48

CHAPTER II

ENERGY STUDIES OF PEST ON TRANSGENIC COTTON

INTRODUCTION

The growth of phytophagous insects mainly depends on the quality of phytal

parts consumed. The quality of the phytal parts varies with plant age, plant structure

and under certain environmental stress conditions (Krips et al., 1999 and Cedola et al.,

2001). The phytophagous insects feed on a wide range of plant families and they are

likely to experience greater heterogenecity in the contents and mixtures of nutrients

found in their natural diets (Rauberheimer and Simpson, 2003). Plant feeding insects

use plant aminoacids to build proteins, which can be used for the synthesis of structural

moieties such as enzymes, for transport and storage or as receptor molecules. Provision

of individual aminoacids like tyrosine connected with hardening of the cuticles;

tryptophan, with the synthesis of visual screening pigments; glutamate, a

neurotransmitter and proline an important energy source depend on the quality of plant

food consumed (Wills, 1999).

Most phytophagous insects obtain carbohydrates needed for their growth

through gluconeogenesis. Behmer (2006) reported that the desert locust, Schistocerca

sp. grew best on a diet containing 20 percent digestible carbohydrates while the flour

beetle, Tenebrio sp. exhibited optimal growth on a diet containing 70 percent

carbohydrates.

49

Phytophagous pests mainly require sterols, fatty acids, fat soluble vitamins and

carotenoids from the plant. In plant feeding pests, cholesterol is the dominant tissue

sterol despite the fact that plants rarely contain it at appreciable levels. Hence

phytophagous pests produce cholesterol by metabolizing the sterols found in their host

plants (Behmer and Nes, 2004). Plant feeding insects require two fat soluble vitamins,

β carotene for the normal pigmentation and vitamin E for boosting the fecundity while

the absence of vitamin E affects normal sperm production (Dadd, 1985).

Environmental factors such as nutrient availability result in considerable

intraspecific variation among plants in susceptibility to herbivory. Nutrient limitation

may alter the pattern of allocation to defence (Bryant et al., 1983 and Wilkens et al.,

1996), affect the nutritional quality of foliage as food for herbivores (Bentz et al., 1995

and Holopainen et al., 1995) or affect the growth of a grazed plant (Fay et al., 1996).

Well-nourished plants tend to grow better under stress than the poorely nourished

(Maschinski and Whitham, 1989). Herbivory reduces plant photosynthetic area

imposing a greater burden on the carbon budget of the plant making them less tolerant

to herbivory (Borowicz, 1997).

Many plants contain defensive compounds that are toxic or interfere with

digestion in the gut of phytophagous insects (Bernays and Chapman, 1994). Plant

feeding insects are quite adept at regulating the intake of important nutrients, and they

employ a range of pre and post ingestive mechanisms to reduce the intake of harmful

50

chemicals and their toxicity (Simpson and Rauberheimer, 2000). Allelochemicals,

water and nitrogen content and physical attributes of foliage are considered to be

determinant factors greatly affecting the quantity and quality of plant as diet, which in

turn suppress insect growth and survival (Appel and Schultz, 1994).

Lepidopteran caterpillars are voracious feeders and usually have very high food

consumption rate (Cunninghan et al., 1999). The consumed food is assimilated and

assimilation is high when the nutritive quality of the host plant is high (Elsayed, 1998).

Consumption of foliage by lepidopteran larvae had been studied in Spodoptera littura

(Fab.) by Chibber et al. (1995); in muga silkworm, Antheraea assama Westwood by

Das et al. (2002); in Lymantria plumbalis Hampson by Rao et al. (1994) and in Bombyx

mori L. by Rajanna and Puttaraja (2000) and Miranda et al. (2003).

Many of the phytophagous pests are sensitive to changes in the nutritional

quality of their host plants. The morphology, growth and development of pests were

affected when the hosts plants are modified by secreted Bt protein in transgenic plants

with genes from Bacillus thuringiensis Berliner. Sublethal effects of Bt proteins on the

host larvae may reduce their nutritional quality for the parasitoid and poor nutritional

quality of the host results in detrimental effects on the development and survival of the

natural enemies (Murugan et al., 2000).

The effect of plant extracts on food consumption and utilization by lepidopteran

caterpillars had been worked out. The effect of neem leaf extract on castor semi-looper,

51

Achaea janata Lin. (Chari and Muralidharan, 1985); neem seed kernel suspension

against S. litura (Sitaramaiah et al., 1986); azadirachtin on tobacco budworm,

Heliothis virescens (Fab.) (Barnby and Klocke, 1987); annona seed extract on S. litura

(Boreddy and Chitra, 2001) and some derived compounds on S. litura had been reported

(Gayathiri et al., 2003).

Effect of chemical pesticides on the consumption and utilization of food have

already been reported. Action of diflubenzuron and trifluron on food consumption,

growth rate and food utilization by Spodoptera littoralis Boisdual larvae was analysed

by Radwan et al. (1986). Nath et al. (1997) investigated the toxic impact of

organophosphorous insecticides on the growth of B. mori. Singh and Nath (2002)

reported the efficacy of sublethal dietary concentration of insecticides on the growth

and development of first instar larvae of maize stalk borer, Chilo partellus Swinhole.

The pests of cotton mainly prefer fruiting parts, especially squares and bolls for

feeding, thus causing direct damage and heavy yield loss in non Bt cotton. The larvae

move to these parts after completing their initial feeding on leaves. The majority of the

newly hatched larvae of cotton pests initially feed by scraping chlorophyll in the tender

leaves and as they grow, move over to the squares and bolls for further feeding and

development (Manjunath, 2006). In the case of Bt cotton, the pests lays their eggs on

leaves and the larvae scrape and feed on the surface of the leaf soon after hatching and

get killed. However the eggs laid directly on the squares, flowers and bolls survive,

depending on the levels of toxins expressed in these parts.

52

Cry1Ac expression levels were the lowest in the ovary of flowers and boll rind

of green bolls. The fruiting parts, especially squares and bolls are the most preferred for

feeding by H. armigera causing direct damage and heavy yield loss (Kranthi,

2006). Manjunath (2006) reported that in Bt cotton plant the expression of Bt protein

was high in the leaves through out the plant life.

Deml et al. (2001) reported that various developmental, feeding and nutritional

indices of phytophagous pests from three orders (Lepidoptera, Coleoptera and

Homoptera) were reduced in response to the consumption of Bt endotoxin. Prutz and

Dettner (2004) observed that the parasitoid, Cotesia flavipes (Cameron) had lower

fitness when reared on C. partellus (Swinhole) (Lepidoptera : Crambidae) hosts that had

fed on Bt corn.

Most of the toxic chemicals and botanicals produced noticable changes in the

physiology of organisms. In most of the organisms, the toxicity response was mainly

through the mobilization of tissue proteins. Proteins were synthesized or broken down

in response to different toxins. During the last larval instar of holometabolous insects,

these proteins nearly accounted for 70 -80 percent of the total soluble proteins by

weight (Tefler and Kunkel, 1991).

Quantitative changes of haemolymph and whole body proteins during the

development of various insects have already been reported. Slovak et al. (1992)

characterized the storage proteins from the haemolymph of Hamestra brassicae L.

53

Ghosh and Chel (2000) studied the electrophoretic protein pattern of haemolymph and

ovary of Indian grass hopper, Oxya hyla hyla Serv. Electrophoretic studies on the

developmental profile of protein in haemolymph, fat body and ovary of the red cotton

bug, Dysdercus cingulatus Fab. were conducted by Mohan and Muraleedharan (2001).

Agarwal et al. (2002) observed the electrophoretic variation in the isoenzymatic pattern

of the mustard aphid, Liapaphis erysimi (Kalt.) in relation to host plants.

The present study was designed to find out food consumption, utilization and

conversion parameters of three instars of S. derogata caterpillars fed with conventional,

F1 and F2 transgenic cotton varieties. The electrophoretic pattern of whole body

proteins of S. derogata larvae raised on F1 and F2 Bt cotton and conventional cotton

plant was also analysed for studying the changes in the protein profile of S. derogata in

response to host plant transgenicity.

54

MATERIALS AND METHODS

Feeding types

S. derogata caterpillars were fed with three types of leaves from conventional,

F1 and F2 Bt cotton varieties - regular leaves (those used for routine feeding), extremely

tender leaves and old mature leaves. Growth was checked by feeding the caterpillars

with tender bolls from conventional, F1 and F2 Bt cotton.

Growth Studies

The growth studies of S. derogata larvae were analyzed based on the methods

developed by Kaushal et al. (1988). Food consumption and utilization of third, fourth

and fifth instar control caterpillars and those exposed to trangenic cotton plant parts

were calculated. The following measurements and calculations were done to find out

the food utilization and growth pattern.

Food consumption = Initial weight of leaves - weight of unconsumed leaves

Assimilation = Food consumed - weight of faecal matter (in mg dry weight)

Tissue growth = Final body weight - initial body weight of larvae (in mg dry

weight)

Approximate digestibility (AD) = %)(in100nConsumptio

onAssimilati×

Tissue growth efficiency (EFD) = %)(in100onAssimilati

growth Tissue×

Ecological growth efficiency (ECI) = %)(in100nConsumptio

growth Tissue×

55

Control S. derogata larvae were kept in well aerated 100 ml plastic containers

and in each container pre weighed leaves were provided ad libitum. Unfed leaf material

was weighed and food consumed by each caterpillar was quantified. The faecal matter

was collected and dried before weighing. The approximate dry weight equivalence was

calculated wherever wet weight measurements were used as such.

Electrophoretic Studies

For electrophoretic studies, whole body tissues of fifth instar S. derogata larvae

reared on conventional cotton, F1 and F2 generation Bt cotton were used.

Protein Determination

Protein concentration of whole tissue determined following the method of

Lowry et al. (1951). Bovine serum albumin served as the standard protein. Protein

determination was done to standardize the amount of tissue to be used for the

preparation of sample for electrophoretic studies.

The following reagents were prepared.

Reagent A : 20 percent sodium carbonate in 0.1 percent sodium hydroxide.

Reagent B : 0.5 percent copper sulphate solution in 1 percent sodium potassium

tartarate solution.

Reagent C : Mixed 50 ml of reagent A with 1 ml reagent B, just prior to use.

Reagent D : Folin Ciocalteau reagent, which was diluted with equal amount of

water before use.

56

The following procedure was adopted.

100 mg of the larval tissue were ground in a mortar and pestle with 8 ml of 20

percent TCA and centrifuged for 15 min at 3500 rpm and the clear supernatant was

discarded. The precipitate was dissolved in 5 ml of 0.1N sodium hydroxide and

centrifuged for 15 minutes. The supernatant was made upto 5ml with 0.1N NaOH.

From this 1.0 ml was taken and treated with 5ml of reagent C for 10 minutes and 0.5 ml

of folin ciocalteau reagent was added and the optical density was read after 30 minutes

at 60 nm in a spectrophotometer. Protein content was read from a preconstructed

standard graph.

Sample preparation

The tissues were homogenised in a glass homogenizer with Teflon pestle in 150

µl of homogenizing buffer (Tris-EDTA, pH 6.8). Homogenization was done in an ice-

bath under freezing conditions. Every sample were homogenized separately.

Homogenised samples were centrifuged at 12,000 rpm for 10 minutes at 4°C in a

cooling centrifuge. Supernatant was taken out and mixed with equal volume of sample

buffer and stored at -4°C in a deep freeze.

Final protein sample

Equal volume of sample buffer containing 0.15 M Tris Hydro Chloride (pH 6.8),

10 percent SDS, glycerol, β-mercaptoethanol and traces of bromophenol blue were

added to the supernatant samples, boiled for 2 minutes and kept on ice immediately to

57

prevent denaturation by over heating. Samples were again centrifuged at 4000rpm for 5

minutes at 4°C before loading into the well in the precasted gel.

SDS-PAGE

SDS-PAGE (Sodium Dodecyl Sulphate Poly Acrylamide Gel Electrophoresis)

was carried out following the method of Laemmli (1970) using separating and stacking

slab gels. Each well was loaded with 50µl sample. A constant current of 60 volts for

stacking and 120 volts for running gel were used for 3 hours. Gels were stained

overnight in coomassie brilliant blue R. 250. After the run, the gels were destained,

stored in 7 percent acetic acid, photographed and subsequently documented in a

computerised gel documentation unit to locate the different densitometric peaks.

58

RESULTS

Food consumption of third, fourth and fifth instar S. derogata caterpillars that

fed conventional cotton regular leaf was 0.50 ± 0.03 mg; 0.6 ± 0.015 mg;

0.71 ± 0.08 mg respectively. Food consumption decreased in caterpillars that fed

transgenic cotton plant parts. The maximum decrease in food consumption (-47.3

percent) was observed in third instar S. derogata caterpillars fed with F1 Bt cotton bolls.

The decrease was minimum (-11.5 percent) in fourth instar caterpillars that fed F2 Bt

cotton bolls. Consumption was maximum in fifth instar caterpillars that fed

conventional cotton tender leaf (0.92 ± 0.02 mg) (Table 2.1).

Based on Tukey analysis, the tender leaf consumption in third instar S. derogata

raised on F1 and F2 Bt cotton was significantly different from conventional cotton raised

S. derogata (Table 2.7). The mature leaf consumption in F1 and F2 Bt cotton raised

fourth instar S. derogata was not significantly different from conventional cotton

(Table 2.8). Boll consumption in fifth instar S. derogata raised on F1 Bt cotton was

significantly different conventional cotton while the fifth instar S. derogata raised on F2

Bt cotton did not differ significantly (Table 2.9).

Assimilation of third, fourth and fifth instar S. derogata caterpillars that fed

conventional cotton regular leaf was 0.41 ± 0.05 mg; 0.49 ± 0.01 mg; 0.60 ± 0.07 mg

respectively. Assimilation decreased in third instar caterpillars that fed F1 Bt cotton old

leaf, 0.32 ± 0.06 mg. Assimilation further decreased in caterpillars that fed on F1 Bt

cotton bolls, third instar, 0.04 ± 0.001 mg; fourth instar, 0.18 ± 0.004 mg; fifth instar

59

0.09 ± 0.005 mg respectively. Assimilation was maximum in fifth instar S. derogata

caterpillars that fed conventional cotton tender leaf (0.80 ± 0.03 mg) (Table 2.2).

Tender leaf assimilation in fourth instar S. derogata reared on F1 and F2 Bt

cotton was significantly different from conventional cotton (Table 2.10). Mature leaf

assimilation in third instar S. derogata fed on F1 and F2 Bt cotton was significantly

lower from conventional cotton (Table 2.11). Cotton boll assimilation in fifth instar

S. derogata raised on F1 and F2 Bt cotton was significantly different from conventional

cotton (Table 2.12).

Tissue growth in S. derogata grown on conventional cotton regular leaf was

0.06 ± 0.008 mg, 0.07 ± 0.008 mg, 0.08 ± 0.008 mg in the third, fourth and fifth instars

respectively. Tissue growth was maximum in S. derogata caterpillars that fed

conventional cotton tender leaf, 0.09 ± 0.005 mg. Tissue growth was closer to

maximum in S. derogata caterpillars that fed F2 Bt cotton tender leaf, 0.07 ± 0.008 mg.

Tissue growth decreased in caterpillars that fed F1 Bt cotton bolls, 0.004 ± 0.0001 mg;

0.006 ± 0.0001; 0.008 ± 0.0001 mg in third, fourth and fifth instars respectively

(Table 2.3).

Tissue growth in third instar S. derogata that fed on tender leaf of F1 and F2 Bt

cotton was significantly different from conventional cotton (Table 2.13). Tissue growth

in F1 Bt cotton mature leaf fed fourth instar larvae was significantly different from

conventional cotton, while it did not differ significantly in F2 Bt cotton fed larvae

60

(Table 2.14). Tissue growth in third instar S. derogata was not significantly different in

F1 Bt and F2 Bt cotton bolls from conventional cotton bolls (Table 2.15).

Approximate digestibility of third, fourth and fifth instar S. derogata caterpillars

that fed conventional cotton regular leaf was 80.8 ± 6.14 percent; 81 ± 1.4 percent; 83.4

± 1.5 percent respectively. Approximate digestibility decreased in caterpillars fed with

F1 Bt cotton bolls. The maximum decrease (-61 percent) was observed in the fourth

instar caterpillars fed with F2 Bt cotton bolls. The decrease was minimum (-2.44

percent) in third instar caterpillars which were fed with F1 Bt cotton old leaf (Table 2.4).

Approximate digestibility of F1 Bt cotton tender leaf raised fourth instar

S. derogata was significantly different from conventional cotton while the approximate

digestibility of F2 Bt cotton tender leaf raised S. derogata did not differ significantly

from conventional cotton (Table 2.16). Approximate digestibility in fifth instar

S. derogata fed on F1 and F2 Bt cotton bolls was significantly lower than conventional

cotton raised S. derogata (Table 2.18).

Tissue growth efficiency in conventional cotton regular leaf was 15.5 ± 0.2

percent in the third, 14.4 ± 0.3 percent in the fourth, 13.6 ± 0.9 percent in the fifth instar

S. derogata caterpillars. Tissue growth efficiency was declined by 9.1 ± 0.7 percent,

9 ± 0.5 percent, 9.1 ± 0.2 percent in the third, fourth and fifth instar caterpillars fed with

F1 Bt cotton tender leaf. The decrease in tissue growth efficiency was maximum in fifth

instar

61

S. derogata caterpillars that fed F1 Bt cotton old leaf, 4.9 ± 0.5 percent. Tissue growth

efficiency was maximum 15.5 ± 0.2 percent in S. derogata caterpillars that fed

conventional cotton regular leaf (Table 2.5).

Tissue growth efficiency of third instar S. derogata raised on F1 and F2 Bt cotton

tender leaf did not differ significantly from conventional cotton reared S. derogata

(Table 2.19). Tissue growth efficiency of F1 and F2 Bt cotton mature leaf fed fifth instar

S. derogata was significantly different from conventional cotton (Table 2.20). Tissue

growth efficiency in fourth instar S. derogata that F1 Bt cotton bolls did not differ

significantly from conventional cotton, while it was significantly different in F2 Bt

cotton bolls (Table 2.21).

Ecological growth efficiency of third, fourth and fifth instar S. derogata

caterpillars that fed conventional cotton regular leaf was 10.2 ± 0.2 percent, 11.7 ± 0.1

percent, 11.5 ± 0.1 percent respectively. Ecological growth efficiency decreased in

caterpillars that fed F1 Bt cotton bolls 3.8 ± 0.2 percent in the third, 3.8 ± 0.8 percent in

the fourth and 3 ± 0.8 percent in the fifth instar respectively. Ecological growth

efficiency was closer to maximum in fifth instar S. derogata caterpillars that fed

conventional cotton tender leaf was 10.1 ± 0.6 percent (Table 2.6).

Ecological growth efficiency in fourth instar S. derogata raised in F1 Bt cotton

tender leaf was significantly different from conventional cotton while it was not

significantly different in F2 Bt cotton (Table 2.22). Ecological growth efficiency in fifth

62

instar S. derogata a fed on F1 and F2 Bt cotton mature leaf was significantly different

from conventional mature leaf cotton fed S. derogata (Table 2.23). Ecological growth

efficiency in third instar S. derogata raised on F1 and F2 Bt cotton bolls was not

significantly different from conventional cotton bolls reared S. derogata (Table 2.24).

The SDS PAGE pattern of broad range marker proteins showed eleven bands

(Table 2.25). The whole body protein profile showed the presence of eight proteins in

S. derogata larvae reared on conventional cotton plant (Table 2.26) and eighteen

proteins in larvae reared in F1 generation Bt cotton plant (Table 2.27). The whole body

protein profile showed the presence of eleven proteins in larvae reared on F2 generation

Bt cotton plant (Table 2.28).

63

DISCUSSION

The energy parameters of third, fourth and fifth instar larvae of S. derogata

varied significantly in response to different stages of Bt and non-Bt cotton leaves and

bolls. Generally in most of the phytophagous lepidopteran pests the food consumption,

assimilation and tissue growth rate decreased significantly in caterpillars fed with a low

quality or with modified leaves. The quantity of food consumed is maximum in

S. derogata caterpillars that fed with non-transgenic tender cotton leaf (0.79 ± 0.04 mg)

whereas the quantity leaf consumption is greatly reduced in S. derogata fed with F1

(0.46 ± 0.03 mg) and F2 (0.68 ± 0.04 mg) generation Bt cotton leaves.

The decrease in the consumption of F1 and F2 Bt cotton leaves by S. derogata is

due to the expression δ-endotoxins in the transgenic cotton leaves. The results of the

present study agree with those of Sanders et al. (2007) who reported that in the adult

parasitoid reared on Bt maize fed Spodoptera frugiperda (J.E. Smith) the consumption

rate greatly decreased and thereby the larvae were significantly small (15-30 percent)

than those reared in hosts fed either of the conventional maize hybrids.

Similarly Saleem et al. (2003) reported that extracts of Peganium harmala, L.

and Aspergillus indica A. Juss when tested at different concentration on 2nd

, 3rd

and 4th

instar larvae of S. littoralis had a deterimental effect on the food intake, consumption

and all associated metabolic parameters compared to the control. Timmins and

Raynolds (1992) reported a similar reduction in the efficiency of food utilization of

Maduca sexta (L.) following treatment with azadirachtin. It was hypothesized that in

64

the absence of adequate supply of aminoacids and other nutrients or any other

modification in the diet resulted in the diversion of metabolic pathways thereby

reducing the growth rate.

Food consumption rate was significantly decreased when the lepidopteran was

exposed to non-target pesticide or any other toxic compounds. Radwan et al. (1986)

noticed that the lowest food consumption level in S. littoralis larvae fed on leaves

treated with diflubenzuron as compared with the larvae fed on trifluron treated leaves or

control larvae. The larvae have shown a proportional relationship between food

consumed and consumption index and growth rates. Higher concentration of botanical

insecticides resulted in reduction in the feeding by S. litura supplied with black gram

leaves treated with neem oil (Venkateswaralu et al., 1988) and a similar effect on

Achaea janata (Fab.) fed with castor leaves treated with Vemidin, neemol and nemidin

was observed by Purohit et al. (1989).

Jeyabalan et al. (1998) investigated the effect of azadirachtin and neem seed

kernel extract on the growth of teak defoliator, Hyblaea purera. The average

consumption of azadirachtin and neem seed kernel extract treated larvae were

significantly lower than control insects and the percentage of feeding and ovipositional

deterring were higher in both the treatments in a dose dependent manner.

In Eligmanarcissus indica (Rothchild) the food consumption rate was greatly

reduced when exposed to neem seed kernel extract. At a neem seed kernel extract

65

concentration of 250 ppm, the food consumption index decreased to 3.6 ± 0.12 while at

1000 ppm it decreased to 1.58 ± 0.24. The relative consumption rate decreased from

1.44 ± 0.56 in the control to 0.17 ± 0.001 in 1000 ppm treatment (Joseph, 2000).

Boreddy and Chitra (2001) reported lower food consumption in S. litura exposed to

sublethal doses of annona seed extract. Growth was affected in S. litura was due to the

utilization of more energy for detoxification of the toxic seed extract.

Abdel-Rahman and Al-Mozini (2007) reported the difference between food

consumption index of the S. litura fed with the leaves of treated with extracts of Rhazya

stricta (Decne) and Solenostemma argel (Delile) after five days of feeding in

comparison with untreated larvae were significant.

Food assimilation and digestion was decreased in all instars fed with Bt cotton

leaves. In fifth instar S. derogata fed with F1 and F2 Bt cotton tender leaves, the

assimilation was greatly reduced (0.56 ± 0.07 mg and 0.70 ± 0.03 mg respectively)

compared to the larvae fed with non Bt cotton leaves (0.80 ± 0.03 mg). The reduction

of assimilation and digestion may results from the formation of covalent bonds with

dietary proteins or digestive enzymes (Abdel-Rahman and Al-Mozini, 2007). Similarly

Senthilnathan et al. (2009) reported that the quality of food ingested and assimilation by

Nilaparvatha lugens Stan on neem treated rice plants was significantly less than on

control rice plants.

66

Tissue growth, approximate digestibility, tissue growth efficiency and ecological

growth efficiency in second, third and fourth instar larvae of S. derogata greatly

reduced when fed with F1 and F2 Bt cotton leaves than control. Ramraj et al. (1992)

reported that oral administration of different sublethal concentrations of Phyllanthus

fraternus Webst extract to fifth instar larvae of Pericallia ricini F. showed a maximum

depletion of all feeding parameters. The consumption rate decreased by 62.9 percent in

800 ppm while assimilation rate decreased by 19.5 percent, ecological growth

efficiency by 99.8 percent and tissue growth efficiency by 99.8 percent and tissue

growth efficiency by 68.6 percent. The results were coincide with the present study.

Joseph (2000) recorded an increase in co-efficient of approximate digestibility

(CAD) and efficiency of conversion of ingested and digested food (ECI and ECD) in

E. indica when exposed to 500 to 1000 ppm of neem seed kernel extract. CAD

increased from 0.33 ± 0.001 (control) to 0.61 ± 0.44 (1000 ppm) and ECD increased

from 0.70 ± 0.040 in control of 1.06 ± 0.174 in 1000 ppm treatment. The increase was

expected to reflect the compensation of feeding inhibition. Inspite of the increase in

CAD, ECI and ECD, the relative growth (RGR) remained significantly low, showing

very clearly wastage of energy due to the influence of toxicants.

Food consumption, assimilation, tissue growth, approximate digestibility, tissue

growth efficiency and ecological growth efficiency were significantly different for the

three instars of B. mori treated with individual as well as binary administrations of

copper oxy chloride and acetamiprid (Bai, 2004). Similar trend was observed in the

67

present study, the effects of Bt toxin on the nutritional response of S. derogata was

more pronounced at the third and fourth instars than fifth instar larvae. The results

coincided with the findings of Saleem et al. (2003) who found that when different

instars of S. littoralis were treated with plant extract of Peganum harmala (Syrian Rue)

the food consumption and assimilation rates greatly reduced in second and third instars

compared to fourth instar larvae. In this study the food consumption of fourth instar

caterpillars that fed F1 Bt cotton regular leaf was 0.55 ± 0.021 mg and it increased to

0.66 ± 0.09 mg in the fifth instar.

In the present study the food consumption and assimilation were greatly reduced

when the S. derogata caterpillars were fed with tender Bt cotton leaf than the mature

leaves. The reason is the expression of δ endotoxin is different in different parts. The

younger leaves expressed more Bt toxin than the older one. The results were supported

by Kranthi et al. (2005) who reported that the Cry1Ac expression was found to be

variable among the hybrids and also between different plant parts. The younger leaves

of Bt cotton plants were found to have the highest levels of Cry1Ac expression followed

by squares, bolls and flowers. Food consumption in third instar S. derogata caterpillars

that fed F2 Bt cotton tender leaf was 0.68 ± 0.04 mg and that fed on F2 Bt cotton old leaf

was 0.43 ± 0.03 mg. Tukey analysis also showed that food consumption in S. derogata

was significantly lower in F1 and F2 Bt cotton tender leaf than conventional cotton.

Many previous studies witness that Bt cotton leaves were most effective against

cotton boll worm and showed that highest level of Cry1Ac expression than flowers,

68

squares and bolls (Greenplate et al., 2000 and Kranthi et al., 2005). This variability in

the expression of Cry1Ac toxin in different parts of Bt cotton can create the variability

in the survival and development of target and non-target pests (Adamczyk and Gore,

2004).

Gore et al. (2001) reported that Cry1Ac toxin expression was variable among Bt

cotton plant parts and the level of expression decreased consistently as the plant aged.

Similar trend was observed in the present study also. Hong et al. (2006) observed that

Bt protein content in functional leaves are different at various developmental stages and

was different among various plant parts. Variation exists in levels of expression of the

toxins in different varieties of commercial Bt cotton (Adamcyk and Gore, 2004) and in

different parts of other transgenic plants (Adamyzk and Summerford, 2001) were

already reported.

Electrophoretic pattern of S. derogata larvae raised on F1 and F2 Bt cotton leaf

showed modifications in the total body protein profile. S. derogata larvae fed with F1

and F2 Bt cotton leaves revealed the presence of 18 bands and 11 bands respectively.

Except 43 KDa protein recorded in F1 Bt cotton fed larvae all other proteins may be the

stress protein with a mw range of 87 KDa to 23 KDa. In F2 cotton fed larvae 9 new

proteins were in the mw of 205 KDa, 204.8 KDa, 98.5 KDa, 97.8 KDa, 64.3 KDa, 62.2

KDa, 43.5 KDa and 29.3 KDa whereas in non-Bt cotton fed S. derogata only 8 proteins

with a mw range of 204.3 KDa to 29 KDa were observed.

69

The proteins recorded in treated group were not observed in control group. The

new proteins observed in the Bt cotton leaf fed larvae is due to synthesis of stress

protein in response to Bt toxin. Number of stress proteins were synthesized in the body

of insect after exposure to many stressors including heat stress (48°C for 4 hrs), a

variety of pollutants, bacterial infections and natural enemies (Gregorc et al., 2004,

2007, Lipinski and Zoltowska, 2005 and Scharlaken et al., 2008). Several stress

proteins have been reported to be broad spectrum in their action, which are produced in

insects in response to the bacterial toxin and of shorter duration in nature. The

expression of stress proteins is triggered only by stressors. The stress protein response

may involve increased expression of one or more proteins (Hranitz et al., 2009).

Kajira and Yamashita (1989) and Davis et al. (1990) reported a significant

increase in the total body protein content in three lepidopterans, S. litura, B. mori and

Lymantria dispar (Lin.)upon methoprene treatment. Bradfield et al. (1990) also

recorded an increase in the number of protein bands in male B. mori larvae. Adams et

al. (1989) and Kempatomm et al. (1990) reported that methoprene brought about an

increase in RNA synthesis which stimulate the vitellogenin gene increasing the

vitellogenin content in Musca domestica J. and Gryllus bimaculatus De Geer. Similar

findings were made by Sohal and Rup (1997) in Lipaphis erysimi (Kaltenbatch).

Padmaja and Rao (2000) recorded the appearance of newer proteins in the final

instar larvae of H. armigera treated with three plant oils. Artemisia annua (Lin.),

Ageratum conyzoides (Lin.) and A. indica (A Juss). The late appearance of some

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proteins in treated insects suggest the interference of the three oils with protein

synthesis. The new proteins were formed in region between 40 and 58.5 KDa. In

contrary Neoliga et al. (2007) reported that the azadirachtin supressed 22,26,40 and 56

KDa head peptides in various treatment of the larvae of H. armigera and also reported

some additional low molecular weight poly peptides of upto 11 KDa in H. armigera

treated with azadirachtin. Jinham and Das (2004) studied the protein profile of

C. cephalonica exposed to carbon dioxide and reported the appearance of new protein

associated with CO2 stress and they also reported that when exposed to double

fumigation, two new proteins were generated compared to the control.

Sohal and Rup (1997) showed that chemical stress produced significant changes

in the tissue protein content, with increase in the number of protein bands and their

relative mobility, indicating synthesis of new proteins to combat stress. Fluctuations in

protein concentration in an insect are based on the rate of protein synthesis, breakdown

and water movement between tissues and haemolymph (Neoliga et al., 2007).

In the present study, when compared to F1 cotton leaf fed larvae of S. derogata

the number of stress proteins were less in F2 Bt cotton fed larvae. It may be due to the

lower expression of Bt genes in F2 Bt cotton plant. Insecticidal protein content in Bt

cotton is also variable with plant age, plant structure or under environmental stress.

Reduction of Bt protein content in later generation of Bt cotton may be due to the over

expression of Bt gene at the earlier stages (Dong and Li, 2006).