukocytes. Neutrophils are known to be the growth of Salmonellae typhi in cells. Decrease in
involved in the phagocytosis of foreign substances in the total white blood cell, lymphocytes and attendant
body during which some of them are ruptured. This may increase in neutrophils on administration of the plant
explain the decrease in neutrophil count on infection extract may be explained by the inhibition of growth of

Journal of Research in Biology (2013) 3(3): 932-939 936


Nwankpa et al., 2013

Salmonellae typhi in the cell. The inhibition of growth of insecticides. J. Exp. Zool., India. 3:41-44.
the microorganism lead to the destruction of excess
Edet EE, Akpanabiatu MI, Uboh FE, Edet TE, Eno
WBC and lymphyocytes released by the cell in response
AE, Itam EH and Umoh B. 2011. Gongronema
to bacterial infection (Nancy et al., 2005). Conversely,
latifolium crude leaf extract reverses alterations in
increase in neutrophil count on administration of the
haemotological indices and weight-loss in diabetic rats.
plant extract may be explained by reduced phagocytosis
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Journal of Research in Biology An International Scientific Research Journal

Original Research

Source of light emission in a luminous mycelium of the fungus


Panellus stipticus
Journal of Research in Biology

Authors: ABSTRACT:
Puzyr Alexey,
Burov Andrey and Mechanism of bioluminescence and light-emitting sources in higher fungi
Bondar Vladimir. remain as an open question for a long time. We investigated the mycelium of
cultivated luminous Panellus stipticus using confocal microscopy. No excitation light
was imposed on the sample. Two types of sources of bioluminescence and their
Institution: location were determined in the substrate mycelium. One were small 0.1-3 m local
1. Institute of Biophysics SB
formations disposed on the surface of hyphae, the other - relatively vast areas in bulk
RAS, Krasnoyarsk.
of the nutrient medium. No luminescence signal was recorded inside the hyphae. This
2. Special Design- may mean that the components of luminescent reaction are spatially separated within
Technology Bureau "Nauka" the cells, or the intracellular conditions block the reaction. The origin and formation of
KSC SB RAS, Krasnoyarsk. the light-emitting structures are discussed.

3. Institute of Biophysics SB
Keywords:
RAS, Siberian Federal
University Krasnoyarsk. Bioluminescence, Panellus stipticus, luminous mycelium, confocal
microscopy.

Corresponding author: Article Citation:


Burov Andrey. Puzyr Alexey, Burov Andrey and Bondar Vladimir.
Source of light emission in a luminous mycelium of the fungus Panellus stipticus.
Journal of Research in Biology (2013) 3(3): 900-905
Email:
andrey@icm.krasn.ru
Dates:
Received: 02 Apr 2013 Accepted: 27 Apr 2013 Published: 06 May 2013
Web Address:
http://jresearchbiology.com/
documents/RA0345.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.

900-905 | JRB | 2013 | Vol 3 | No 3


Journal of Research in Biology
An International Scientific
Research Journal www.jresearchbiology.com
Puzyr et al., 2013

INTRODUCTION widely used to investigate non-luminous fungi


Bioluminescence in fungal cells, which involves (Riquelme and Bartnicki-Garcia, 2008; Roberson et al.,
the emission of light generated by a chemical reaction, 2011; Steinberg and Schuster, 2011).
has long attracted attention of scientists (Harvey, 1952; In this report the mycelium of luminous
Shimomura, 2006; Desjardin et al., 2008). Researchers Panellus stipticus was studied using confocal
studying bioluminescence of fungi focus their efforts on microscopy to determine and localize the source of light
three key areas: (i) methods of cultivation under emission. In our opinion it is important to find in
laboratory conditions and characteristics of the light luminous fungi structures (or formations), which are the
emission (Weitz et al., 2001; Prasher et al., 2012; Dao, light-emitting sources, and their location. On the one
2009; Mori et al., 2011), (ii) the molecular organization hand, this can provide additional knowledge about
of luminescence system and light emission mechanism morphology of luminous fungi, on the other - might give
(Shimomura, 2006; Airth and McElroy, 1959; insight into molecular-cellular organization of fungal
Kamzolkina et al., 1983; Oliveira and Stevani, 2009; luminescent system and mechanism of light emission.
Bondar et al., 2011), (iii) - application of fungal
luminescence in analytical techniques (Weitz et al., MATERIALS AND METHODS
2002; Mendes and Stevani, 2010). In this work we studied the culture of
There has been little research conducted to Panellus stipticus luminous fungus (Bull:Fr.) Karst.,
determine sources of luminescent light in the fungal IBSO 2301 (Figure 1). The mycelium was grown in
structures. To the best of our knowledge, only the plastic Petri dishes at temperature 22 on a commercial
mycelium of Panus stipticus and Armillaria fusipes, nutrient medium Potato Dextrose Agar (HiMedia
growing on agar were investigated for light source Laboratories Pvt., India), or on richer medium containing
detection (Berliner and Hovnanian, 1963). The used in 1 liter: 10 g of glucose, 5 g of peptone, 3 g of yeast
photographic process allowed to record light from a extract, 3 g of malt extract, 20 g of agar-agar. The
single hypha. specimens exhibiting the highest light intensity were
However, a low resolution of the technique selected for the experiments.
limited by the emulsion grain size denied localizing the For confocal microscopy, a confocal laser
source of light. The authors of this, obviously, pioneer scanning microscope (LSM-780 NLO, Carl Zeiss,
work, suggested that the light was emitted over the entire Gottingen, Germany) equipped with a high sensitivity
cell. Given the size of the objects under study, such GaAsP was used. Bioluminescence was recorded in the
research should employ methods of microscopic accumulation mode with the 491631 nm filter. The laser
investigations. Calleja and Reynolds, who studied was turned off (laser power = 0.0%) so that no excitation
Panus stipticus and Armillaria mellea by optical light was imposed on the sample. This was done to avoid
microscope with EMI 4-stage image intensifier tube, fungal autofluorescence - emission of light by biological
came to the conclusion that light emission in an substances such as flavins, lipofuscins and porphyrins
individual hypha was limited to a segment removed from when excited by ultraviolet, violet, or blue light (Zizka
the apical point (Calleja and Reynolds, 1970). Absence and Gabriel, 2008).
of later works related to structural and morphological Images were processed using ZEN 2010 software
studies of mycelium of luminous fungi with microscopy (version 6.0; Carl Zeiss). To prepare a specimen for
is astonishing as all known microscopic methods are microscopy a fragment of agar with mycelium was cut
901 Journal of Research in Biology (2013) 3(3): 900-905
Puzyr et al., 2013

Figure 1 View of culture of Panellus stipticus (IBSO 2301) growing on agar in natural light (A) and in the dark (B).

out and transferred to the cover glass. of hyphae could be assumed, finding of luminescent
areas in the agar came as a surprise. It is uncontroversial
RESULTS AND DISCUSSION that the recorded bioluminescent signals result from the
Figure 2 shows a 3D projection of the mycelium interaction of mixing light components synthesized by
by producing a Z-stack with 82 sections, 0.208 m thick the fungal cells. Luminescent signals were recorded by
each. No bioluminescence was detected from the aerial the confocal microscope only when these components
mycelium. The light emission was recorded from the were outside the cells. No bioluminescence inside
surface of specimen to a depth of ~ 16 m with hyphae may mean that inside the cells the components of
maximum intensity localized at the depth of Z= 6-8 m luminescent reaction are spatially separated and do not
where the main body of mycelium was located. Only interact with each other, or the intracellular conditions
isolated signals were detected at Z=8-16 m that (pH, oxygen concentration, presence of inhibitors, etc.)
confirmed that the agar did not contribute to the observed block the reaction.
bioluminescence. One could argue that the surface of glowing
Two types of sources emitting luminescent structures should be either hydrophobic or they have a
signals could be distinguished. One light source were membrane enclosing the internal volume. Only under
small 0.1-3 m local formations, associated with the these conditions components necessary for the
substrate hyphae, the other vast areas in bulk of agar luminescent reaction do not mix with the water phase
(Figure 3). Light intensity recorded in the agar was much contained within the nutrient medium. This suggestion is
higher than that of the local sites in the area of hyphae. based on the sharp boundaries exhibiting by both small
The use of the larger magnification (Figure 4) and bright local formations on the walls of hyphae and vast areas in
field microscopy (Figure 5a) suggests that the local the nutrient agar (Figure 5b).
luminous sites are cellular excretions located on the So far it is not clear whether the luminous
hyphae surface while vast luminescent areas are formed structures containing components necessary for the
by their aggregation in agar. emission are formed within the fungal hypha or on/in
While presence of luminous sites on the surface their surface. In the first case it requires a transport
system providing for the mechanism excreting the

Journal of Research in Biology (2013) 3(3): 900-905 902


Puzyr et al., 2013

Figure 2 Fragment of 3D pattern of bioluminescence produced by P. stipticus.

20m
5m

Figure 3 Confocal luminescence image of the Figure 4 Confocal luminescence image of an


P. stipticus mycelium. individual hyphae.

forming structures outside the cell. This is plausible are moved on the outside surface of the hyphae by
because the Golgi apparatus, that synthesizes secretory a mechanism analogous to the mechanism of transport
vesicles containing products of vital functions and via the Golgi complex. They can be also assumed to
excretes them from the cell, is well known. In the second form directly on/in outside surface of the hyphae by
case on/in the wall cell there should exist structural structural elements of the cell possessing secretory
elements performing specialized secretory function. function. Such enclosed structures make possible to
On the basis of the results above we hypothesize concentrate the necessary components within a small
the following. Cells of P. stipticus synthesize and volume. Separation of luminous structures from the
localize the components required for bioluminescence in surface of hyphae and their subsequent diffusion into the
structures which can originate within the cell and then bulk of the nutrient medium produce the vast
903 Journal of Research in Biology (2013) 3(3): 900-905
Puzyr et al., 2013

Figure 5 Confocal luminescence (A), bright field (B) and overlay (C) images of the substrate. Scale bar = 20 m.

areas of luminescence in the agar. Program of the Government of Russian Federation


Measures to Attract Leading Scientists to Russian
CONCLUSION Educational Institutions (grant No 11. G34.31.058); by
Confocal microscopy due to its high resolution the Program of SB RAS (project No 71).
and ability to record low light signals offers new
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ACKNOWLEDGEMENTS
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Shimomura O. Bioluminescence: chemical principles Submit your articles online at www.jresearchbiology.com


and methods. Singapore: World Scientific, 2006. Advantages
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Weitz HJ, Ballard AL, Campbell CD, Killham K. Complete Peer review
2001. The effect of culture conditions on the mycelial Affordable Charges
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growth and luminescence of naturally bioluminescent Extensive indexing
fungi. FEMS Microbiol Lett. 202(2):165-170. You retain your copyright

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