18

Chapter-II
KHAPRA BEETLE AND ESTIMATION OF LOSSES
2.1 INTRODUCTION

Khapra beetle Trogoderma granarium Everts and other beetles of the Dermestidae
family are considered to be the most destructive stored product pests. The pest had been
given status as an A2 quarantine organism for EPPO (OEPP/EPPO, 2007). Besides it is
considered as a pest of quarantine concern for CPPC, COSAVE, JUNAC, NAPPO and
OIRSA.

The continued occurrence of T. granarium on produce imported from countries where it

is native, and the potential for spread due to increasing trade and transportation of
cereals, made it a persistent threat to EPPO countries. The WTO committee on SPS had
also prohibited the importation of wheat, maize, rice, sorghum, chickpeas, beans,
sunflower seeds, sesamum seeds, soybeans, ground nuts, linseed and similar grains and
seeds and the flours and meals thereof in order to protect domestic production and to
prevent the introduction and spread of this notorious pest. This restriction is applicable to
products originating in or consigned from various countries including Austria, Serbia,
Spain, Switzerland; Afghanistan, Bangladesh, Chinese Taipei, Cyprus, India, Iran, Iraq,
Israel, Korea, Lebanon, Pakistan, Saudi Arabia, Sri Lanka, Syria, Turkey, Yemen,
Angola, Algeria, Egypt, Gambia, Libya, Mali, Mauritania, Morocco, Mozambique,
Niger, Nigeria, Senegal, Somalia, Sudan, Tunisia, Zimbabwe, Indonesia, Malaysia and
all other countries in which the Khapra beetle has been reported (EPPO, 1981).

The import restrictions are supported by the facts that feeding by Khapra beetle larvae
reduces the quality, grade and weight of grain. In India, average damage levels ranged
from 6-33 percent of grain in a single storage season, with maximum damage of 73
percent (Rahman et al., 1945). Loss of weight in wheat ranged from 2.2 to 5.5 percent.
Under optimal conditions of 36 °C, 15 percent infestation level caused 2.6 percent loss in
weight and 24 percent in viability of wheat grains in few months of storage (Prasad et al.,
1977). The weight loss of grain and cost of necessary treatment may result in less profit
for the wholesalers. Severe infestations of grain by Khapra beetle may make it
unpalatable or un-marketable. Grain quality may decrease due to depletion of specific
nutrients. Infestation levels of 75 percent in wheat, maize, and sorghum grains results in
 19

significant decreases in crude fat, total carbohydrates, sugars and true protein contents;
and increases in moisture, crude fiber and total protein contents (Jood and Kapoor, 1993;
Jood et al., 1993, 1996a). Besides, substantial loss of vitamins e.g. thiamin, riboflavin
and niacin occurs due to the infestation (Jood and Kapoor, 1994). Significant increases or
health hazardous non-protein nitrogen, total nitrogen and total protein have also been
recorded (Jood and Kapoor, 1993). Whereas level of uric acid exceeds the acceptable
limits due to T. granarium and other insect pest’s infestations. Total lipids,
phospholipids, galactolipids, and polar and non-polar lipids have also been reported to
decline significantly at infestation levels of 50 percent and 75 percent (Jood et al.,
1996b). On the other hand, antinutrient polyphenol and phytic acid have been reported to
increase significantly (Jood et al., 1995). Morison (1925) suggested that barbed hairs of
larvae that rub off and remain in the grain may present a serious health hazard if
swallowed. Cast skins may cause dermatitis in people handling heavily infested grains
(Pruthi and Singh, 1950).

Theses quality deteriorating characteristics of T. granarium have assigned the pest a

status of a noxious pest and a technical barrier to trade. Keeping the destructive nature
and its significance for the global food security and safety in view, the present
investigations were carried out with the following objectives.

Objectives:
1. Estimation of quantitative losses caused by the infestation of T. granarium larvae.
2. Evaluation of qualitative losses with special reference to crude protein, fiber and fat
contents.
3. Varietal preference of T. granarium toward different major varieties.
 20

Chapter-II
2.2 REVIEW OF LITERATURE

The Khapra beetle Trogoderma granarium Everts, particularly in its larval stage of
development, is the world's most destructive pest of stored grain and stored vegetable
products. Numerous research reports corresponding to extent of damage have proved that
T. granarium is one of the serious threats to food security of the countries, which are
already facing food shortages. Larvae of this pest feed and contaminate the food grains
through their cast skins, urine, excreta rendering the commodities unsuitable for human
consumption; ultimately causing critical food security and safety problems. In adverse
conditions, the pupae can survive in dormant phase for at least three years and so can
easily spread during foodstuff handling and transport. Work of some notable researchers
is summarized as under.

2.2.1 Quantitative Losses Caused by Khapra Beetle

The Khapra Beetle is one of the world’s most damaging pests of whole and ground
cereals, oilseeds, dry fruits, copra and other stored products. Its immense economic
significance is due to its potential to cause huge loss in stored grains through voracious
feeding and heating of grains, in larval ability to withstand starvation for up to 3 years as
well as in its ability to live on food with very low moisture content.

El-Lakwah et al., (1989) observed that T. granarium flourishes in unheated premises in

most countries of Africa, the Middle East and southern Asia. It normally occurs at places
where temperature is relatively high. That is why Khapra beetle is considered as the most
serious pests in hot and dry areas including Sahel region of Africa and parts of the Indian
sub-continent (Banks, 1978; Haines, 1981). Studies have further revealed that the pest is
extremely cold-hardy and tolerant of both high temperature and extremely low relative
humidity.

Rees (1998) reported that larvae as well as adults of T. granarium are similar in
appearance to T. variabile but T. granarium is better able to survive and breed on cereals
and other stored products. Devastating infestations can occur, especially in bagged grains
and seed stores with a loss ranging 2-70 %. The larval stages of the pest are more tolerant
to contact insecticides and fumigants. Pruthi and Singh (1950) maintained that Khapra
 21

beetle larvae typically attack the embryo point or a weak place in the pericarp of grain or
seed, but in case of higher infestation they feed other parts too. Young larvae feed on
damaged seeds, while the older larvae are capable to feed on whole grains. Howe (1952)
revealed that larvae wander in and out of sacked material, weakening the sacks, which
may ultimately tear resulting in spillage and spoilage of grains. Rahman et al., (1945)
observed that feeding by Khapra larvae reduce weight and grade of grain. They reported
severe damage caused by larval feeding to the tune of 6 to 33 percent in a single storage
season, with maximum damage of 73 percent. Hafiz and Hussain (1961) recorded 10-18
percent losses due to insects, rats and mold in Pakistan. Girish et al., (1975) recorded an
average weight loss of 7 to 22 percent caused by T. granarium, T. castaneum, R.
dominica, S. oryzae, Sitotroga cerealella and Ephestia cautella during six months of
storage. Bains et al., (1976) described distribution and pest status of T. granarium and
other stored product pests in wheat grain stores in the Indian Punjab, on the basis of a
survey conducted during 1971-72. They reported grain damage ranging 9 to 14.4 percent
and the weight loss ranging 1 to 3 percent. The incidence was higher in bulk-stored
wheat than in bags. Bhardwaj et al., (1977) collected 180 samples of wheat from all 12
districts of the Indian Punjab, during December 1975 and January 1976. They found that
the samples were commonly infested with T. granarium, T. castaneum, S. oryzae, R.
dominica, S. cerealella and O. surinamensis. Laboratory investigations of the samples
revealed that the moisture content of the grain averaged 11 percent, germination 82.5
percent, infested grains 5.1 percent, germ eaten 2.6 percent and weight loss 2.5 percent
after about 8-10 months' storage. Prasad et al., (1977) also found that khapra larvae
caused 2.2 to 5.5 percent weight loss in stored wheat. They found that 15 % infestation
level caused 2.6 % weight loss coupled with 24 percent loss in viability under optimum
condition at f 36 °C.

Such significant quantitative losses and cost of control negatively affect profitability for
grain dealers, flour millers and wholesalers. T. granarium normally attacks as a major
pest except in arid climates where other primary pests are inhibited by the dryness
(Proctor, 1994). Khan and Cheema (1978) determined 2.3 percent storage loss of wheat
in some parts of the Punjab province of Pakistan. Kraszpulski (1985) reported that T.
granarium feeds mainly on products of plant origin. Optimum growth and development
of the pest takes place at temperatures ranging 32 to 35 °C. He further reported that
larvae of Khapra beetle are much resistant to insecticides than other stored product pests.
Ahmed (1988) during his investigation reported 10-15 percent loss of wheat grains due to
 22

the attack of Khapra beetles, lesser grain borers, rice weevils and granary in cereal
storage sector of Pakistan.

Hemed et al., (1992) worked on population buildup and losses caused by different
combinations of T. granarium, S. cerealella and R. dominica to maize grains under
laboratory conditions. They found that S. cerealella was more successful grain feeder
than T. granarium and R. dominica. Seifelnasr (1992) used baited insect traps to survey
and identify stored grain insects in the main grain production and storage area in central
Sudan. T. granarium, R. dominica, O. surinamensis, O. mercator, T. castaneum,
T. confusum, S. oryzae, Cryptolestes ferrugineus, S. cerealella and Tenebroides
mauritanicus were collected in the traps. T. granarium and R. dominica ranked 1st and
2nd in abundance, respectively at all sites. The percentage losses of sorghum were
strongly related with the mean numbers of insects trapped at different sites and ranged
between 2.5 and 7.6 percent. Khan and Kulachi, 2002, collected 220 samples of wheat
grains from different locations of D.I. Khan. They recorded average losses to the tune of
3.4 and 6.5 percent by C&W and TGM methods, respectively within five months storage.
They also observed black pointed grains, broken/shriveled grains and green/immature
grains, which were counted as 0.6, 3.1 and 0.7 percent, respectively. While an average
amount of foreign matter was found 30.3 percent. On an average 1.93 percent grains
were found infested with T. granarium, R. dominica and Tribolium spp.

Aheer and Ahmed (1993) evaluated feeding preference of khapra larvae and lesser grain
borer for twelve wheat varieties / lines including 85054, 86175, Shalimar-88, 85205,
86371, 85276-2, 84021, 86299, Punjab-85, 83035, 86369 and 84133-6. They observed
significant differences in number of bored grain and grain weight loss caused by
T. granarium and R. dominica among the varieties. A wheat variety, 86175 proved less
susceptible to R. dominica and exhibited minimum damaged grains (0.6 percent) and loss
in weight (0.34 percent). In case of T. granarium, a variety 86299 was found relatively
more resistant. It showed minimum loss in weight (0.58 percent). Rao et al., (2004)
screened twenty-eight wheat cultivars for their resistance to T. granarium. In these
investigations mean number of progeny beetles per 20 grams of different cultivars ranged
10.0 to 36.9. The wheat varieties Kalyansona, HS 240, WTN 50 and UAS 2023 were
found as the most resistant, while MACS 2846, Sonalika, Raj 6062, DT 46 and GW 1188
proved to be the most susceptible. Correlation studies between various grain
characteristics and progeny beetle emergence revealed that grain size and hardness
 23

played an important role in resistance and susceptibility of wheat grain to Khapra beetle.
The maximum percentage loss in weight was recorded in MACS 2846 (63.6) and
minimum in Kalyansona (19.6).

2.2.2 Qualitative Losses of Wheat, Food Safety and the Khapra Beetle

Morison (1925) disclosed that barbed hairs of khapra larvae that rub off and remain in
the grain may prove a serious hazard to human heath if swallowed. Pruthi and Singh
(1950) concluded that cast skins may cause dermatitis in people handling heavily infested
grains. Diekmann (1996) stated that out of 20 species of storage pests, T. granarium has
been declared as a quarantine pest in most of the countries of the world due to its food
safety concerns. Perez et al., (2003) also reported that insect fragments in wheat flour are
major concerns of the milling industry because consumers demand high quality and
wholesome products. Due to the reason FDA has established a defect action level of 75
insect fragments per 50 g of flour. Today flour millers of the United States regularly test
their flour to comply with this federal requirement and to deliver sound flour to their
consumers. Parashar (2006) reported that T. granarium larvae are one of the most serious
stored grain pests but the adults are harmless. The larvae start feeding from embryo point
and later consume the entire kernel/seed, which makes the grain hollow and only the
husk remains. In case of severe infestations, infested grains are filled with frass, cast
skins and excreta, which badly deteriorates quality of the grains. Hence, there is an
increasing trend among grain buyers towards zero-tolerance to these contaminants.
Countries such as Canada have a legally defined zero tolerance for stored-grain insects
(Canada Grain Act, 1975). The larvae are often found on edges of jute stacks and make
the infested store unhygienic. Khare et al., (1974) reported that insect infestation in
stored wheat grain reduce germination and produce unpleasant odors, dirty appearance
and abhorrent taste due to contamination with insect fragments and excrement. They
reported that commonly found stored product insect pests include T. granarium,
T. castaneum, S. oryzae, R. dominica, S. cerealella, O. surinamensis, Cryptolestes
pusillus and Laemophloeus minutus.

Yadav (1980) conducted an experiment to study the effect of temperature and humidity
changes on the protection of stored wheat against insect infestation. He observed that 9.5
percent moisture facilitates the development of S. oryzae. That is why majority of wheat
samples taken were infested by this pest. R. dominica was found in 8.2 percent of
 24

samples and T. granarium for which high levels of humidity are known to be
unfavourable, was present in only 2.8 percent of samples. Raju (1984) reviewed the
biotic and abiotic factors that cause approximately 20 percent losses of stored foodstuffs
and stored grains in India as compared with 5 percent in Europe. It was found that
T. granarium, T. castaneum, S. oryzae, S. cerealella, R. dominica and Laemophloeus
minutus were the most common species found in food stuffs. He reported that the grain
with > 0.5 percent kernel infestation was unfit for milling, and the wheat flour containing
over 10 mg uric acid/100 g was unacceptable to consumers. Ramzan and Chahal (1985)
conducted laboratory studies with three levels of initial infestation (1, 2 and 3 pairs) of T.
granarium, T. castaneum and S. oryzae. Their results enunciated significant increases in
kernel damage and loss in seed viability with the proportional increase in infestation
level and storage period.

Yadav and Singh (1985) evaluated host-suitability of lentil flour for the development of
eight stored product insects; out of which T. granarium took four times longer in lentil
flour as compared to wheat flour. Similarly, T. castaneum, L. serricorne, and Stegobium
paniceum developed about twice as slowly on lentil flour as compared to wheat.
Warchalewski et al., (1993) studied varietal preference of S. granarius, T. confusum and
E. kuehniella towards 9 wheat varieties. It was found that kernel hardness, falling
number, non-protein nitrogen content and protein quality instead of quantity appears to
contribute towards increased wheat grain resistance. Gharib (2004) carried out studies on
the susceptibility of thirteen Egyptian wheat varieties to infestation by the Khapra beetle
and lesser grain borer. Their results revealed significant differences in growth index,
percent weight loss, damage and germination (%). among the tested varieties. Hala et al.,
(2005) confirmed results of Gharib, 2004 and Samir et al., (2005) by reporting food
preference of T. granarium and S. oryzae towards different wheat and maize varieties.
Their results showed that mean number of eggs/female as well as mean numbers of the
emerged individuals increased when the insects were allowed to feed on their preferred
food grains. Wheat varieties generally proved as more preferred food to the both insects
either in non-choice or free choice methods. Wheat variety Sakha 8 followed by Sakha
93 and maize variety Tri-H 322 were reported as the best preferred for the both insects,
while Giza 168 and Okrani were the least preferred. Poplawska et al., (2001) also carried
out investigations on feeding preferences of Khapra beetle towards grain of four barley
cultivars: Their results revealed that the development of T. granarium larvae on barley
grain and malt took longer as compared to wheat grain. Samir et al., (2005) reported
 25

T. granarium and S. oryzae as serious primary insect pests of the stored wheat and maize
grains. They reared the both insect pests on five varieties of wheat and maize and
reported significant differences in larval, pupal stages, oviposition period and female
longevity as well as number of eggs/ female. As far food preference is concerned,
T. granarium preferred wheat and maize nearly at the same rate, but S. oryzae preferred
wheat over maize grains. Pingale et al., (2006) subjected soft wheat to artificial
infestation by T. granarium C. oryzae L. and Ephestia cautella W. They reported that
weevils caused heavy reduction in weight of the grain. But the moth, which feeds only on
the germ portion, caused more reduction in viability. Analysis of results exhibited
significant increase in the acidity of fat and a decrease in the thiamine content due to
insect damage; but the effect on other constituents of the grain, such as total nitrogen and
reducing sugars was not significant. Some workers had reported that dietary preferences
of T. granarium vary not only from commodity to commodity but also within different
varieties of the same commodity. Sayed et al., (2006) performed experiments to
determine varietal resistance of wheat against T. granarium and R. dominica. For this
purpose they used twelve wheat varieties viz., Pak-70, Tandojam-83, Mehran-89, TJ-
0787, Sarsabz, Soghat, Sonalika, AZS-4, Zardana, Yekora, Abadgar and Anmol. Their
results revealed that population build up in both insect treatments was the lowest in
variety Mehran-89, whereas the highest population was recorded in case of TJ-0787. On
the basis of percent grain weight loss, the most resistant variety to both insect species
was found to be Mehran-89, while the least resistant varieties recorded were TJ-0787 and
Sarsabz against T. granarium and R. dominica, respectively. The remaining varieties
were considered as moderately resistant to susceptible. It was finally concluded that the
moisture content play a highly significant role in population growth, percent weight loss
and percent grain damage.

Krzymanska and Golebiowska (1987) investigated the effects of feeding by 6 species of

stored products beetles on the chemical composition of wheat grains. It was found that all
the pests exhausted the starch content. However feeding by the adults and larvae of
S. granarius and S. oryzae and the larvae of T. confusum and T. granarium caused an
increase in protein content, whereas larvae of O. surinamensis, T. granarium and T.
confusum reduced lipid content. In case of fatty acids, feeding by Sitophilus spp. reduced
palmitic and oleic acids but increased linoleic and linolenic acids; R. dominica,
T. confusum and T. granarium caused an insignificant increase in linoleic acid. All
species except Sitophilus damaged the germ. Sinha and Sinha (1990) collected fifty
 26

wheat samples including 25 free from infestation and the remaining 25 infested by stored
product insects. Aspergillus flavus infection was found common (87 percent) in insect
damaged samples; whereas 25 percent in insect-free samples. Of the four major pests
recorded, S. oryzae was the dominant insect species, followed by T. castaneum,
R. dominica and T. granarium. Lopes et al., (1991) subjected grains of Maize variety
BR-300 to infestation level of 5, 20, 30 or 50 percent with S. zeamais. Results did not
reveal significant differences in dry matter digestibility or nitrogen absorption among the
different infested meals but Nitrogen retention was significantly greater for the 5 percent
infested maize (33.77 %) than recorded for the other 3 infestation levels.

Ahmed et al., (1992) conducted a survey to assess the quality characteristics of wheat
grains and compared it with the existing Fair Average Quality (FAQ) standards. A total
of 824 samples were collected and their physical characteristics and insect infestation
were determined. They recorded significant differences among different samples in
relation to presence of foreign matter, moisture content, insect-damaged grain and broken
grain percentages. The incidence of live or dead T. granarium, T. castaneum, S. oryzae
Sitotroga cerealella and R. dominica was recorded in about 7 percent of samples. The
results proved that the current FAQ standards do not describe the quality of wheat
produced. Hence, need for a new grading system was suggested. Sinha and Sinha (1992)
found S. oryzae and T. castaneum as the dominant species found in maize samples when
stored for 4-12 months. Other species included O. surinamensis, R. dominica, T.
granarium and S. cerealella. They found that incidence of Aspergillus flavus as well as
the level of aflatoxins was comparatively higher in insect damaged maize samples than
undamaged samples. Toxigenic strains of A. flavus were isolated from both S. oryzae and
T. castaneum. Jood and Kapoor (1992a) evaluated protein quality of wheat grains having
25, 50 and 75 percent infestation caused by mixed populations of T. granarium and R.
dominica by feeding the infested grains to rats and studying their growth and nitrogen
balance. They found that feeding rats fed on grain with 50 and 75 % resulted in marked
decreases of food intake, body weight gain, food efficiency ratio, protein efficiency ratio,
nitrogen absorption, net protein utilization and dry matter digestibility. These parameters
showed negative association with infestation levels. However 25 percent level of grain
infestation did not affect the parameters significantly. In other experiments Jood et al.,
(1992a, 1993a) used maize and sorghum instead of wheat and reached the same
conclusion as drawn earlier. Jood and Kapoor (1992b) conducted an experiment wherein
they reported that protein and starch digestibility of wheat, maize and sorghum are
 27

affected significantly (P<0.05) and adversely at 25, 50 and 75 percent grain infestation
caused by T. granarium and R. dominica individually as well as in mixed form. They
observed that T. granarium, primarily a germ feeder, reduces protein digestibility of
wheat and maize more than R. dominica or a mixed population of the both insect species.
The reduction in digestibility was found dependent on the distribution of proteins and
starch in seed components as well as feeding preferences of insects. Similarly substantial
changes in the contents of calcium, phosphorus, zinc, iron, copper and manganese were
observed when wheat, maize and sorghum grains were subjected to artificial infestation
by T. granarium and R. dominica (Jood et al., 1992b). In case of R. dominica they
observed significant increases in the mineral contents of wheat and maize at 75 %
infestation level, but these increases were not significant in case of T. granarium. On the
other hand, R. dominica did not significantly change the mineral content of maize.
However a significant reduction was noticed in case of T. granarium. Mixed populations
of both species showed intermediate changes in the mineral contents of the three cereal
grains. Storage of un-infested grains up to 4 months did not induce any appreciable
changes in the levels of the six minerals. In another experiment, Jood et al., (1993b) used
T. granarium and R. dominica to investigate the effect of insect infestation on
organoleptic characteristics of stored cereals. ‘Chapattis’ made from infested and
uninfested grain flours were evaluated for color, taste, texture, aroma and appearance by
a panel of semi-trained judges. Their results revealed no significant differences in the
scores for color, appearance, aroma and texture at all infestation levels for the three
cereals and the scores even did not differ significantly from the uninfested grains.
However at 50 and 75 percent infestation levels, scores for the taste of Chapattis were
significantly lower than uninfested grains. Bitter taste also resulted in poor overall
acceptability of Chapattis prepared from flours infested at 50 and 75 percent levels.
While investigating changes in the levels of total nitrogen, total protein, non-protein
nitrogen and uric acid contents of wheat, maize and sorghum grains having 25, 50 and 75
percent infestation caused by T. granarium and R. dominica separately and in mixed
population Jood and Kapoor (1993c) recorded a significant increase these contents with
the increase in infestation.. But there was significant reduction in nitrogen and true
protein contents of three cereal grains at 75 percent infestation level though the decrease
was non-significant at the 25 and 50 percent levels of grain infestation as compared to
control. Both insect species produced uric acid contents above acceptable limits (10
mg/100 g) even at 50 percent infestation levels in the three cereal grains. Variations in
contents of nitrogenous compounds during storage (1, 2 and 4 months) were found
 28

marginal. Further studies conducted by Jood et al., (1993d) on the effect of insect
infestation on total soluble sugar, reducing sugar, non-reducing sugar and starch contents
of wheat, maize and sorghum grains indicated that these parameters are adversely
affected at 25, 50 and 75 percent insect infestation caused by T. granarium and R.
dominica, separately as well as mixed population. R. dominica caused significant
reduction in available carbohydrates at 50 and 75 percent infestation levels whereas T.
granarium achieved similar effect only at 75 percent. Mixture of both insect species
caused intermediate losses. Storage of cereal grains up to 4 months resulted in substantial
increase in sugars and decrease in starch content, but storage for a shorter period did not
cause any significant changes in levels of carbohydrates. Warchalewski and Nawrot
(1993) carried out investigations on activity of total wheat extractable proteins of 2
spring and 4 winter wheat cultivars relative to alpha-amylase inhibition of S. granarius,
T. confusum and Ephestia kuehniella in vitro, and also on differences between these
cultivars relative to various other wheat grain properties. Results exhibited a positive
correlation between alpha-amylase inhibition in E. kuehniella and growth parameters,
while kernel hardness of wheat was a determinant in the development of S. granarius.
Increased levels of non-protein nitrogen had a negative effect on growth of all the 3
species. Charjan et al., (1994) studied certain physiological, mycological and
biochemical changes in stored wheat induced by the infestation of S. oryzae and R.
dominica. It was found that the incidence percentage of fungal flora, total nitrogen, uric
acid, nitrogen and free fatty acid of grains increased considerably at the end of the third
and fourth month of storage. The 100-grain weight, germinability, non-reducing sugars,
reducing sugars and total water soluble sugars decreased with an increase in insect
infestation as compared to the control. Jood and Kapoor (1994) again reported substantial
losses in thiamine, riboflavin and niacin contents of wheat, maize and sorghum grains at
25, 50 and 75 % infestation levels induced by T. granarium and R. dominica separately
and as mixed population. As far amino acids are concerned, Jood et al., (1995) also
observed a significant decrease in essential amino acids of wheat, maize and sorghum
due to grain infestation caused by mixed populations of T. granarium and R. dominica
(50:50). Non-essential amino acids were also adversely affected. Jood et al., (1995)
declared a great increase in anti-nutrient polyphenol contents at 75 percent infestation
level. However increase was meager in case of anti-nutrient phytic acid. In an other
experiment, Jood et al., (1996a) reported that infestation of wheat, maize and sorghum
grains caused by T. granarium and R. dominica individually or in mixed populations
resulted in substantial reductions in the contents of total lipids, phospholipids,
 29

galactolipids, and polar and nonpolar lipids. Losses of lipids showed significantly
negative correlations with the levels of insect infestations. T. granarium was found
comparatively more destructive than R. dominica. Jood et al., (1996b) finally found that
severe infestations of grain by Khapra beetle may make it unpalatable or unmarketable.
Grain quality may decrease due to depletion of specific nutrients. Wakil et al., (2003)
studied the nutritional losses of wheat due to the attack of T. granarium, T. castaneum, S.
oryzae and R. dominica. Their results revealed more nutritional losses in laboratory
infested wheat grains as compared to the infested samples taken from the public stores.
They reported a positive correlation among the damaged wheat and protein & fat
contents, whereas, negative correlation was found between carbohydrate contents and
insect damage. Fogliazza and Pagani (2003) conducted experiments using Sitophilus spp.
R. dominica, Tribolium spp., Trogoderma spp., O. surinamensis, E. kuehniella and S.
cerealella. They found that the insects infesting kernels (i.e. Sitophilus. spp. and R.
dominica) negatively affected the rheological properties more than flour pests. Both
kernel and flour pests showed negative effects on baking quality.

2.3 Food Security and Storage Losses

Food security of the world is linked with production of food crops as well as minimizing
the factors which negatively influence the produce. There is no second opinion that
stored grains pests cause considerable economic losses to cereal grains each year all over
the world. The damage caused by insects is comparatively greater than those caused by
other pests because of their ability for rapid population increase. There are over 750,000
known species of insects out of which 100 species had been found infesting stored
products. Detailed investigations had further revealed that only 20 species (excluding
psocids) out of the 100 are of major importance with worldwide distribution (Andrew,
2004). Rahman et al., (1945) reported that an average loss sustained by the Indian grain
storage sector ranged 6-33 percent in a single storage season with a maximum damage at
73 percent. Whereas storage losses in Australia averaged 10 percent with an end result of
2 percent additional cost of control (Bengston, 1997). Girish et al., (1974) carried out
survey of storage losses caused by the stored product insect pests during 1973-74. Their
results revealed that significant losses of grain were caused due to infestation by T.
granarium, T. castaneum, S. oryzae, R. dominica, S. cerealella, O. surinamensis and E.
cautella. The loss in weight after storage for six months varied from 0.06 to 9.7 percent
and the loss in viability ranged from 7 to 22 percent. Significant differences in the
 30

susceptibility to damage were also shown by different varieties of wheat. Golebiowska et

al., (1976) observed that stored product beetles including T. granarium, S. granarius , S.
oryzae, R. dominica, T. confusum and O. surinamensis cause significant reduction in
germination and nutritional composition of the infested grains as well as significant
increases in weight loss and the number of damaged grains thus posing a serious threat to
food security. Caliboso (1977) estimated 34 percent loss caused to corn stored for eight
months in government warehouses in Philippine. Golop and Hodges (1982) reported
losses of stored grains in Tanzania as high as 30 percent during 3 to 6 months storage
period due to the attack of insect pest. It is generally accepted that 5-15 percent of the
total weight of all cereals, oilseeds, and pulses is lost due to many factors including
insect attack during post harvest operations (Anonymous, 1989). Chelkowski (1991) also
reported that 10 to 30 percent of total world grain production is lost after harvest. The
losses are more pronounced in the developing countries with a common average of 10–
15 percent (Lucia & Assennato, 1994). Neethirajan et al., (2007) reported that storage
pests cause considerable quantitative as well as qualitative losses; ultimately deteriorate
nutritional contents and lower crop value. Heavily infested grains become unfit for seed
purposes and its products become unsuited for human consumption. Thus there is a
census among notable workers that the insect infestation during storage may make grains
totally inedible through associated microbial spoilage and contamination resulting in
heavy financial losses (White, 1993).

In Pakistan, first schematic survey of losses was conducted by Chaudhary (1980) who
reported an aggregate loss of 15.3 percent during various post-harvest operation of wheat
in the country. Jilani (1981) also observed 10 to 15 percent post harvest loss of food
grains in Pakistan, which were chiefly caused by the attack of insect pests. His report was
later on supported by findings of Ahmed (1984). In the same year Ahmad and Afzal
(1984) recorded 22.7 percent post harvest loss of wheat in Pakistan, out which 9.5
percent occurred during storage period and the remaining 13.2 percent during harvesting
and threshing practices. Farmanullah (1985) recorded 0.22 percent handling & spoilage
losses and 2-33 percent quality loss of stored wheat in public sector godowns at D. I.
Khan. On the basis of a country wide survey, storage loss of wheat in Pakistan was found
in the range of 3.5 to 25 percent (Irshad & Baloch, 1985). Mohammad (1986) reported
that losses of wheat grains stored for 4 months in house type godowns of Pakistan were
2.03, 8.18 and 1.35 percent determined by S.V.W, T.G.M. and G.M. methods of loss
assessment, respectively. The corresponding figures for 7 months storage period were
 31

recorded as 3.02, 9.41 and 2.06 percent, respectively. Whereas six months wheat storage
resulted in average weight loss of 1.99, 6.33 and 2.01 percent, determined by S.V.W,
T.G.M. and G.M. methods, respectively (Khan, 1986). Baloch (1986) recorded 4 and 7
percent annual storage losses at farm level and in the public sector, respectively
depending upon ecological conditions, biological factor (insects, molds, rodents and
birds), storage type, storage management, etc. Among the stored grain insect pests,
T. granarium, T. castaneum, R. dominica, S. oryzae and Sitotroga cerealella had been
found most destructive causing a minimum of 5-10 percent loss annually (Baloch et al.,
1994). These findings are sufficient to establish that stored grain insect pests are serious
threat to the global food security.
 32

Chapter-II
2.4 MATERIALS AND METHODS

The present investigations were carried out in Stored Grain Research Laboratory of
Entomology Department of Pir Mehr Ali Shah, Arid Agriculture University Rawalpindi
during 2004-2008. Main objectives of the experiment were to determine quantitative and
qualitative losses caused by artificial infestation by Trogoderma granarium Everts and
relative resistance of wheat varieties against feeding by the pest. The materials and
methods employed in this study are briefed as under:

2.4.1 Collection of Insects

Mixed age cultures of the T. granarium were collected from farm houses as well as
stores of the Punjab Food department located at various places in Rawalpindi district.
The cultures were reared on healthy wheat grains apparently free from insect infestation.
To further ensure exclusion of any undetected population of insects, the wheat samples
were subjected to phosphine fumigation before using the grains as rearing medium (Jood
et al., 1992b). The fumigated grains were put in three glass jars (20 ×15 cm) each
containing one kg wheat. The jars were covered with muslin cloth with the help of rubber
bands and were placed in the laboratory at 30±2 °C and 65±5 % R.H. for conditioning.
After a fortnight, when moisture level of the grains ranged from 10-12 %, which is
suitable for insect growth and development (Pingale and Girish, 1967), the grains were
used as rearing medium.

2.4.2 Mass Rearing of Insects

From the mixed age cultures collected from various destinations, Khapra beetle were
separated at the pupal stage and the pupae were then reared in an incubator at 32± 2 °C
and 65±5 % R.H. The newly emerged adults (24-48 hrs after emergence) were used for
mass rearing. For this purpose, ten pairs (10 males+10 females) of adults were introduced
in the jars having cleaned, healthy and fumigated wheat with 12 % moisture contents.
The cultures were maintained in the incubator at 32±1 °C and 65±5 % relative humidity
for a period of three months for mass rearing. Later on uniform size larvae of the insect
were used in the experiment.
 33

2.4.3 Collection of Wheat Varieties

Nine wheat varieties commonly grown in Pakistan vis-à-vis BWP-97, Manthar, Bhakkar-
2000, BWP-2000, GA-2002, Inqlab-91, DWR-97 Panjnad, and Wafaq-2001 were
collected from the Regional Agricultural Research Institute, Bahawalpur, Punjab,
Pakistan. One kg sample from each variety was taken and fumigated with Aluminium
phosphide tablets to nullify the possibility of previous infestation if any. The samples
were then cleaned by sieving through 3/8, 3/16, 1/8 and 1/12 inch mesh sieves. From this
cleaned wheat, working samples weighing 25 grams from each variety were drawn
(Proctor, 1994) and subjected to analyses for determination of moisture contents, insect
damaged, broken and healthy grains. Similarly 50 grams samples from each variety were
also drawn for analyses of crude protein, crude fat, crude fiber and ash contents.

2.4.4 Inoculation of Wheat Samples

200 gram wheat grain sample from each variety was taken in 250 ml glass jars and
twenty uniform size larvae of about 1-2 weeks old were taken from the culture and
introduced in each jar as triplicates. All the jars were then covered with muslin cloth with
the help of rubber bands. The jars were later on placed in an incubator under semi-
warehouse environment at 25± 2°C, 55±5 % R.H. and 12:12 hrs continuous light:dark
conditions for a period of six months to realize the filed conditions. Doors of the
incubator were opened for a period of 30 minutes on alternate days to ensure proper
aeration and to avoid accumulation of carbon dioxide produced as a result of biotic
respiration. After a period of 6 months, the jars were taken out and further analyses were
made for progeny development as well as physical and biochemical changes induced by
the insect infestation.

2.4.5 Determination of Moisture contents

Moisture of the grain was determined to investigate the correlation among moisture
content of the grain and levels of T. granarium infestation, progeny development, weight
loss, weight of frass, crude protein, crude fat, crude fibre, ash contents, insect damaged,
broken and healthy grains etc. For this purpose, a U.S. made grain moisture tester
(Farmex-MT3) was used.
 34

2.5 Progeny Development

After weighing, the entire grain sample taken from each variety was sieved through
sieves of different mesh sizes ranging 1/8, 1/12, 3/16 and 3/8 inches. Live as well as dead
larvae, pupae and adults were counted for each replication of the corresponding wheat
variety under the laboratory magnifying glass 10³.

2.5.1 Weight Loss

Using sieve set, the infested grain in each jar was subjected to sieving to separate insect
skin debris, grain dust and other excretion added due to T. granarium infestation. A
sample weighing 25 gram was drawn from the cleaned wheat for assessment of percent
weight loss. For this purpose number and weight of damaged and undamaged grain was
recorded and put in the following equation for determination of weight loss (Gwinner et
al., 1996).

Percent Weight Loss = (Wµ × Nd) – (Wd × Nµ) × 100

Wµ × (Nd + Nµ)

Wµ = weight of undamaged grains

Nµ = number of undamaged grains
Wd = weight of damaged grains
Nd = number of damaged grains

2.5.2 Weight of Frass

While determination of weight loss, weight of exuviae, dead as well as live adult and
immature stages of Khapra beetle, flour dust and other excretion produced during the
larval infestation was recorded for each variety.

2.6 Comparative Resistance of Wheat Varieties

Progeny development, percent infested grains and weight loss was selected as criteria for
determination of comparative resistance of wheat varieties against T. granarium
infestation.
 35

2.7 Insect Damaged, Broken and Healthy Grain Count

After removing the frass, samples of cleaned grain each weighing 25 gram was drawn
from each replication of the respective wheat variety. The grains were segregated and
counted for insect damaged, broken and healthy grains by using the following equations.

Percent Insect damaged grains = No. of insect damaged grains × 100

Total number of grains in the sample

Percent Broken grains = No. of broken grains × 100

Total number of grains in the sample

Percent Healthy grains = No. of healthy grains × 100

Total number of grains in the sample

2.8 Biochemical Analyses of Wheat Samples

Nutritional changes of the infested grains induced by infestation of T. granarium larvae

were studied for crude protein, crude fat, crude fiber and ash contents using ICC and
AOAC methods (Helrich, 1990). For this purpose, infested wheat samples were cleaned
and sieved to remove insect body parts. Later on 50 gm grains in triplicate were taken for
determination of crude protein, fat, carbohydrates, fiber and ash contents.

2.8.1 Determination of Crude Protein

Crude protein is a conventional expression for the total content of nitrogenous

compounds of the analyzed product, calculated by multiplying the corresponding total
nitrogen content by factor of 5.7 for wheat. Whereas nitrogenous compounds were
determined by oxidizing the organic matter of the sample with concentrated sulfuric acid
in the presence of a catalyst: the product of the reaction (NH4)2SO4 was treated by alkali;
free ammonia was distilled and titrated (AOAC Method No. 979.09).

2.8.2 Determination of Crude Fiber

Wheat samples were gelatinized in the presence of heat stable alpha amylase, and then
enzymatically digested with protease and amyloglucosidase to remove digestible protein
and starch. Four volumes of ethanol were added to precipitate soluble dietary fiber. Total
residue was filtered off and washed with ethanol and acetone. The residue was weighed
after drying. The remaining material was analyzed for protein and ash content,
 36

respectively. Subtracting the amounts measured for protein, ash and a blank control from
the dry weight of the filtered residue yielded a value for total dietary fiber content (ICC
Standard No.156, 1994).

2.8.3 Determination of Ash

A five gram well mixed sample was weighed into a shallow, relatively broad dish that
had been ignited, cooled in a desiccator and weighed soon after reaching at room
temperature. The sample was later on ignited in a furnace at 550 °C (dull read ) until the
light gray ash resulted, which was cooled in a desiccator and weighed after reaching at
room temperature (AOAC Method No. 923.03).

2.8.4 Determination of Crude Fat

AOAC method No. 920.39 was followed for determination of crude fat contents

2.8.5 Determination of Carbohydrates

The starch content is the portion of the sample that can be determined as glucose after
enzymatic decomposition with amyloglucosidase. AOAC method No. 979.10 was used
for determination of carbohydrates.
 37

Chapter-II
2.9 RESULTS AND DISCUSSION

2.9.1 Moisture Contents

Analysis of variance depicted significant differences between moisture content of grain

before and after infestation (Appendix-4). The moisture recorded before infestation
revealed that there was no statistical difference in moisture contents of DWR-97, Panjnad
and Wafaq-2001 with mc values of 8.44, 8.34 and 8.08 percent, respectively. Similarly
there was fond no statistical difference between moisture content of wheat varieties
BWP-97 and Manthar, which depicted mc values of 10.47 and 10.41 percent,
respectively. Moisture content in case of Inqlab-91, GA-2002 was though statistically
similar with each other as well as with BWP-2000 but was found higher than those of
DWR-97, Panjnad and Wafaq-2001. Wheat variety Bhakkar-2000 exhibited a distinct
moisture content of 9.87, lower than those of BWP-97 and Manthar, but higher than
moisture contents of the remaining wheat varieties (Table-1). There was found a
significant increase in moisture content of all the wheat varieties after subjecting them to
artificial infestation of T. granarium larvae for a period of March 2006-August 2006. As
is evident from the Table-1, variety Wafaq-2001 showed minimum increase in moisture
content from 8.08 to 8.41 percent after infestation. The varieties Inqlab-91, DWR-97 and
Panjnad statistically proved similar with each other in respect of increase in moisture
content but ranked lower than those of BWP-97, Manthar, Bhakkar-2000 and BWP-
2000. Maximum moisture content was recorded in case of Bhakkar-2000 with 2.09
percent increase after infestation. Similarly Jood et al., (1996) also observed that 75
percent infestation level of T. granarium and R. dominica cause a significant (P < 0.05)
increase in moisture contents of the infested grains as compared to control and lower
levels of infestation. The results are also in line with the findings of Ravan et al., (1987),
Jood and Kapoor (1993) and Jood et al., (1993). The increase in moisture contents over
time may be due to the absorbance of atmospheric moisture by the grain, biotic
respiration as well insect excretion.

2.9.2 Progeny Development, Weight Loss and Weight of Frass

It is evident from the Table-2 that maximum progeny development was observed in
wheat variety BWP-97 showing 792.7 larvae per 200 gram wheat. Progeny development
 38

Table 1 Moisture Content of Wheat Varieties before and after infestation.

Wheat Varieties Moisture content (%)

Before infestation After infestation
BWP-97 10.47a 11.79 a
MANTHAR 10.41a 11.32 ab
BHAKKAR-2000 9.87 b 11.96 a
BWP-2000 9.00 c 10.37 cd
GA-2002 8.59 cd 10.67 bc
INQLAB-91 8.55 cd 9.693 de
DWR-97 8.44 d 9.633 e
PANJNAD 8.34 d 9.487 e
WAFAQ-2001 8.08 d 8.41 f

Means followed by the same letter in each column are not significantly different by
Duncan's multiple range test (P = 0.01)

Table 2 Progeny development, Weight loss and Weight of frass of Wheat

Varieties after infestation.

WHEAT VARIETIES Progeny Development Weight Loss Weight of

(No.) (%) Frass
(g)
BWP-97 792.70 a 20.25 a 24.47 a
MANTHAR 752.00 a 19.53 a 23.21 a
BHAKKAR-2000 668.70 b 18.27 ab 20.64 b
BWP-2000 624.0 b 16.24 bc 19.25 b
GA-2002 551.70 c 15.47 bc 17.03 c
INQLAB-91 440.00 d 15.37 bc 13.58 d
DWR-97 472.00 d 14.10 c 14.57 d
PANJNAD 280.70 e 13.81 c 8.663 e
WAFAQ-2001 189.70 f 6.223 d 5.857 f
Mean 530.16 15.47 16.36

Means followed by the same letter in each column are not significantly different by
Duncan's Multiple Range Test (P = 0.01)
 39

in case of Manthar-2000 was though statistically similar to that recorded in BWP-97 but
was lower having 752 larvae per 200 gram wheat. The varieties Bhakkar-2000 and BWP-
2000 were also found statistically similar in respect of progeny development with 668.7
and 624 larvae. Minimum progeny development of 189.7 was recorded in case of Wafaq-
2001.

Whereas weight loss caused after 6 months of artificial infestation by T. granarium

larvae revealed BWP-97 as one of the most susceptible wheat varieties with mean loss of
20.25 percent. Other varieties vis-à-vis Manthar, Bhakkar-2000, BWP-2000, GA-2000,
Inqlab-91, DWR-97, Panjnad and Wafaq-2001 followed BWP-97 with an average weight
loss of 19.53, 18.27, 16.24, 15.47, 15.37, 14.1, 13.81 and 6.2 percent, respectively. Data
pertaining to the weight of frass also depicted approximately similar ranking except that
weight of frass recorded in case of DWR-97 was higher as compared to that recorded in
case of Inqalab-91. There was found a positive correlation among progeny development
and infestation percentage, weight loss as well as weight of frass. The results are in
conformity with the previous findings reported by Ahmed et al., (1976) and Navarro et
al., (1978) who observed a high degree of positive correlation among the progeny
development and the infestation level, grain damage and weight loss of the infested
grains. Bhardwaj et al., (1977) observed 2.5 percent weight loss against 5.1 percent
infestation. Khattak et al., (2000) while working on the effect of T. granarium infestation
on twelve rainfed wheat lines also found that correlation among progeny development,
damaged grains and weight loss was positive and highly significant (P < 0.01). Grain is
living entity, which is affected by biotic and a biotic factors resulting in qualitative and
quantitative loss (Ahmed, 1995; Singh et al., (1997). Khan and Kulachi (2002) also
reported a positive correlation between the progeny development of T. granarium,
T. castaneum and R. dominica and the losses caused by them in terms of grain weight
loss. Our results are also in line with those of Syed et al., (2006) who correlated T.
granarium and R. dominica infestation with significant weight loss of different wheat
varieties.

2.9.3 Insect Damaged, Broken and Healthy Grains Recorded after Infestation.

The results pertaining to percent damage to wheat grains caused by T. granarium larvae
in different wheat varieties under natural storage conditions are presented in Table-3.
Mean values did not reveal statistically significant differences among various varieties of
 40

Table 3 Insect damaged, Broken and Healthy grains Recorded after infestation.

WHEAT Insect damaged Broken Grains Healthy Grains

VARITIES grains % (%) (%)
Before After Before After Before After
infestation infestation infestation infestation infestation infestation
BWP-97 2.00 a 43.37 a 2.66 a 16.72 a 95.34 a 39.91 f
MANTHAR 0.66 bc 42.87 a 2.00 a 15.02 ab 97.33 a 42.10 f
BHAKKAR-2000 0.00 c 38.44 b 4.00 a 13.65 b 96.00 a 47.91 e
BWP-2000 0.00 c 34.21 c 1.66 a 11.27 c 98.34 a 54.52 d
GA-2002 1.33 ab 34.45 c 1.66 a 11.17 c 97.00 a 54.38 d
INQLAB-91 1.66 a 30.27 d 2.66 a 10.43 c 95.68 a 59.30 c
DWR-97 1.66 a 35.94 bc 3.33 a 6.913 d 95.00 a 57.15 cd
PANJNAD 0.00 c 24.67 e 2.00 a 5.237 d 98.00 a 70.10 b
WAFAQ-2001 0.00 c 19.97 f 3.33 a 4.973 d 96.67 a 75.06 a
Mean 0.81 33.79 2.59 10.6 96.59 55.6

Means followed by the same letter in each column are not significantly different by
Duncan's Multiple Range Test (P = 0.01)

Fig. 3 Anatomy of a Wheat grain Fig. 4 Nutritional Composition of kernel

Source: AWB, 2006.

 41

wheat in respect of broken and healthy grains before infestation. However, there were
found significant differences among the wheat varieties in respect of insect damaged
grains before subjecting to the artificial infestation. Maximum number of damaged grains
was recorded in variety BWP-97 with 2 percent infestation followed by Inqlab-91,
DWR-97, GA-2002 and Manthar with 1.66, 1.66, 1.33 and 0.66 percent infestation,
respectively. The remaining four varieties did not show any sign of grain damage.
Comparison of mean values before and after infestation showed highly significant
variations among different wheat varieties in respect of damaged, broken and healthy
grains. As is evident from the Table 3, maximum damaged grain was found in BWP-97
with 43.37 percent, statistically resembling to Manthar with 42.87 percent insect
damaged grains. Minimum insect damaged grain was found in Wafaq-2001 with 19.97
percent. Same trend existed in insect broken grains and vice versa with the healthy
grains. These findings are in consistent with those of Pingale (1964), Badawy and Hassan
(1965), Shah (1969), Azeem et al., (1976), Hameed et al., (1984), Irshad and Baluch
(1985), Ahmad et al., (1986), Irshad et al,. (1988), and Khattak et al., (2000) who
observed a positive correlation between infestation caused by T. granarium and te
damage caused to wheat grains. Lately Syed et al., (2006) carried out investigation to
evaluate the comparative resistance of wheat varieties against infestation of T. granarium
and R. dominica. Their results revealed that grain damage and weight loss percentage
was mainly dependent upon the progeny development, which was also dependent on the
varietal preference of the pests used in the investigations.

2.9.4 Biochemical Changes Caused by T. granarium Infestation

Analysis of variance presented in Appendix-5 exhibited highly significant differences

among different biochemical parameters before and after infestation of T. granarium
larvae. However no visible effects of feeding by different larval strains were observed in
respect of protein, fat, carbohydrate, ash and fiber contents of wheat grains after the
infestation. Generally the feeding preference of the larvae toward different varieties was
found definitely diverse. It is worth mentioning that there was found a positive
correlation between larval infestation and protein, fat, fiber and ash contents irrespective
of wheat varieties. On the contrary there was found a negative correlation between
carbohydrate and infestation caused to stored grains irrespective of wheat varieties.
Quality of flour made from the infested wheat may depend upon the nature and feeding
behaviors of pests attacking the grain. If we look at wheat grains they appear as seeds,
 42

but closer examination shows them to be true fruit. Wheat grains when milled produce
fine powdery flour. A single grain makes about 20,000 particles of flour.

While making whole meal flour all components of kernel are included, but in extracting
white flour the seed coats and the germ are not used. In fact, they are crushed but taken
out as small flakes, by sieving through nylon or silk mesh. The outer part of the kernel is
composed of different layers of intercrossing cellulose (Fig. 3). Chemically a wheat grain
is composed of cellulose, minerals (bran) and protein with a high biological value.
However, due to low sifting rates of the flour, the protein is almost lost (Fig. 4). The
peripheral part of the wheat caryopsis is chiefly composed of indigestible and irritating
parts (cellulose and lignin) as well as minerals (ashes) that can interact, creating
undesirable compositions during the technological production processes of both dried
and fresh pasta. From a technological standpoint, the low sifting of the flour and the
semolina determines a "rational" sacrifice of the biodynamic components present in the
external layers of the caryopsis (the protein in the aleuronic layer). A good percentage of
these components are, however, present in more highly sifted flour (and semolina). In
modern mills (high milling) the sifting rate determines the type of flour and semolina.
The relationship between the principal chemical characteristics of the flour and the
sifting rate are very close, even if these characteristics always depend on the intrinsic
characteristics of the grains that are milled. That is, with a 50 percent sifting rate, the
parts farthest from the floury kernel or the layers nearest to the cortical part of the grain
are excluded from the flour. More over, for the flours subjected to a 72 percent or 80
percent sifting rate, the percentage of the parts closest to the external layers of the
caryopsis is obviously greater. To clarify this basic but important concept, the cross-
section of the wheat caryopsis indicates the concentration of the chemical elements that
most interest the flour and the semolina intended for use in making pasta. In short it is
the type of insect pest i.e. germ feeder, endosperm feeder, grain borer or external feeder
which decide deficiency of some nutritional components, which they eat out. The flour
that comes out from the grain of wheat is used in making chapatti, Nan, bread, biscuits,
cakes, confectionery, pudding and pies. Normally, the wheaten flour is rich in
carbohydrates (for energy), protein (for growth and development), the essential B
vitamins (for good health, good nerves and good digestion) and important minerals like
iron (for healthy blood) and calcium (for strong bones and teeth). General composition of
a 100 percent whole wheat bread is given in Fig. 5.
 43

2.9.4.1 Crude Fiber

It is evident from that Table-4 that fiber contents generally increased after infestation on
over all weight bases. A maximum increase of 1.4 percent was observed in the variety
BWP-97 followed by Manthar, Inqlab-91, Bakhar-2000, GA-2002, DWR-97, BWP-
2000, Wafaq-2001 and Panjnad with 1.36, 0.92, 0.84, 0.86, 0.587, 0.56, 0.25 and 0.21
percent, respectively. The difference observed in level of fiber contents may be attributed
to the feeding behaviour of the T. granarium larvae (Jood et al., 1992). It has been
reported that T. granarium larvae primarily prefer to feed on germ portion as compared
to bran or endosperm. That is why bran portion is least effected under the normal course
of infestation resulting in an increase in fiber contents on over all weight basis (Salunkhe
et al., 1985). The results are also in line with the work of Hameed et al.,1984; Jood and
Kapoor 1993, Jood et al., 1993, 1996a) who observed an increase in fiber contents of
wheat grains after subjecting to T. granarium infestation. They also found an inverse
correlation among susceptibility of the stored grains to insect pests and the fat, protein,
fiber and ash contents. Our findings are in consistence to the previous work as Wafaq-
2001 with the highest fiber contents of 1.953 proved to be the most resistant wheat
variety with only 6.2 percent weight loss. The results are also in line with findings of
Samuels and Modgil (2003) who concluded that with the increase in infestation and the
storage period; moisture, ash, crude proteins, crude fiber, non-protein nitrogen and uric
acid contents of wheat grains are significantly increased at (P<0.05).

2.9.4.2 Crude Protein

The results pertaining to protein enunciated highly significant differences between the
crude protein contents of wheat grains before and after infestation by the T. granarium
larvae. Maximum increase of 2.83 % was observed in case of BWP-97 and Manthar
followed by GA-2002, Panjnad, Inqalab-91, Bhakkar-2000, Wafaq-2001 and BWP-2000
with 1.65, 1.64, 1.11, 1.10, 0.57 and 0.01 percent, respectively (Table-4). In case of
Panjnad, no change in content of crude protein was observed. Increase in the content of
crude protein apparently seems illogical as larvae of T. granarium preferably eat on grain
germ, which contains protein. Hence there should have been an inverse relationship
between infestation level and the protein content of the wheat grain. But we found a
positive correlation between the two parameters against the hypothesis. A survey of
literature revealed apparently contradicting results. Some showed increase in protein
contents under the influence of T. granarium infestation. While other indicated reduction
 44

38 percent Water 38 percent Digestible Carbohydrates

8 percent Fiber

10 percent Protein

4 percent Fat

2 percent Vitamin

Fig. 5 The Nutritional Value of 100 % Whole Wheat Bread

Table 4 Biochemical Changes Induced in Wheat Varsities by T. granarium

Infestation

WHEAT CRUDE CRUDE FAT CRUDE FIBER ASH CONTENT CARBOHYDRATE

VARITIES PROTEIN
Before After Before After Before After Before After Before After
BWP-97 9.2 g 12.03 d 1.36 e 2.76 a 2.14 b 3.45 b 0.80 g 1.53 bcd 81.04 a 74.4 b
MANTHAR 9.2 g 12.03 d 1.51 d 2.87 a 2.36 a 3.79 a 0.86 f 1.41 e 77.69 c 73.3 bc
BHAKKAR-2000 9.84 f 10.94 e 1.66 bc 2.50 b 2.14 b 3.43 b 0.98 d 1.39 e 75.79 d 72.20 c
BWP-2000 10.93 e 10.94 e 1.59 cd 2.15 c 1.99 c 3.20 c 1.00 d 1.49 d 79.85 b 74.97 b
GA-2002 11.48 d 13.13 c 1.50 d 2.18 c 2.15 b 3.46 b 0.91 e 1.50 cd 73.76 e 68.50 d
INQLAB-91 12.03 c 13.14 c 1.947 a 2.87 a 2.24 ab 2.94 d 1.53 ab 1.66 a 80.7 ab 78.05 a
DWR-97 12.03 c 12.03 d 1.713 b 2.30 bc 1.74 d 2.63 e 1.44 c 1.56bcd 68.07 g 65.93 e
PANJNAD 12.58 b 14.22 a 1.983 a 2.19 c 1.58 e 2.23 f 1.50 b 1.58 bc 69.97 f 67.3 de
WAFAQ-2001 13.10 a 13.67 b 1.953 a 2.21 c 2.11 bc 2.27 f 1.55 a 1.59 ab 69.97 f 67.2 de
Means 11.15 12.45 1.69 2.44 2.05 3.04 1.17 1.52 75.21 71.32

Means followed by the same letter in each column are not significantly different by
Duncan's multiple range test (P = 0.01)
 45

in the protein contents. For example, Jood and Kapoor (1992a) carried out investigations
on protein and starch digestibility of wheat, maize and sorghum grains affected by
T. granarium and R. dominica. They observed that T. granarium, primarily a germ
feeder, reduce protein digestibility of wheat and maize more than do R. dominica or a
mixed population of both insect species. In the same year they evaluated protein quality
of wheat grains having 25, 50 and 75 percent infestation caused by mixed populations of
T. granarium and R. dominica by rat growth and nitrogen balance studies. They observed
that feeding of a diet containing insect-infested wheat grain (50 and 75 %) results in
marked decreases of food intake, body weight gain, food efficiency ratio, protein
efficiency ratio, nitrogen absorption, biological value, net protein utilization and dry
matter digestibility. These parameters showed negative association with infestation levels
(Jood and Kapoor, 1992b). Further experiments carried out by Jood and Kapoor (1993)
exhibited contradicting results wherein they recorded a significant increase in the levels
of total nitrogen, total protein, non-protein nitrogen and uric acid contents of wheat,
maize and sorghum grains having 25, 50 and 75 percent infestation caused by
T. granarium and R. dominica separately and in mixed population. They also observed a
significant reduction in protein nitrogen and true protein contents of the three cereal
grains at 75 percent infestation level. The picture became clearer by the findings of
Samuels and Modgil (2003), who stated that an increase in insect infestation and storage
period though significantly increase crude proteins, moisture, ash, crude fiber, non-
protein nitrogen and uric acid contents; yet, true proteins, crude fat, calorific value,
weight and density of wheat grains are significantly reduced at (P<0.05). That is why Aja
et al., (2004) suggested that while determining the effect of insect infestation on protein
contents of stored grains, gluten index should be measured as an index of protein quality.
Later studies revealed that increase in contents of crude protein in the infested grains is
actually due to production of non beneficial rather harmful proteins such as cast skins,
exuviates, dead insects, wings, legs and other body parts of the insects that come along
with the infested grain samples. On the other hand true protein or beneficial proteins are
factually reduced. Besides the increase in protein percentage may also be due to
significant depletion of carbohydrates that constitute major part of a wheat grain.

2.9.4.3 Carbohydrates

Findings of the present investigations enunciated a negative effect of T. granarium

infestation on the carbohydrate contents of wheat grains. Maximum reduction to the tune
 46

of 6.59 percent carbohydrate was observed in BWP-97 having maximum progeny

development and weight loss; followed by GA-2002 (5.26), BWP-2000 (4.88), Manthar
(4.36), Bhakkar-2000 (3.59), Wafaq-2001 (2.78), Inqalab-91 (2.71), Panjnad (2.67) and
DWR-97 (2.14 %), respectively (Table-4). These results showed a positive value of the
correlation coefficient between progeny development and carbohydrate contents valuing
0.678 with the infested grains. It is obvious from the values of correlation co-efficient
that reduction in carbohydrate was not solely dependent upon the progeny development.
There are some other factors, which might have contributed toward reduction in
carbohydrate contents. Such factors are varietals resistance as well as insect preferences
toward specific wheat varieties. The results are in conformity with the findings of Jood
and Kapoor (1992a) who carried out investigations on the effect of storage and insect
infestation on starch and protein digestibility of cereal grains. They found that T.
granarium significantly reduce starch digestibility of wheat and maize. Further studies
carried out by them also revealed a significant reduction in carbohydrate contents of
wheat, maize and sorghum when artificially infested with T. granarium and R. dominica.
The results are also in line with the findings of previous workers such as Hemeed et al.,
(1984) who observed a significant decrease in carbohydrate contents of wheat grains due
to attack of T. granarium larvae. These results were later on confirmed by Jood et al.,
(1996).

2.9.4.4 Crude Fat Contents

It is evident from the Table-4 that maximum increase in crude fat was observed in case of
BWP-97; whereas it was found minimum in case of Panjnad. There was found a highly
positive correlation (0.89) between increase in fat percentage and weight of frass after the
infestation. The phenomenon is contradictory to the facts that T. granarium primarily
feeds on germ of the wheat grain, which is store house for protein and lipids (Fig. 4).
Under these circumstances higher infestation should have been resulted in reduction of
fat content of the wheat grains. But the present findings have revealed opposite results,
which are in conformity with those observed by Hameed et al., (1984). Later studies
revealed opposite results indicating significant decrease in crude fat contents with
increase in the infestation level. In an experiment, infestation levels of 75 percent in
wheat, maize, and sorghum grains caused by T. granarium and R. dominica resulted in
significant decreases in crude fat, total carbohydrates, sugars, protein nitrogen, and true
protein contents and increases in moisture, crude fiber, and total protein (Jood and
 47

Kapoor 1993, Jood et al., 1993; 1996a). Samuels and Modgil (2003) also reported
significant decreases in crude fat, weight, density, calorific value and true proteins with
increase in the insect infestation and storage period. In case of present investigations
increase in fat is simply a percent increase resulting from depletion of carbohydrates etc
due to infestation and hence is not an actual increase in fat content.

2.9.4.5 Ash Contents

The results have revealed significant increase in ash contents of the infested grains. A
maximum of 0.73 percent increase was observed in wheat variety BWP-97 followed by
GA-2002, Manthar, BWP-2000, Bhakkar-2000, Inqalab-91, DWR-97, Panjnad and
Wafaq-2001 with 0.59, 0.55, 0.49, 0.41, 0.13, 0.12, 0.08 and 0.04 percent, respectively.
Increase in ash content is commonsensical because of feeding preference of T. granarium
larvae that bore into germ and prefer to eat germ as compared to the bran portion, which
is chief store house of minerals in case of wheat grains (Salunkhe et al., (1985). Our
findings are in line with those of Hameed et al., (1984) who recorded significant increase
in ash contents in wheat grains attacked by the T. granarium larvae. Later on Jood et al.,
(1992b) also concluded that T. granarium primarily a germ feeder appears to eat a
portion of bran, thus offsetting the increase in mineral composition of wheat grains,
which stores minerals mostly in its bran portion. Our results are also in conformity with
those of Samuels and Modgil (2003) who found a significant increase in ash, moisture,
crude proteins, crude fiber, non-protein nitrogen and uric acid with increase in infestation
and storage period.

2.9.5 Conclusions

Results of the present investigations have revealed more or less similar feeding behavior
of all the test strains of T. granarium. The larvae fed on both germ as well as endosperm
of the grains. In most of the cases, entire grain was consumed and partially eaten husk
was only remaining of the healthy grain. In some grains larvae were found camouflaging
themselves inside the shell. A huge quantity of larval exuviates was an indication of the
infestation severity among different varieties used in the present investigation. Flour dust
was also visible on lower part of the jars. Some live and dead adults of T. granarium
were also observed among the grains. Apparently, there was found a varying quantity of
exuviates, flour dust, live & dead adults and cast skins on different wheat samples
indicating different levels of susceptibility or resistance. Color change as well as foul
 48

odor was also observed in severely infested samples. Statistical analyses of the data
showing various physical, quantitative, qualitative and nutritional characteristics of
different wheat varieties influenced by the artificial infestation of T. granarium larvae for
6 month storage period under normal condition is presented in Appendix-4 & 5 and
Tables 1, 2, 3 & 4. Overall correlation among different parameters has been presented in
Table-5.

Literature has revealed that the insect resistance mechanisms of cereal grains are
complex and depend on physico-chemical and biochemical properties of the grain and on
the subsequent biochemical and physical adaptation of post-harvest insects to these
properties (Baker, 1986; Warchalewski et al., 1989; Dobie, 1991; Warchalewski and
Nawrot, 1993; Warchalewski et al., 1993). Stored grains may have high resistance to
insect pests because of the lack of vital nutrients or the presence of compounds that
adversely affect insect development (Taylor and Medici, 1966; Medici and Taylor, 1966;
Yetter et al., 1979; Nawrot et al., 1985; Gatehouse et al., 1986; Dobie, 1991; Baker et
al., 1991; Huesing et al., 1991; Warchalewski and Nawrot, 1993; Pueyo et al., 1995;
Zhang et al., 1997; Piasecka-Kwiatkowska, 1999; Piasecka-Kwiatkowska and
Warchalewski, 2000a, b). In general, insects tend to develop more slowly on resistant
grain varieties. Various studies on wheat grain resistance had been condcuted, but there
were few attempts to determine the particular factors that govern resistance (Dobie,
1991). The present studies aimed to evaluate the relative resistance of the commoly
grown wheat varities of Pakistan as well as effect of T. granarium infestation on physico-
chemical and biochemical chracteristics of the stored wheat revealed Wafaq-2001 as the
most resistant variety against T. granarium.
 49

Table 5 Correlation Matrix showing relationship of different Physical and Biochemical changes of Wheat grains Induced by Larval
Infestation of Khapra beetle T. granarium.

Parameters Progeny
development
Progeny Moisture
development 1.000 content
Weight of
Moisture content 0.900 1.000 Frass
Weight
Weight of Frass 1.000 0.900 1.000 Loss
Weevilled
Weight Loss 0.920 0.835 0.920 1.000 Grains
Broken
Weeviled grains 0.968 0.864 0.968 0.911 1.000 Grains
Healthy
Broken Grains 0.954 0.920 0.954 0.876 0.888 1.000 Grains
Crude
Healthy Grains -0.989 -0.907 -0.989 -0.923 -0.986 -0.952 1.000 Protein
Crude
Crude Protein 0.962 -0.981 0.962 -0.887 -0.921 -0.954 0.957 1.000 Fat
Crude
Crude Fat 0.892 -0.749 0.893 -0.724 -0.868 -0.823 0.875 0.826 1.000 Fiber
Crude
Crude Fiber 0.494 0.524 0.494 0.310 0.408 0.667 -0.512 -0.530 -0.408 1.000 Ash
Carbo-
Crude Ash -0.901 -0.813 0.494 -0.767 -0.821 -0.877 0.863 0.885 0.940 -0.492 1.000 hydrate
Carbohydrate 0.678 0.621 0.678 0.658 -0.821 0.794 -0.642 -0.645 -0.466 0.644 -0.553 1.000
 50

Chapter-II
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