INDUCTION OF HEAT SHOCK PROTEINS UNDER TEMPERATURE STRESS IN SILKWORM BOMBYX MORI L. RACES ANALYSED BY SDS PAGE

DISSERTATION SUBMITTED TO UNIVERSITY OF MYSORE IN PARTIAL FULFILMENT FOR THE AWARD OF DEGREE OF MASTER OF SCIENCE IN SERICULTURE TECHNOLOGY

GK. RAJESH Reg. No. 03S-20010

CENTRAL SERICULTURAL RESEARCH AND TRAINING INSTITUTE CENTRAL SILK BOARD, MINISTRY OF TEXTILES, GOVT. OF INDIA SRIRAMPURA, MANANDAVADI ROAD, MYSORE 570 008 MAY 2004

DECLARATION

Hereby I declare that the dissertation entitled Induction of Heat Shock Proteins Under Temperature Stress in Silkworm Bombyx mori L. Races Analysed by SDS PAGE embodies the results of the work done by me at the Molecular Biology Laboratory, Central Sericultural Research and Training Institute, Mysore, under the guidance of Dr. S. Chitra, Senior Research Officer and Dr. S. Sreekumar, Senior Research Officer. I further declare that this dissertation or part thereof has not been the basis for award of any other degree, diploma, fellowship or similar title.

GK RAJESH (Asst. Sericulture

Officer,

SERIFED,

Kerala) Mysore 16-05-2005

Final Year Msc. Student. Reg. No. 03S-20010 Training Division, CSR&TI, Mysore

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3

CONTENTS Introduction

1-9

Review of Literature

10 - 19

Materials and Methods

20 - 33

Results and Discussion

34 - 45

Summary

46

Reference

47 - 54

Figures and Graphs (between pages)

33 & 34

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Chapter 1 INTRODUCTION It is well known that temperature has a pervasive effect on insects. Nearly every aspect of an insect’s life is influenced by temperature, from direct effects on the kinetics of enzyme relations, to defining the limits of physiological function and behavior, and ultimately to shaping of evolutionary pathways. As a group, insects, more than any other eukaryotic taxon, have evolved not only to survive but to flourish in a wide variety of thermal environments. The versatility of insect species to temperature tolerance is illustrated by the examples of the larvae of a dipteran insect Polypedilum vanderplanki. Inhabiting Nigeria, which can tolerate temperature as low as –270°C (Hinton, 1960) and the larvae of coleopteran insects Rhyzopertha

dominica. and Sitophylus oryzae. Which can tolerate temperature as high as 80°C (Evans, 1981). Silk is a valuable commodity. Silk products have been held at high esteem throughout the world ever since its invention. The bulk of silk is obtained from the domesticated mulberry silkworm. Bombyx mori, the most important source of natural silk is by nature a delicate insect which is quite sensitive to temperature fluctuations. The species was domesticated during 2700 BC (Jacqes, 1995). Since then, the insect has been being reared under optimum conditions created by man. Such careful domestication over centuries has apparently deprived the insect of opportunities to acquire thermotolerance. However the sericigenous fauna distributed across the world show significant variations among themselves with respect to the degree of thermotolerance exhibited, apart from productivity and quality of silk. 1.1. Silkworm Races The best known classification of silkworm races is in terms of the number of generations that a race can live each year. Some races hatch only once a yearknown as univoltine. Those hatch twice a year are bivoltines and those hatch several times are polyvoltines. Uni and bivoltines thrive in the temperate zone and polyvoltines thrive in the tropical zone (Vijay, 1985). A sericulture belt running parallel to the tropic of cancer circles the earth.

The Temperate Zone is extended up to 50° N latitude and hosts uni/ bivoltine silkworm races, which are capable of producing one or two generations only in a year. This zone include Japan, China, Korea, Northern India, Burma, Iran, Turky, the southern fringe of Russia, Lebanon, Syria, Cyprus, Greece, Romania, Bulgaria, 5

Hungary, Yugoslavia, Spain, Italy and Poland. The tropical zone extend up to 10° S latitude and host polyvoltines silkworm races, which can produce more than two generation per year. This zone includes Southern China, Thailand, Central and Southern India, Central Africa, Brazil and Peru (Nanavati, 1990). Bivoltine silk has an edge over polyvoltine silk. Silk reeled from the polyvoltine cocoons is very often of the ‘E’ grade quality compared to ‘A’ and ‘B’ grade quality silk reeled from bivoltine cocoons. Bivoltine silk is also much thicker than polyvoltine silk. Fabrics made of bivoltine silk are comparatively popular throughout the world. Bivoltine cocoons also have a thicker shell and are amenable to being used in sophisticated semi-automatic reeling machines. Apart from silk quality another important factor prompting a switch over from polyvoltine to bivoltine is the high productivity of bivoltine hybrids. A number of countries in the tropics are indeed making efforts at switching over from polyvoltine to bivoltine (Vijay, 1985). Given the challenge posed by countries in the temperate zone which can produce high quality bivoltine silk at comparatively cheaper rates, India is not only facing stiff competition in the international silk market but also struggling to keep the domestic silk industry upright from being flooded with imported silk and to find market for the silk yarn produced by Our farming community. The challenge is big and the only possible way to face it appears to be - producing bivoltine silk. During 1980’s India emerged as worlds second largest producer of silk (Nanavati, 1990). Since independence, India has increased its silk production from 969 metric tones in 1950’s to 13970 metric tones in 2003-04 [Anonymous, 1990; 2004). This improvement has been possible partly due to the Tropical Sericultural

Technology developed during 1970’s. The major thrust of Tropical Sericultural Technology was towards the introduction of bivoltine silkworm, breeding of new silkworm breeds especially suited our tropical climate and development of new rearing technology. But only 40% of the potential of bivoltine silkworms could be realized at the farmer’s level. The yield gap analysis showed that in comparison to the polyvoltines, the bivoltine silkworms are much less adapted to the tropical condition (Datta and Chatterjee, 1992). A breakthrough was achieved under the Bivoltine Sericulture Technology

development Project (1991-099), wherein many productive and qualitatively superior bivoltine hybrids were developed at Central Sericultural Research and Training Institute, Mysore by utilizing Japanese commercial hybrids as breeding 6

resource material (Basavaraja, et.al., 1995). But the full potential of these new highly productive bivoltine hybrids could be realized only under optimum conditions of quality food and environment. Therefore these hybrids have been identified for rearing during climatically favorable months (August to February) in South India. The hot climatic condition of tropics prevailing in summer is not conducive to rear these productive bivoltine hybrids (Suresh Kumar et.al., 1999). As a result of this and other sub-optimal conditions prevailing in Indian farms only 40% of the genetic potential of these breeds is realized (Vijay, 1985). 1.2. Breeding for Robustness Among many factors attributed to poor performance of the bivoltine strains under tropical condition, the major aspect is that many quantitative characters such as viability and cocoon traits decline sharply when temperature is higher than 28°C, since bivoltine breeds are fixed in temperate climatic conditions. The risk of hybridization of polyvoltine to bivoltine could not be taken due to the delay in fixation of economic characters. The need for high temperature resistant strains was so important, the veteran silkworm geneticist Y. Tazima started a breeding experiment, by using Pure

Mysore and C134a as parent breeds, but did not yield expected outcome. Tazima concluded with an apprehension whether the heavy cocoon trait was negatively correlated with that of high temperature resistance (Tazima and Ohnuma, 1995). The only way out was to evolve tropical bivoltine breeds. Thus a breeding technique was evolved to select the robustness genes along with its modifiers in high temperature conditions (Datta, 2003). Consequently in 1988 a temperature tolerant bivoltine hybrid namely CSR18 X CSR19 was developed and authorized for commercial exploitation throughout the year (Sureshkumar, et.al., 2002). Though the introduction of CSR18 X CSR19 in the field during summer months had considerable impact, the productivity level and returns realized did not match to that of other CSR hybrids. Therefore the acceptance level of this hybrid with the farmer was not up to the expected level (Sureshkumar et.al., 2005). The front ranking breeders in the field themselves agree to the fact that it is a difficult task to breed such bivoltine breeds which are suitable to high temperature environment and yet productive (Sureshkumar et.al., 2005). Therefore means other than the conventional breeding methods are to be adopted to attain the goal. Need based breeding strategies have to be formulated based on genetic principles, with 7

the aid of modern biotechnological tools. It may be possible to quantify the factors responsible for the expression of temperature tolerance due to polygenes or any specific set of genes. Resistance to high temperature has been recognized as a heritable character in silkworm and the possibility for temperature tolerant silkworm races were suggested (Kato, et.al., 1989). Thorough understanding of the phenomenon of temperature tolerance in silkworm is an essential pre requisite for attaining any results in this direction. 1.3. The Phenomenon of Heat Shock Heat shock is the thermal injury caused by a sudden increase in temperature. At cellular level a number of abnormalities are produced in response to heat stress. pH and ionic concentration of the cell, biological molecules (proteins, DNA, RNA, lipids and carbohydrates) and cell structure are vulnerable to heat stress. At high temperature, pH of the body fluid drops. Normal pattern of protein synthesis halts. Transfer RNA and ribosomal RNA loose conformational integrity leading to degradation. DNA looses ability to function properly. Lipids and carbohydrates in the body are affected by temperature shock. Cell structure may collapse due to aggregation of intermediate filamentous proteins at the nucleus instead of forming the cytoskeleton. Increase of temperature leads to increase in kinetic energy of macromolecules, decrease of ionic bonds, hydrogen bonds, Van der - Waals bonds etc. and increase its hydrophobic interactions; leading to loss of its shape. Protein denaturation may cause adhering of denatured proteins to DNA and restrict enzymatic access to DNA causing large-scale DNA damage. Thermal death of a multi cellular organism is not usually the consequence of massive cell death per se. but is due to the loss or disruption of cells in a certain critical tissue. The more complex the biological system, the more susceptible it is to high temperature stress. Macromolecules are tolerant than cells, cells more tolerant than tissues and tissues more tolerant than the whole organism. The organism after experiencing the thermal stress may be still alive but may not survive and reproduce, as reviewed by Denlinger and Yocum (1998). Many scientists reported the effect of temperature on silkworm. It has been reported after substantial studies that silkworm produce good quality cocoons only within a temperature range of 22-27°C (Krishnaswamy, et.al., 1973). Quantitative characters such as cocoon weight, shell weight, pupal weight, silk weight, filament length, filament thickness and survival rate of larvae are important parameters to be considered in sericulture. Many of these characters are directly affected by exposure to temperature above 30°C. High temperature could decrease the rate of 8

utilization of protein by silkworms, as reviewed by Sureshkumar, et.al., (2005). Fifth instar larvae exposed to high temperature show low survival rate due to the low feeding activity resulting in the physiological imbalance and poor health of larvae. Exposure to high temperature during the later developmental stages considerably reduce the survival rate, cocoon quality and fecundity (Pillai Venugopala and Krishnaswamy, 1980; 1987). A study conducted with thermo-tolerant bivoltine pure breeds, their single hybrids foundation crosses and double hybrids revealed that a high temperature of 36°C ( at 85% RH) caused deleterious effects on their rearing performance (Sureshkumar et.al., 2003). Yield per 10,000 larvae, cocoon weight, shell weight and shell ratio were low in all the high temperature treated batches. However polyvoltine races reared in tropical countries are known to tolerate slightly higher temperature. It is also true with cross breeds, which have been evolved especially for tropical climate as reviewed by (Sureshkumar et.al., 2003). The deleterious effects of high temperature is more pronounced in productive hybrids, than the robust hybrids (Sureshkumar et.al., 1999). Thermotolerance can be attained by several routes; genetic adaptation, long-term acclimatization and rapid heat hardening. The potential for genetic adaptation was demonstrated by Cavicchi in 1995 as reviewed by Denlinger and Yocum (1998), in lines of Drosophilla melanogaster maintained for 15 years at 10°C, 25°C or 28°C. Heat shock survival was greatest in 28°C flies, and lowest in 18°C. The difference in thermo-tolerance persisted even when the three lines were reared at the same temperature for one generation, thus implying a genetic basis for the difference in thermo-tolerance. Physiological adaptation to environmental changes of short duration requires plalstic responses primarily through the ability to acclimate to the new conditions. High temperature acclimation studies on Drosophilla buzzati proved that selection on acclimated individual can increase thermal resistance (Krebs and Loeshcke, 1996). Further, Kilias, and Alahiotis (1985) concluded that short term indirect selection for heat sensitivity or resistance resulted in diverse correlated responses in behavioural, biochemical and fitness components in Drosophilla

melanogaster, and even short duration selective regimes can induce significant adaptive and evolutionary changes. The third manifestation of increased thermo-tolerance - rapid heat hardening can be elicited by a brief exposure to an intermediately high temperature which in turn provides protection from subsequent and more severe high

9

temperature. It is this response that has been the focus of attention for the huge army of workers who investigate the heat shock response. 1.4. Heat Shock Response Ritossa (1962) reported that heat and the metabolic inhibitor dinitrophenol induced a characteristic pattern of puffing in the chromosomes of Drosophila. This discovery eventually led to the identification of the heat-shock or stress proteins (these names will be used interchangeably) whose expression these puffs represented, the cloning of the genes encoding these proteins, and elucidation of the regulation of expression of these genes. Beginning in the mid-1980's, investigators recognized that many hsps function as molecular chaperones and thus play a critical role in protein folding, intracellular trafficking of proteins, and coping with proteins denatured by heat and other stresses. Accordingly, the study of stress proteins has undergone explosive growth (as reviewed by Feder 1996).

Although heat has received more attention than other hsp-inducing stresses, it is by no means the only inducer. Inducing stresses include ethanol, heavy metals, hypoxia, hyperoxia, changes in pH, free radicals, various poisons and toxins, ischemia, osmotic shock, ionizing radiation, pathogen stress and many others. To date, no stresses have been reported not to induce heat shock proteins. Studies examining stress protein expression in the wild or in response to laboratory simulations of natural stress regimes are still few. Nonetheless, even these few studies are sufficient to demonstrate that patterns of stress protein expression can be correlated with species' natural thermal environments; that is, cells and species from warm environments undergo a stress response at warmer temperatures than counterparts from cool environments (Lindquist, 1981; Burton et.al., 1988; Rebagoodman and Martin Bank 1998; Denglinger Yocum 1998 and Feder and Hoffman 1999).

Thus the term heat shock proteins (Hsps) is a bit of a misnomer, and it is more accurate to refer to these proteins as stress proteins. Even this term is somewhat misleading, because many members of these families play important role in cell function at normal temperature. It has been proven in Drosophilla that the small heat shock proteins Hsp 23 and Hsp 26 exhibit distinct spatial and temporal patterns of constitutive expression during normal development in the absence of stress (Marin et.al., 1993). It is also known that Hsp 70, one of the most prominent products of the heat shock genes is a molecular chaperone, involved in normal protein folding (Becker, et. al., 2004). Yet the term heat shock proteins is so deeply 10

entrenched in the literature and so accurate a descriptor of the response at high temperature that, it is likely to persist for years to come as reviewed by Denlinger and Yocum (1998). Heat shock factor (HSF) is present in the cytoplasm as a latent monomeric molecule that is unable to bind to DNA. Under stressful conditions, the flux of nonnative proteins (which are non-functional, prone to aggregation, protease-sensitive, and bind to chaperones) leads to phosphorylation (P) and trimerisation of HSF. The trimers translocate to the nucleus, bind the promoter regions of heat shock protein (hsp) genes and mediate hsp gene transcription. The activity of HSF trimers is downregulated by hsps (e.g. Hsp70) and the heat shock binding protein 1 (HSBP1) that is found in the nucleus.

Regulation of transcription of heat shock protein genes by heat shock factor http://www-ermm.cbcu.cam.ac.uk Pockley AG (2001)

The Hsps are highly conserved and occur in several well-characterized families, most named by molecular weight (eg: the Hsp 70 family). These proteins are now known to be only part of a large machinery that manages unfolded proteins within the cell. Almost every organism studied from bacteria, plants, invertebrates and vertebrates including man are able to synthesize Hsps in response to various stress (Mascarenhas and Schuler, 1983; Lyashko VN et.al., 1994; Denglinger Yocum 1998; Feder and Hoffman 1999). During initial synthesis, intra-cellular transport and under the influence of cell stress proteins may not be in their native conformation. In non native proteins, residues can be exposed and interact, which can lead non-native proteins to aggregate. Such aggregations can be harmful if not lethal to cell. Hsps and their constitutively expressed cognates function as molecular chaperones. They recognize and bind to non-native proteins, thus impeding aggregation as reviewed by Feder and Krebs, 1997.

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Heat-shock proteins are classified into families on the basis of sequence homology and typical molecular weight as hsp 110, hsp 100, hsp 90, hsp 70, hsp 40, hsp 10 and small heat- shock protein families. In eukaryotes many families comprise multiple members that differ in inducibility, intra cellular localisation and function Feder and Hofman (1999). 1.5. Heat Shock Proteins by size, function and cellular localization Size (kDa) 27-28 60 70-73 90 100-104

Major Function

Cellular Localization

Stabilization of microfilaments Cytokine signal transduction Protein Assembly Protein folding and translocation

Cytosol and nucleus

Protein translocation Receptor regulation Protein folding

Mitochondria Cytosol, nucleus, endoplasmic reticulum, mitochondria Cytosol, nucleus, endoplasmic reticulum Cytosol

(Moseley, 1997)

The threshold temperature for hsp induction is correlated with the typical temperature at which species live. Thermophilic species have a higher threshold than the psychrophilic species. Many species exhibit charecteristic and distinctive patterns of hsp expression (or non expression) during the various stages of development, including gametogenesis, embryogenesis and metamorphosis. Hsps are among the most ancient and highly conserved of all proteins. The archea or archebacteria are the most extremophilic and most primitive organisms. The archeal genome encodes homologues of most hsps represented in other prokaryotes and eukaryotes, aswell as their consensus promoter sequences as reviewed by Feder and Hofman, 1999. The hsp 70 proteins are found to be highly conserved, showing 60-78 % identity among eukaryotic proteins and 40-60% identity between the E.Coli hsp 70 and the eukaryotic proteins as reviewed by Craig and Gross, 1991. Extensive studies have been conducted on the heat- shock response in insects such as the fruit fly-Drosophila, Chironomous, Lymantria dispar, the tobacco hornworm-Manduca sexta, the desert ant-Cataglyphis, the fleshfly-Sarcophaga

crassipalpis, Locusta migratoria etc. (Burma and Lakhotia, 1984; Carretero et.al., 1986; Whyard et.al., 1986; Burton et.al., 1988; Fittinghoff and Riddiford, 1990; Joplin et.al., 1990; Denlinger et.al., 1992; Gehring and Wehner, 1995). The volume of literature available on the heat shock response of the silkworm Bombyx mori is not very huge. Most of the work done was carried out on cell and tissue cultures or organisms from temperate climate. There is dire necessity for 1. understanding the 12

molecular mechanism of temperature tolerance in silkworm. 2. Identification of the various families of hsps synthesized and the threshold temperature which induce their expression. 3. Understanding the differential expression pattern of various hsps in bivoltine and polyvoltine races and 4. To locate the genes responsible for the heat inducible hsps and subsequent steps to introgress the same into the bivoltine genome either by conventional breeding or by use of molecular techniques. The present study was taken up to understand the nature of heat induced protein synthesis in tropical and temperate silkworm strains as a preliminary attempt.

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Chapter 2 REVIEW OF LITERATURE The review of literature is considered under three heads viz., History & Basics of Hsp Induction, Hsp synthesis & its kinetics in Insect systems and The role of specific heat shock proteins. 2.1. History & Basics of Hsp Induction Smith in 1957 reported temperature tolerance and acclimatory response in

Drosophila subobscura. Survival was increased in individuals exposed to a prior sub lethal temperature. He also reported two kinds of acclimation – Developmental acclimation (in pre adults) and transitory acclimation (in adults). Ritossa in 1962 reported that when Drosophila larvae were shifted from 27- 37°C, several new puffs appeared in the polytene chromosomes. Further the same puffs could also be induced by tretment with dinitriphenol and sodium salicylate. Dingley and Smith (1968) conducted radio active protein labelling studies in heat shocked Drosophilla subobscura, after stopping protein synthesis by feeding cycloheximide. They concluded that neither acclimatization nor recovery from exposure to high temperature requires the synthesis of new protein. Even after the stoppage of protein synthesis, the experimental animals could survive and acclimate to high temperature. However, these findings which were made in 1968 were much before the functions of Hsps were clearly elucidated and the constitutive presence of Hsps in cells was not known. Tissiers et.al., in 1974 discovered heat shock proteins in Drosophila melanogaster. The predominant protein synthesis at 37°C was of 70 kDa. Nagata M (1976) conducted studies on silkworms injected with radio active leucin to demonstrate specific activities of tissue proteins at different stages of development and concluded that the rate of protein synthesis of larval body wall, mid gut and whole body are high at feeding period and low at moulting period. Lindquist (1980) reported that in response to a simple elevation in temperature the entire programme of gene expression in Drosophilla melanogaster is altered in a rapid and highly orchestrated manner. Within a few minutes of heat treatment transcription at most previously active regions of a genome is greatly reduced and is vigorously initiated at a small number of previously quiescent sites. Cells cultured at 23°C when exposed to higher temperatures first increased protein synthesis in 14

certain specific regions and then declined, as synthesis shifted to the production of a small number of characteristic heat shock proteins. Nath and Lakhotia (1989) conducted studies on Heat shock response in a population of Chironomus striatipennis exposed to seasonal varying ambient temperatures and found inhibition of general chromosomal transcription and activation of a specific set of heat shock loci on heat shock. Heat shock at 37°C was not as effective in inhibiting general transcription, as at 39°C. They also observed that during summer months a more or less continuously high ambient temperature keeps the animals under a mild heat shock condition and thus they accumulate an optimum level of Hsps. They concluded that threshold level for the heat shock response in a given species is in relation to the general environmental temperature to which the organisms is exposed in nature. Fittinghoff and Riddiford (1990) conducted a classical study on heat sensitivity and protein synthesis in the tobacco horn worm Munduca sexta under heat shock. They observed that as in other insects the heat shock response of Munduca sexta involves induction of synthesis of heat shock proteins very similar in size to that of

Drosophilla. But Munduca maintained synthesis of most non-heat shock proteins during heat shock except at lethal conditions. This result was in difference with the trend observed in Drosophilla, where the normal protein synthesis was repressed during heat shock protein synthesis. Fittinghoff suggestted the possibility that the heat induced shut down of normal protein synthesis may be a unique feature of Diptera and not found in other insect orders. Klemenz et. al., (1991) reported that α-B-Crystallin, the eye lens protein, in fact is a small Hsp, which not only bear a squence homology with other small Hasps, but also is expressed under heat shock in cells. They speculated that apart from the transparent nature of the protein, its capacity to render the lens cells (which are devoid of nuclei and particularly vulnerable) with the required stress tolerance is the reason for its recruitment as the material for eye lens. Lyashko et.al., (1994) performed a systematic broad-scale analysis of the heat shock response at the cellular level in ethnically and ecologically different human populations. They showed that the fibroblasts isolated from Turkmen living in the hot desert after severe heat shock exhibited intensive synthesis of hsps, where as only trace synthesis of hsps was observed in the Russians living in the moderate climatic regions of Russia. The survival percentage of cells were also high in the first group.

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Gilchrist and Huey in 1999 suggested that genetic variation for `knock-down’ temperature in Drosophilla is maintained by natural selection after conducting experiments with 32 generations of selection of Drosophilla melanogaster. They also suggested that the `knock-down’ temperature was the product of one or two genes of large effect. Karunanithi et.al., (1999) recorded synaptic activity in Drosophilla exposed to high temperature. They observed that expression of Hsp-70 was maximal when synaptic neuro-protection was observed and the protective effects declined with the diminished presence of Hsp-70. Singh and Lakhotia in 2000 studied the pattern of heat induced synthesis of Hsps in grass hoppers, cockroaches and the gram pest Heliothis armigera and found that the threshold temperature for eliciting Hsp synthesis was between 37°C and 42°C in all the cases. Fernando and Heikkila in 2000 cloned the Xenopus hsp family member3 hsp 30c gene into a plasmid vector and over expressed in the bacteria E.coli. The bacteria which expressed the recombinant protein were more thermo-tolerant to a severe thermal challenge of 60°C than either non transformed bacteria or those containing only the plasmid vector. Scaglia et.al., (2003) Compared Haemolymph electrophoretic pattern of Ascia

monuste orseis larvae (Lepidoptera: Pieridae) parasitized by Cotesia glomerata (Hymenoptera: braconidae) with the differences in total protein synthesis and found that new proteins of molecular weight 90, 98, and 110 kDa were synthesized by the parasitised larvae. 2.2. Hsp synthesis & its kinetics in Insect systems Petersen and Lindquist (1988) genetically engineered Drosophilla cells to produce a modified Hsp70 mRNA that behaves exactly as the wild-type message which was stable during heat shock but degraded during recovery indicating that when heat shocked cells are returned to normal temperatures, Hsp synthesis was repressed and normal protein restored. Whyard et.al., (1986) reported that Locusta migratoria adults given a prior heat shock of 45°C upto 4 hours can survive a subsequent heat shock of 50°C for 2 hours, whereas without prior heat shock they die.

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Whyard et.al., (1986) reported that under heat shock conditions, vitellogenin in the fat bodies of Locusta migratoria exposed to 45°C was only 55% of that observed under normal conditions. Total protein synthesis remained unchanged. He concluded that the reduced proportion of vitellogenin synthesis may not be due to a translational control mechanism but may represent a non-specific competition for available ribosoms by Hsp and vitellogenin messages. Joplin and Denlinger in 1990 reported developmental and tissue specific control of 70 kDa hsp in Sarcophaga crassipalpis. They demonstrated that Hsp 65 and 72 in the flesh fly are immunologicaly related to the Drosophilla Hsp 70 family and the flesh fly Hsp 92 was related to Drosophilla Hsp 83. Joplin and Denlinger in 1990 reported a control on Hsp expression in Sarcophaga

crassipalpis, which is a temperature dependent switch. At low heat shock temperature, Hsp 72 was expressed while at high temperature, Hsp 65 was primarily expressed. They also noticed that the differential temperature control were restricted to a particular stage of development of the brain of the organism. Smith and Yaffe in 1991 Conducted mutation studies in the yeast Saccharomyces

cervisiae and demonstrated that a mutation, hsf1-m3 resulted in in a general block in heat-shock induction but does not affect the acquisition of thermotolerance. Thus they argued that high level induction of the major hsps is not a pre requisite for acquiring

thermotolerance

if

hsps

are

constitutively

present.

Acquired

thermotolerance might result instead from a temperature- dependent change in the activity or structure of preexisting hsp and perhaps, in the alteration of other proteins. Joplin et. al. (1990) studied developmental and tissue specific heat shock proteins in the flesh fly Sarcophaga crassipalpis. Two major polypeptides of 65kDa and 72kDa were induced at 40-43°C. They observed that these two proteins showed developmental and tissue specific expression. Hsp 65 was expressed in both the tissues of brain and integument of third instar larvae at 43°C but hsp 72 was expressed at 43°C throughout the rest of developmental period. Nath and Lakhotia (1989) conducted studies on tropical Chironomus, Chironomus

striatipennis and compared the behaviour of its temperate counterpart. they observed that the heat shock response has an important role in the homeostatic mechanism of the tropical species, which respond optimally at 39°C instead of 37°C as in the temperate Chironomus. The heat shock response of the tropical species not only serves as a short term protection mechanism against a sudden elevation of 17

environmental temperature, but also has a significant long term role in permitting the organism to adapt to the severe summer temperature of the tropics. Singh and Lakhotia in 1995 found that subsequent to heat shock the malpighian tubules of Drosophilla larvae did not induce synthesis of any of the common Hsps. Employing immono-cytochemical localisation of Hsp 70 they concluded that the non-induction of common Hsps in the malpighian tubules of Drosophilla was not due to auto regulation by constitutively high levels of Hsp 70 or Hsc 70. Joy and Gopinathan in 1995 reported the appearance of 93, 70, 46 and 28 kDa protein bands consequent to high temperature exposure in Bombyx mori. in both bivoltine and multivoltine strains, but with variying kinetics.The isolated hemocyte of multivoltine race exhibited the induction of 70 kDa protein.They concluded that fat body is the reservoir and active synthetic tissue of the haemolymph proteins. The proteins are synthesized in the fat body tissue and find its way to haemolymph. Jean in 1981 experimented with stage dependant synthesis of hsps in early embryos of Drosophila melanogaster and showed that hsps are synthesized only after treatment at blastoderm and later stages. Normal protein synthesis stops after heat shock at all stages. Lindquist in 1981 studied the regulation of protein synthesis during heat shock and found that in Drosophila melanogaster, regulation takes place at transitional level. The mechanism causes to translate the hsmRNA and specific repression of translation of pre existing mRNAs. In contrast to this in yeast there was no system to sequester pre-existing messages from translation but most of the normal signals disappear rapidly from the cell. Velazques et. al. (1983) observed that hsps are synthesized in rapidly growing cells at normal temperature also. They detected hsp70 mRNA in rapidly growing cells but the concentration was less than 1/1000th the level achieved after induction by heat shock. Carvalto and Rebello (1987) when shifted cells of Aedes albopictus from 28°C to 37°C, two major proteins with molecular weight of 82kDa and 66kDa were synthesized in cells derived from lag or stationary phase. Actinomycin D treated cells failed to induce any hsp. Yocum in 2001 developed partial clones of Hsp 70A and Hsp 70 B in the Colorado potato beetle Leptinotarsa decemleneata, using RT-PCR and studied their expression. He found that Hsp 70A and Hsp 70 B were differentially expressed 18

during diapause and temperature stress. He established a role for the thermal history of the diapausing insect in expression of Hsp 70 and that the regulation of heat shock response is not a simple on-off response but, is finely tuned to developmental and environmental conditions. Pelham (1986) speculated that hsps participated in several ATP dependant reactions and two such major proteins are hsp 70 and hsp 90. He further stated that members of these families of proteins are involved in assembly and disassembly of proteins and protein containing structures. Stephanou in 1987 studied the heat shock response in two different species of the fruit fly namely Ceratitis capitata and Drosophila melanogaster and showed that the regulation of hsp synthesis take places at the translational level. Lindquist (1980) observed that in Drosophilla heat shocked cells four sHsps had a narrow range of induction. hsp27 and 23 reached maximum levels at 35°C. hsp70 was produced in much larger amount than any of the other proteins. While its induction was detectable at most temperatures, it was maximally induced over a rather narrow range centered around 37°C. Completed transcripts of heat shock mRNAs are produced within 4 min. of temperature elevation; within 8-12 min. These messages have been processed, transported to the cytoplasm and translated into protein. The vigour and speed of the response are greatly enhanced by the fact that synthesis of the normal complement of proteins and mRNAs is sharply and rapidly curtailed. Fittinghoff and Riddiford (1990) observed in Manduca Sexta that the Hsps are induced maximally at 40°C survival of larvae of 1h at 42°C is nearly 100% and tissue survival at 42°C is nearly as high. At higher temperatures synthesis of large Hsps decline and survival dropped. Fittinghoff and Riddiford (1990) suggested that both phylogenetic and environmental considerations influence normal protein synthesis during stress. Also the heat induced shut down of normal protein synthesis as observed in Drosophilla may not be universal for all insect orders. Theodorakis et.al., (1999) suggested that thermo-tolerant cells limit Hsp70 expression by transcriptional and pretranslational mechanisms, perhaps to avoid the potential cytotoxic effect of these proteins. Krebs and Feder (1998) established that the relationship between changes in Hsp70 and thermo tolerance is not linear in Drosophilla. 19

2.3. The role of specific heat shock proteins Lindquist (1980) observed that a gradual rise in temperature greatly extended the temperature range of the response in Drosophilla. She explained that the cells in this case had the opportunity to synthesize substantial quantities of heat shock RNAs and proteins before they are subjected to the stress of extreme temperature. When such cells were returned to 25°C, they recovered normal protein synthesis a great deal sooner than cells exposed to rapid temperature elevations. Synthesis of Hsp declined when heat shocked cells were shifted to lower temperatures, while synthesis of other Hsps was unaffected. She suggested that during heat treatment normal cellular messages are not degraded, but are maintained in the cell ready to function in translation after cells have been recovered for a certain period at normal temperatures. Also when Drosophilla cells were shifted from high temperature to low temperature an increase in normal cellular protein synthesis was detected. She observed at least three mechanisms involved in stress response in Drosophilla viz., specific genes are selected for transcription, specific messages are selected for degradation and certain classes of messages are discriminated against in translation. Schlesinger (1986) reviewed the functions of hsps. Bacteriophage lambda uses several E. coli hsps for its replication. Recent studies showed that several eukaryotic DNA viruses such as adenovirus, herpes virus, simian virus 40 and polyoma viruses induce the synthesis of hsp70 early in infection. Some RNA viruses also induce hsp70 and hsp90 in infected chicken cells. In the case of bacteriophage lambda use of hsps by viral replication system shows that this induction reflects a stress from the infection. Loomis and Wheeler in 1980 studied heat shock response in Dictyostelium and found that on shifting cells from 22°C to 30°C there was a rapid synthesis of some 82 kDa, 70 kDa, 60 kDa and 43 kDa proteins. The relative rate of synthesis of most other proteins decreased especially actin. Stephanou, et.al., in 1983 induced a set of at least 8 specific polypeptides in response to heat shock in the Mediterranean fruit fly Ceratitis capitata. The major polypeptides identified were of molecular weight 69 kDa, along with the other polypeptides of 87 kDa, 20 kDa, 16 kDa, 14 kDa, 13 kDa and 12 kDa. Heat pretreatment at milder temperature significantly enhanced the survivability and hsp synthesis. Whyard et.al., (1986) showed that fat bodies from heat shocked adult females

Locusta migratoria produce a set of at least 6 specific polypeptides with molecular 20

weights of 81, 73, 68, 42, 28 and 24 kDa in response to heat shock at 39-47°C for 1.5 h. As reviewed by Singh and Lakhotia in (1995) the Hsp 70 protein of Drosophilla can decay in vivo as well as in vitro at a much faster rate; degradation, which is mediated by a proteolytic action of Hsp 70 protein upon itself, may sometimes occur rapidly, even during the course of electrophoresis. Singh and Lakhotia in 2000 Observed that cockroaches collected from drains showed good expression of 70 kDa and 64 kDa proteins even without heat shock, where as the laboratory reared ones did not. They suggested the scope for utilizing the constitutive expression of Hsp 70 as a biomarker for pollutants present in drains. Singh and Lakhotia in 2000 studied the pattern of Hsp synthesis in many insects and found that induction of Hsps did not hinder the ongoing protein synthesis at high temperature. But the profiles of Hsps induced varied in a tissue specific manner. Krebs and Feder in 1997 found that all tissues of Drosophilla melanogaster subjected to heat shock expressed Hsp 70 but the kinetics varied. Gut tissue, Fat body and malpighian tubules required a long recovery period after heat shock for expressing of Hsp 70. They also observed that those tissues which lagged in Hsp 70 synthesis sustained more injuries and the sensitivity of larval gut to heat shock limited larval thermo-tolerance. Roccheri et. al., (1981) Subjected developing sea urchins to an elevated temperature of 31°C and found increased stimulated synthesis of bulk proteins of which the major one was 70 kDa. They observed that hsps were synthesized only if the embryos are heated after hatching. They observed that the stages which synthesis hsps can survive and those cannot synthesis hsps cannot survive high temperature. Further they demonstrated that hsps are not produced in presence of the inhibitor actinomycin D. Yocum and Denlinger in 1992 showed that the flesh fly Sarcophaga crassipalpis developed long term (>48 h) thermotolerance after a brief exposure to supra optimal temperature and expression of thermo-tolerance is dose-dependent on both the duration of the exposure and temperature of the pretreatment, illustrating that prolonged thermotolerance does not require continuous expression of Hsps.

21

Karunanithi et.al., (2002) heat shocked transgenic Drosophilla melanogaster strains containing 12 extra copies of Hsp-70 gene and studied the pattern of Hsp-70 synthesis along with synaptic thermo-tolerance. They found that pre-synaptic function was selectively enhanced in the transgenic strains. They speculated that the Hsp-70 confers synaptic protection by preservation of second messenger mediated cellular pathways, inhibition of protease dependent events and preservation of synaptic residual proteins. Yocum (2003) isolated fragments of three diapause-associated transcripts from the Colorado porato beetle Leptinotarsa decemlinieta. After gene analysis he reported that Hsps play important role in diapause. Hsp-90 genese are down regulated during diapause and Hsp 23 & Hsp-70 genes are up regulated during diapause. Zatsepina et.al., (2001) after conducting studies with a Drosophilla melanogaster strain from sub-equitorial Africa, concluded that adaptation via natural selection is sufficiently strong to overcome even the immense Phylogenetic inertia of the heat shock response. They found that evolution at high temperature has lead to decreased expression of Hsp 70 in the African thermo tolerant strain, which expressed decreased Hsp 70 synthesis at high temperature. Evegnev et. al. (1987) studied heat shock response in Bombyx mori

cells.

Temperature elevation induced active transcription of heat shock mRNAs in infected cells. But at the level of translation headstock treatment failed to induce hsp synthesis and was not able to inhibit production of polyhedrin in such cells. Joy and Gopinathan in 1995 conducted a pioneering study on the heat shock response in the mulberry silkworm Bombyx mori L. When exposed to high temperature ranging from 37-47°C showed varying levels of heat tolerance at different developmental stages as egg, larva, pupa and adult. Out of these, the later larval instars and pupal stages were found to be tolerant to high temperature for longer durations. Two multivoltine strains C.nichi and Pure Mysore larvae were found to be more tolerant to high temperature, than the bivoltine strains. At 39°C 2 h exposure, the bivoltine and multivoltine were equally tolerant. The difference in tolerance was observed at the temperature range 41-43°C. Lee et.al., in 2003 cloned a genomic DNA fragment containing a promoter region for the gene encoding an HSC70-4 homologue, the structure of which was deduced from the partial cDNA sequences. The deduced amino acid sequence with 649 residues was 89% and 96% identical to those from Drosphilla melanogaster hsc-4 and Muduca sexta HSC-70-4 respectively. The expression analysis demonstrated that mRNA transcription occurred in all tissues examined and was not stimulated by 22

heat shock. Thus HSC70-4, the molecular chaperon is found to be ubiquitously expressed in every tissue of Bombyx mori. Landaise et.al., (2001) completely sequenced the Hsp90 cDNA from the lepidopteran insects Bombyx mori and Spodoptera frugiperda and found a high consistency with known phylogeny at both high and low taxonomic levels between the two. They performed transcription analysis of hsp90 gene and found that the induction of the gene occurs only at 42°C (14°C above physiological growth conditions) gene of S. frugiperda.

23

Chapter 3 MATERIALS & METHODS 3.1. Experimental Animals The experiment was conducted during 2004-05 at Central Sericultural Research and Training Institute, Srirampura, Mysore, India. A temperature tolerant polyvoltine silkworm breed namely Nistari and a non-tolerant bivoltine breed namely CSR2 were selected for the study (Fig.1 &2). Ten disease free layings of each race were obtained from the pure line stocks maintained by the Silkworm Genetics Laboratory during September 2004.

The

facilities available at the Silkworm Genetics Laboratory were used for incubation of the layings and rearing of the worms upto cocooning. Thorough disinfection of the rearing house, equipments and surroundings was carried out twice, before the experiment. Utmost care was taken to ensure hygiene and sanitation. The layings were incubated at 24-25°C and 80-85% RH till hatching. Towards the end of the incubation period at the blue spot stage, the eggs were kept in dark boxes to ensure uniform and timely hatching.

The hatched larvae were brushed into

uniform sized plastic trays and reared upto cocooning by following the standard method (Krishnaswamyet.al.,1973) with recommended temperature and humidity conditions (Young silkworms: 27-28°C and RH 85-90%; Late age: 24-25°C and RH 70-75%), feeding V1 variety mulberry leaf. 3.2. Heat Shock Treatment Heat shock treatment was administered on the 2nd day of 3rd, 4th and 5th instar larvae. The moulted larvae of each of the three instars were allowed to feed for a full day after moult in order to recover from any stress induced by starvation during moult. On the second day of 3rd, 4th and 5th instar, 150 larvae from each race were exposed to two high temperature treatments, viz., 36°C and 40°C for two different durations, viz., 1 h and 6h. As male and female larvae can be separated only after 4th moult, 3rd and 4th instar larvae were treated without any sex separation, whereas during the 5th instar, the worms were sexed before temperature treatment. After the 4th moult, the larvae were sex separated by observing for the reproductive primordia on the ventral side of the abdomen. The male larva has one spot of the `Herold’ imaginal bud in the centre line of the ventral side of the 12th abdominal segment.

The 24

female has four spots of `Ishiwata’ imaginal buds at the ventral side of the 11th and 12th segments (Bungo,1978). A pair of magnifying goggles fitted to a headgear and a table lamp were used for this purpose. Exposure to high temperature was given in SERICATRON (CHUO- Japan) Environment chamber with precise and automatic control facilities for uniform maintenance of temperature and humidity available at the Bivoltine Breeding Laboratory of CSR&TI, Mysore (Sureshkumar et.al., 2003) Fig.3. A constant relative humidity of 80±1% was maintained throughout the treatment period. The larvae were allowed to feed on fresh mulberry leaf during the high temperature treatment. Suitable control batches for each race were separately maintained at room temperature. Samples were collected immediately after the treatments as well as at specific intervals during recovery periods. 3.3. Sample Collection All the materials used such as eppendorf tubes, containers, microtips, needles, dissection paraphernalia, etc., were thoroughly disinfected and autoclaved before use. Distilled De-ionized autoclaved water was used for all operations (Kauffman et. al., 1995). From the 3rd and 4th instar larvae, gut juice and haemolymph samples were collected from six randomly selected individuals (each serving as a replication) of each treatment and pooled. From the 5th instar larvae gut juice and haemolymph samples were collected from 6 male and 6 females separately. Samples from treated and control larvae were collected separately, prior to heat shock, immediately after heat shock (0h recovery) and after allowing recovery periods of 1 h, 6h, 24h and 48h. Gut juice was collected by exposing the larvae briefly to Chloroform. The vomited gut juice was collected directly into pre-cooled eppendorf tubes. Collected gut juice was centrifuged at 5000 rpm using a cooling centrifuge at –4°C for 5 min. to remove leaf particles and debris. The samples were stored at –80°C till further use with proper labelling (Kauffman et. al., 1995). Haemolymph was collected by pricking one of the first prolegs of each larva with a sterile needle. The oozing haemolymph was collected into autoclaved precooled eppendorf tubes containing a pinch of Phenyl thiourea (C7H8N2S, mw: 152.21). PTU was used to prevent coagulation of the insect blood. Collected 25

haemolymph was centrifuged at 5000 rpm using a cooling centrifuge at -4°C for 5 min. to remove haemocytes and cellular debris and stored at -80°C till further use, with proper labelling (Joy and Gopinathan, 1995). One hundred larvae from each treated batch and control of all the three instars were separately maintained under uniform conditions of atmosphere and food till cocooning. Observations till pupation were made for mortality, moulting behaviour, cocooning, pupation, post cocooning mortality, etc. 3.4. Preservation of samples According to Peter B. Kaufman, proteins and enzymes are relatively unstable macromolecules as compared with DNA. Their structures or activities are sensitive to a variety of factors or parameters involved in isolation and purification procedures. The half-life of a protein largely depends on the storage temperature. It is suggested that, the best conditions are at 4, -20 or -80°C or in liquid nitrogen (-196°C) depending on particular uses (55). All collected samples were kept at 80°C for preservation and care was taken to keep the samples in ice whenever handled (Kauffman et. al., 1995). 3.5. Estimation of Total Proteins The total protein content of the samples was determined by using a protein estimation kit by Lowry’s method. (GENEI, Bangalore. Protein Estimation Kit, Cat #: KT-18). The method is based on combination of biuret reaction and Folin-Ciocalteau reaction. In the first step of the reaction, protein binds to copper in alkaline medium and produces Cu ions. In the second step Cu ion catalyzes. Oxidation of aromatic aminoacids by reducing phosphomolybdotungstate to heteropolymolybdenum blue. This reaction produces strong blue colour, which predominantly depends upon tyrosin and tryptophan content of protein and to a lesser extend, cysteine and other residues in protein. 3.5.1. Kit Components Protein standard BSA

5 x 5 mg

Solution I

Copper Sulphate solution (5 ml)

Solution II

Alkaline tatarate (2 x 250 ml)

Solution III

Folin Reagent (60 ml)

26

3.5.2. Preparation of Standard Protein and Reagents


Standard Protein: One vial containing 5 mg BSA was reconstituted with 1 ml ddH2O to get 5 mg/ml. 0.1 ml of this solution was diluted with 0.9 ml distilled water to get 0.5 mg/ml just before use.




Complex forming Reagent: To one volume of Solution I 100 volume of Solution II was added just before use.




Foline Reagent: Solution III was used directly.

3.5.3. Assay


BSA Standard Graph Preparation: Progressive concentrations of the BSA such as 0, 5, 10, 15, 20, 25, 30, 35, 40, 60, 80, 100 & 200 µg/2 ml were prepared by diluting with water. 2 µl from each sample was diluted to 2 ml with water for assay.




2 ml Complex forming reagent was added to the BSA standard as well as to the samples. Mixed well and kept for 10 min.




0.2 ml of Solution III was added with vortexing and incubated for 20-30 min.




The optical density was read on Spectrophotometer at 660 nm and the readings were recorded.




A calibration curve was constructed by plotting optical density readings on Y axis against BSA standard protein concentrations.




The values `X’ from the graph corresponding to the optical density reading for individual samples were recorded. The sample protein concentration were calculated using the formula,

Sample Concentration =

Value from graph in µg (X) Volume of sample in µl (2 µl)

27

3.6. Sodium Dodecyl Sulphate Poly Acrylamide Gel Electrophoresis (SDS-PAGE) The prepared samples were subjected to SDS PAGE analysis by following standard protocols (Sambrook and Russel, 2001). Poly acrylamide gel (PAG) had been known as a potential embedding medium for sectioning tissues as early as 1954. Two independent groups Davis and Raymond employed PAG in electrophoresis in 1959. It possesses several electrophoretically desirable features that made it a versatile medium. Poly acrylamide gel separates protein molecules according to both size and charge. It is a synthetic gel, thermo-stable, transparent, strong, relatively chemically inert, can be prepared with a wide range of average pore sizes, can withstand high voltage gradients, feasible to various staining and destaining procedures and can be digested to extract separated fractions or dried for autoradiography and permanent recording. (Anbalagan, 1999). 3.6.1. Chemicals used All the chemicals used in the present experiment were of analytical grade and procured from SRL (SISCO Research Laboratories, Mumbai, India), unless otherwise specified. The various chemicals used and their role are discussed below. (Anbalagan, 1999)


Tris (tris (hydroxy methyl) aminomethane) (C4H11NO3; mw: 121.14). It has been used as a buffer because it is an innocuous substance to most proteins. Its pKa is 8.3 at 20°C and reasonably a very satisfactory buffer in the pH range 7.0 – 9.0.




Glycine (Amino Acetic Acid) (C2H5NO2; mw: 75.07). Glycine has been used as the source of trailing ion or slow ion because its pKa is 9.69 and mobility of glycinate are such that the effective mobility can be set at a value below that of the slowest known proteins of net negative charge in the pH range. The minimum pH of this range is somewhere around 8.0.




Acrylamide (C3H5NO; mw: 71.08). It is a white crystalline powder. While dissolving in water, autopolymerisation of acrylamide takes place. It is a slow spontaneous process by which acrylamide molecules join together by head on tail fashion. But in presence of free radicals generating system, acrylamide 28

monomers are activated into a free-radical state. These activated monomers polymerise quickly and form long chain polymers. This kind of reaction is known as Vinyl addition polymerisation. A solution of these polymer chains becomes viscous but does not form a gel, because the chains simply slide over one another. Gel formation requires hooking various chains together. Acrylamide is a neuro toxin. It is also essential to store acrylamide in a cool dark and dry place to reduce autopolymerisation and hydrolysis.


Bisacrylamide

(N,N’-Methylenebisacrylamide)

(C7H10N2O2;

mw:

154.17). Bisacrylamide is the most frequently used cross linking agent for poly acryl- amide gels. Chemically it is thought of having two-acrylamide molecules coupled head to head at their non-reactive ends. Bisacrylamide was preserved at 4°C.


Sodium Dodecyl Sulphate (SDS) (C12H25NaO4S; mw: 288.38). SDS is the most common dissociating agent used to denature native proteins to individual polypeptides. When a protein mixture is heated to 100°C in presence of SDS, the detergent wraps around the polypeptide backbone. It binds to polypeptides in a constant weight ratio of 1.4 g/g of polypeptide. In this process, the intrinsic charges of polypeptides becomes negligible when compared to the negative charges contributed by SDS. Thus polypeptides after treatment becomes a rod like structure possessing a uniform charge density, that is same net negative charge per unit length. Mobilities of these proteins will be a linear function of the logarithms of their molecular weights.




Ammonium per sulphate (APS) (N2H8S2O8; mw: 228.2). APS is an initiator for gel formation. APS was stored at 4°C.




TEMED (N, N, N’, N’-tetramethylethylenediamine) (C6H16N2; mw: 116.21). Chemical polymerisation of acrylamide gel is used for SDS-PAGE. It can be initiated by ammonium per sulphate and the quarternary amine, N, N, N’, N’-tetramethylethylenediamine (TEMED). The rate of polymerisation and the properties of the resulting gel depends on the concentration of APS and TEMED. Increasing the amount of APS and TEMED results in a decrease in the average polymer chain length, an increase in gel turbidity and a decrease in gel elasticity. Decreasing the amount of initiators shows the reverse effect. It is recommended that lowest catalysts concentrations that will allow polymerisation in the optimal period of time should be used. APS and TEMED are used,

29

approximately in equimoloar concentrations in the range of 1 to 10 mM. TEMED was stored at 4°C.


Bromo Phenol Blue (BPB) (3’, 3’’, 5’, 5’’-Tetrabromophenolsulphonephthalein) (C19H10Br4O5S; mw: 669.99). BPB is the universal marker dye. Proteins and nucleic acids are mostly colourless. When they are subjected to electrophoresis, it is important to stop the run before they run off the gel. BPB is the most commonly employed tracking dye, because it is viable in alkali and neutral pH, it is a small molecule, it is ionisable and it is negatively charged above pH 4.6 and hence moves towards anode. Being a small molecule it moves ahead of most proteins and nucleic acids. As it reaches the anodic end of the electrophoresis medium electrophoresis is stopped. It can bind with proteins weakly and give blue colour.




Glycerol (C3H8O3; mw: 92.09). It is a preservative and a weighing agent. Additon of glycerol (20-30 or 50%) is often recommended for the storage of enzymes. Glycerol maintains the protein solution at very low temperature, without freezing. It also helps to weigh down the sample into the wells without being spread while loading.




Coomassie Brilliant Blue (CBB) (C45H44N3NaO7S2; mw: 825.97). CBB is the most popular protein stain. It is an anionic dye, which binds with proteins non-specifically. The structure of CBB is predominantly non-polar. So is usually used (0.025%) in methanolic solution (40%) and Acetic Acid (7%). Proteins in the gel are fixed by acetic acid and simultaneously stained. The excess dye incorporated in the gel can be removed by destaining with the same solution containing no dye. The proteins are detected as blue bands on a clear background. As SDS is also anionic in nature, it is reported to interfere with staining process. Therefore, large volume of staining solution is recommended. Approximately 10 times the volume of the gel.




Methanol (CH4OH; mw: 32.04).




Acetic Acid Glacial (C2H4O2; mw: 60.05).




Butanol (C4H10O; mw: 74.12). Water saturated butanol is used as an overlay solution on the resolving gel.

30




Beta Mercapto Ethanol (HS-CH2CH2OH; mw: 78.13). BME was procured from LKB, Bromma, Sweden and was stored at 4°C.

3.6.2. Preparation of the monomer solutions Distilled de-ionized water (ddH2O) was used throughout the experiment, as the primary ingredient in solutions or buffer systems, since unpurified water contains a lot of micro-organisms or proteases, that can cause protein degradation.


30% Acrylamide-Bisacrylamide solution: For preparing 100 ml AcrylamideBisacrylamide solution, 29 g Acrylamide and 1 g Bis-acrylamide were dissolved in 40 ml warm ddH2O, made upto 100 ml, filtered through Whatman paper and stored at 4°C in dark bottle.




1.5 molar Tris (pH 8.8): For preparing 100 ml of 1.5 M tris, 18.15 g Tris buffer was dissolved in 70 ml ddH2O. pH adjusted to 8.8 using concentrated Hydrochloric Acid (HCl), made upto 100 ml with ddH2O, filtered, autoclaved and stored at room temperature.




0.5 molar Tris (pH 6.8): For preparing 100 ml of 0.5 M tris, 6.057 g Tris buffer was dissolved in 70 ml ddH2O. pH adjusted to 6.8 using concentrated Hydrochloric Acid (HCl), made upto 100 ml with ddH2O, filtered, autoclaved and stored at room temperature.




10% SDS: 10 g SDS was dissolved in 100 ml ddH2O and stored at RT.




10% APS: 0.1 g APS was dissolved in 1 ml ddH2O and stored at 4°C in an eppendorf tube. APS was freshly prepared for every week.

3.6.3. Preparation of buffers for PAGE A buffer is a solution consisting of a conjugate base and a conjugate acid group that is able to resist pH change to varying degrees. It is strongly recommended that specific buffer conditions be maintained during the extraction and purification of proteins since, the structures or activities of proteins/enzymes are very sensitive to environmental pH changes (Kauffman, et.aI., 1995). Laemmli

buffer system, a modification of Davis-Ornstein buffer system was used in the current experiment. It is a dissociating buffer containing SDS and is reportedly the most widely used electrophoretic buffering system today (Anbalagan, 1999). 31




5X Reservoir Buffer or Tank Buffer or Glycine Buffer: For making 1000 ml of Tank buffer, 15.1 g Tris buffer, 72 g Glycine (pH 8.3) and 5 g SDS were dissolved in 800 ml ddH2O and made upto 1000 ml. The solution was stored at room temperature. For electrophoresis, the 5X buffer was diluted to 1X with ddH2O. For the standard gel tank, 120 ml 5X buffer was diluted to 600 ml. For the mini gel tank, 50 ml 5X buffer was diluted to 250 ml. For running native gel, separate tank buffer was prepared, without adding SDS, the other contents being the same.




Sample buffer: 0.605 g Tris buffer was dissolved in 40 ml ddH2O and pH was adjusted to 6.8 with conc. HCl and made upto 50 ml using ddH2O. To prepare 50 ml sample buffer, 32.5 ml of the above mentioned Tris solution was taken and 5 ml of 10% Glycerol, 10 ml of 10% SDS and 25 ml BPB were added and dissolved fully. This stock solution was aliquoted into 1 ml aliquotes in eppendorf tubes and preserved at 4°C. Just before use, 50 µl Beta Mercapto Ethanol per ml was added to each tube, cyclo-mixed and stored at 4°C. For native gels, the sample buffer was prepared without the SDS component.




Phosphate Buffered Saline (PBS): PBS was prepared by dissolving 2 g Sodium Chloride (NaCl), 50 mg Potassium Chloride (KCl), 340 mg Disodium Phosphate (Na2HPO4) and 60 mg Potassium Phosphate (KH2PO4) in 200 ml distilled water, made upto 250 ml after adjusting pH to 7.2 with Hydrochloric Acid (HCl) used after autoclaving.

3.6.4. Preparation Staining & Destaining Solutions


Staining Solution: To prepare 1000 ml staining solution, 2.5 g CBB (R250) was completely dissolved in 100 ml Methanol. After complete dissolution, another 250 ml methanol, 450 ml ddH2O and 100 ml glacial acetic acid were added and mixed thoroughly. The resultant solution was filtered through Whatman paper and stored at room temperature.




Destaining Solution: For preparing 1000 ml destaining solution, 450 ml methanol, 450 ml ddH2O and 100 ml glacial acetic acid were mixed together and stored at room temperature.

32

3.6.5. Assembly of Gel Casting Unit A Slab Gel Vertical Electrophorectic system manufactured by GENEI, Bangalore was used for electrophoresis. Standard sized gels (16 x 14 cm) were used for separation of haemolymph proteins and mini gels (8 x 7 cm) used for separation of gut juice native proteins. The standard sized apparatus is constructed of perspex and consist of a base (20.5 x 20.5 x 0.9 cm) supporting a vertical section (21.3 x 17.8 x 0.9 cm) which has a notch 3 cm deep and 14 cm long cut in one of the 17.8 cm edges. The other 17.8 cm long edge is cemented and screwed into the base of the apparatus 12.5 cm from one of the base edges. The base of the upper buffer reservoir and 3 sides are formed from 0.3 cm thick pespex to give a chamber, 14.6 cm long, 6 cm wide and 7 cm deep. This is cemented to the vertical section. The lower buffer reservoir is 17.8 cm long, 6 cm wide and 6 cm deep and consists of a sheet of 0.3 cm thick perspex, 29.8 cm long and 6.5 cm wide, moulded to form the three sides of the reservoir and then cemented to the lower part of the vertical section and to the base of the apparatus. Two perspex blocks 2.5 x 0.9 x 0.9 cm are also cemented, 12 cm apart to the base of the lower reservoir to form supports for the glass sandwich. Removable platinum wire electrodes are placed in the lower and upper buffer reservoirs. The dimensions of the glass plates are 17 x 19.5 x 0.3 cm and the second plate (notched) is the same size, but with a notch 2 cm deep and 14 cm long cut in one of the 17 cm edges. The two glass plates are placed together to form the gel holder with teflon spacers (22 cm long, 1 cm wide and 0.1 cm thick) running down each vertical side of the sandwich. Sample wells are formed in the gel during polymerisation using a teflon comb, having 13 teeth. Glass plates were thoroughly washed and rinsed first with distilled water then with absolute alcohol and wiped clean.

The spacers after cleaning with

alcohol were greased using Metro Ark 211 compound. The glass plates were assembled with the spacers appropriately placed in between. A slight modification to the conventional method was introduced by avoiding the bottom spacer, so that 2 to 2.5 cm extra gel length was obtained, which provided a better separation of proteins and well resolved bands. The glass plates were sealed with cellophane tapes, leaving no gap for any potential leakage. Once assembled, the set up was reinforced with special clips.

33

3.6.6. Preparation of Poly acrylamide Gel DISC electrophoresis utilizes gels of different pore sizes. The name DISC was derived from the discontinuities in the electrophoretic matrix and coincidentally from the discoid shape of the separated zones of ions (Anbalagan, 1999). There are two layers of gel namely staking gel or spacer gel and resolving gel or separating gel.


Staking gel or spacer gel: It is a large pore poly acrylamide gel (4%). This gel is prepared with Tris buffer pH 6.8 of about 2 pH units lower than that of electrophoresis buffer. These conditions provide an environment for Kohlrausch

reactions, as a result, proteins are concentrated to several fold and a thin starting zone of the order of 19 microns is achieved in a few minutes. This gel is cast over the resolving gel. The height of the staking gel region was always maintained more than double the height and the volume of the sample to be applied. The ingredients for making staking gel for standard and mini gel were as follows:

Ingredients

Standard Gel

Mini Gel

ddH2O

3.6 ml

2.35 ml

Acrylamide-Bisacrylamide

0.8 ml

0.53 ml

Tris (pH 6.8)

1.5 ml

1.00 ml

10% SDS

60 µl

40 µl

10% APS

60 µl

80 µl

TEMED

06 µl

02 µl

After addition of each ingredient, the mix was thoroughly swirled to ensure complete mixing.


Resolving gel or Separating Gel: This is a small pore polyacryl amide gel (3 - 30%). The Tris buffer used is of pH 8.8. In this gel, macro molecules separate according to their size. In the present experiment, 8%, 10% and 12% Resolving gel were used for separating different range of proteins. 8% gel for 24 – 205 kD proteins, 10% gel for 14-205 kD proteins and 12% gel for 14-66 kD proteins. The composition are as follows:

34

Standard Gel

Ingredients

Mini Gel

8%

10%

12%

10%

ddH2O

11.5 ml

09.95 ml

08.2 ml

4.6 ml

Acrylamide-Bisacrylamide

06.7 ml

08.25 ml

10.0 ml

2.7 ml

Tris (pH 8.8)

06.3 ml

06.3 ml

06.3 ml

2.5 ml

10% SDS

250 µl

250 µl

250 µl

100 µl

10% APS

250 µl

250 µl

250 µl

100 µl

TEMED

15 µl

15 µl

15 µl

06 µl

TEMED was added and thoroughly mixed immediately before pouring into the glass sandwich. Extreme caution was taken to ensure no air bubbles in the matrix.


Native Gels: For native gels, the SDS fraction was avoided, all other ingredients being the same. The resolving gel approximately 25 ml (for standard gel) prepared was

poured in between the glass plates immediately after the addition of TEMED with constant swirling without causing any air bubbles getting trapped in the solution. After pouring, the whole set up was placed on a leveled table. An overlay solution of water saturated butanol (1 ml) was applied over the resolving gel to get flat gel surface. The overlay solution also prevents the gel mixture from getting exposed to atmospheric air and thereby helps to form the gel completely. A polymerisation time of 30 min. was given. On complete polymerisation, the overlay solution was poured out. Using a piece of blotting paper, the remaining space in between the glass plates was wiped clean and the staking gel mixture was poured above the resolving gel and a clean teflon comb was inserted leaving a gap of approximately 1 cm from the resolving gel. The

polymerization

reaction

is

exothermic.

Heat

is

generated

as

polymerization begins and this heat in turn drives the reaction faster. As a result, gelation occurs rapidly once the polymerisation is initiated. Polymerization carried out at low temperatures (0 - 4°C) result in turbid, porous and inelastic gels. Where as at high temperature, polymer chains formed are shorter and elastic. In order to obtain transparent gels with good consistency, 23 - 25°C is to be maintained (Anbalagan, 1999). An air conditioner was used to keep the room temperature at 23 - 25°C during gel preparation and electrophoresis.

35

A polymerization time of 30 - 40 min was allowed. After complete polymerization, the comb was removed very carefully, using both hands, so that the well formation was not disturbed. Using cut blotting paper strips, the wells were cleaned. The cellophane tape sealing was removed and the bottom was wiped clean. The wells and the line of separation between the staking and resolving gels were marked using marker pen (on the rectangular glass plate) for easy identification. The sandwich was inserted into the electrophoretic tank with the notched plate facing inward, exposing the wells into the upper tank. The glass plate was screwed tight into the electrophoretic tank. The upper and lower tanks were filled with the tank buffer ensuring that no air bubble is formed either on the bottom of the gel surface or inside the wells. The electrodes were connected to the power pack and the power pack was briefly switched on to ensure that the electric circuit is complete. 3.6.7. Sample Preparation The samples were taken out of -80°C and allowed to thaw on ice (Kauffman, et.al., 1995). After thawing, the samples were mixed properly and centrifuged in cooling centrifuge at -4°C at 5000 rpm for 5 min. Samples of 2 - 5µl were mixed (as per requirement) with 5 - 8 µl PBS and 10 µl Sample buffer and boiled for 4 min. at 96°C and were briefly spinned down. Samples for native gels were prepared similarly, but with sample buffer lacking in SDS and without boiling. The total prepared sample quantity was 20 µl in all the cases. The protein marker used was of the range 3.5 - 205 kD of Conc. 4.5 mg/ml supplied by GENEI, Bangalore. 4 µl marker was used with other components being the same that of the sample. For native gels, a pre-stained protein marker was used. As a rule, the marker was always loaded in the first well from the left hand side. 3.6.8. Sample Loading & Electrophoresis The prepared samples and marker were loaded carefully into the predetermined wells using a 20 µl pipette quickly. Electric connections were established and a current of 20 mA was applied till the dye front reached the resolving gel and increased to 40 mA till end of the electrophoresis. Native gel electrophoresis was carried out at 4°C. After loading the samples, the electrophoretic apparatus was placed inside a refrigerator, maintained at 4°C.

36

Once the electrophoresis was complete, the power was disconnected, the glass sandwich taken out and the glass plates separated, using a spatula. The gel stuck to one of the glass plates was carefully removed and put in the staining solution for staining overnight. After staining, the gel was taken out and de-stained repeatedly 3 to 4 times in de-staining solution. The de-stained gels were placed on a visible trans-illuminator and photographed.

37

Chapter 4 RESULTS AND DISCUSSION 4.1. Results The Protein samples collected were subjected to total protein estimation and electrophoretic separation by SDS PAGE. The samples were chosen for electrophoresis in such a way as to compare the changes in protein levels at specific intervals of recovery under each treatment. Haemolymph samples were run in both 12% and 8% gels to see the differential accumulation of polypeptides of varying molecular weight. Gut juice samples were subjected to native SDS-PAGE analysis to take care of any possible disintegration of

proteins during

electrophoresis (Singh and Lakhotia, 1995). In general 12-15 bands were observed in haemolymph profiles of CSR2 and Nistari races. This is in agreement with established results (Joy and Gopinathan, 1995). 5-6 bands were found in the gut juice protein profile. The protein marker used for comparison showed bands at approximately 205, 97, 68, 43, 29 and 14 kDa. The protein profiles of both the races under various treatments when compared with control indicated deviations mostly in the 68-97 kDa region apart from the case of new bands. Therefore the variation at 68-97 kDa region was considered for comparison of various treatments. The variations in protein profiles visualized in the gels were compared with the changes in the corresponding values obtained from total protein estimation. This was done by following the methodology suggested by Scaglia et.al., (2003). Both the races showed similar protein banding patterns, though the intensity of the bands varied with treatments. Induction of new protein in the region was clearly discernible in many treatments in both the races but their kinetics varied. Also observed was induction of absolutely new bands in both high and low molecular weight regions. The results obtained are presented and discussed below. 4.1.1. Changes in haemolymph protein profiles in 3rd instar larvae CSR2: Haemolymph protein banding patterns of the 3rd instar CSR2 worms subjected to various treatments are given as Fig. 4 & 5. The 3rd instar CSR2 worms subjected to 36°C treatment for 1h displayed a slight decrease in protein levels in the 68-97 kDa region immediately after the treatment. After a period of recovery of 1h the protein levels raised above the control. Further at the end of 24h the protein levels dropped below the control drastically and again rose almost on par with the 38

control by 48h. The worms subjected to 40°C 1h treatment immediately showed a hike in the protein concentration at 68-97 kDa region continued the same trend throughout the 1h recovery period and dropped below the control at 24h and again raised to become more or less equal to control. In case of 36°C 6h treatment the protein levels hiked immediately after the treatment. This hike persisted throughout the 1h recovery period, dropped below the control at 24h and again hiked above control by 48h. The worms treated at 40°C for 6h showed a hike in protein concentration immediately after the treatment, continued the same trend through the 1h recovery period dropped below the control at 24h and again rose above the control. Here the response of the worms to 36°C 1h and 40°C 1h treatment was different from that to 36°C 6h and 40°C 6h treatment. The downshift in protein content at 24h was more in the second case and less in the first case. The subsequent hike at 48h was high (above control) in second case and low (below control) in the first case. Nistari: Haemolymph protein banding patterns of the 3rd instar Nistari worms subjected to various treatments are given as Fig. 6 & 7. The Nistari worms treated to 36°C for 1h exhibited absolutely no change in the protein levels in comparison with control immediately after the treatment. But after 1h the proteins at 68-97 kDa region dropped slightly. At the end of 24h it further diminished to a drastic level. By the end of 48h again improved on par with the control. Worms treated to 40°C for 1h showed slight decrease in protein concentration immediately after treatment. It went on diminishing to the low level (similar that of 36°C 1h) by 24h and improved to become more or less similar to control at 48h. The worms subjected to 36°C 6h treatment showed a minor increase in protein levels immediately after treatment, further increased after 1h then fell drastically to the same level as that of the above treatments by 24h and then increased to a level slightly higher than the control. The worms treated with 40°C for 6h exhibited a sudden hike in protein levels at 68-97 kDa region immediately after the treatment. Subsequently it fell to reach the same level as the control then drastically reduced to a level same as the 36°C 6h treatment. By 48h the protein level raised to reach above the control. In the case of Nistari also the effects produced by 36°C 6h and 40°C 6h were drastic compared to the 1h treatments. The 1h treatments produced only minor effects in Nistari as compared to CSR2. The long duration treatments were also on par with the 1h treatments till 24h recovery period. But the hike at 48h was more pronounced. Haemolymph protein profiles of treated CSR2 worms did not show any new bands apart from the 12-15 bands generally observed though increase in protein 39

concentration in the 68-97 kDa region was apparent as discussed above. In case of Nistari new bands appeared approx.at 29 kDa, 80 kDa, 90 kDa and 97-205 kDa regions. 90 kDa proteins appeared in treatments viz., 36°C 1h, 40°C 1h, their 1h recovery period, 36°C 6h and 40°C 6h (Fig.-6, lanes 3-8). 97-205 kDa proteins appeared in treatments viz., 36°C 1h 6h recovery, 40°C 1h 6h recovery, 36°C 6h 1h recovery and 40°C 6h 1h recovery (Fig.6, lanes 9-12). 29 kDa protein appeared in 36°C 1h 48h recovery (Fig.7, lane 8). 80 kDa proteins appeared in treatments 40°C 6h 24h recovery and 36°C 1h 48h recovery (Fig.7, lanes 6 and 8).

4.1.2. Changes in haemolymph protein profiles in 4th instar larvae CSR2: Haemolymph protein banding patterns of the 4th instar CSR2 worms subjected to various treatments are given as Fig. 8 & 9. The CSR2 worms treated at 36°C 1h showed a sudden downshift in the protein levels immediately after the treatments but the levels increased above the control after 1h, continued at the same level (above the control) through the 24h recovery period but downshifted below the control by 48h. The larvae exposed to 36°C for 6h and 40°C for 1h showed a similar trend. The only difference was that the downshift started at 1h recovery that continued to 48h recovery period was steady. In contrast to this the 40°C exposure for 6h triggered a hike in the protein levels remained so through the 24h recovery period and then fell below the control at 48h. Nistari: Haemolymph protein banding patterns of the 4th instar Nistari worms subjected to various treatments are given as Fig. 10 & 11. All the treated worms showed a sudden downshift in protein levels (below the control) immediately after the treatment. After 1h the protein levels raised visibly above the control. At this point of time the protein levels showed duration specific kinetics i.e., different for shorter and longer duration treatments. The 6h treatments caused gradual increase of protein level till 24h and then caused downshift below control by 48h. Whereas the 1h treatments caused a gradual downshift of the proteins till 24h and then caused a hike. The 36°C 1h treatment became on par with control at 48h. Whereas the 40°C 1h treatment hiked above control. Here the observations can be summed up as follows. During 24h recovery period the 6h treatments induced hike and 1h treatments induced a dip in protein levels. Whereas at 48h the 6h treatments induced a dip in proteins below control and 1 h treatments induced a hike in protein levels above control.

40

In contrast with the observations made in the 3rd instar the haemolymph protein profiles of Nistari worms did not show any additional bands apart from the 12-15 bands generally observed though increase in protein concentration in the 6897 kDa and 205 kDa region was apparent. In case of CSR2 new bands appeared at 35 kDa region in case of 36°C and 40°C treated larvae after 24h and 48h recovery (fig.9 lanes-2, 3, 5, 6, 8, 9, 11 &12). 4.1.3. Changes in haemolymph protein profiles in 5th instar larvae In 5th instar the larvae were sex separated to observe the differential response towards temperature stress. CSR2: Haemolymph protein banding patterns of the 5th instar CSR2 male and female worms subjected to various treatments are given as Fig. 12-14. The CSR2 male worms subjected to 36°C 1h treatment showed an instant downshift in protein levels subsequent to temperature shock. The protein levels increased to become more or less equal to control by 24h and then slightly dropped below the control after 48h. 40°C 1h treatment elicited more or less similar trend in the protein levels. With 36°C 6h treatment the worms showed a sudden increase in protein concentration, the same trend continued through 1h and dropped below control by 24h further diminished by 48h. Whereas the 40°C 6h treated male worms showed a downshift in the protein levels after 1h recovery subsequent to initial hike. Further it still fell at 24h then hiked above the control to reach a level higher than that exhibited by 36°C 6h treatment. The female larvae of CSR2 treated with 36°C 1h and 40°C 1h showed an immediate downshift in protein levels after temperature treatment and a subsequent hike above the control within 1h recovery. They further exhibited a mild lowering in the protein, which persisted through the 24h recovery period. Then the 36°C temperature treated worms showed an improved protein level and reached a level well above the control. Whereas the high temperature treated ones remained below the control. In contrast both the 36°C 6h and 40°C 6h treated worms showed an immediate hike in the protein levels subsequent to treatment then drastically dropped at 1h and remained at the same level through the 24h recovery period. Then at 48h the 40°C treated larvae showed a drastic downshift in protein content at 68-97 kDa. Nistari: Haemolymph protein banding patterns of the 5th instar Nistari male and female worms subjected to various treatments are given as Fig. 15-17. The male 41

worms treated with 36°C 1h and 40°C 1h showed a sudden downshift in protein content at 68-97 kDa region. Subsequently it improved towards the control levels through the 1h period but found to be diminished after 24h. The extent of diminution was greater in 40°C 1h treated batch. Then they exhibited different behavior at 48h. 36°C temperature treated batch showing a higher protein level above the control and the 40C° temperature treated batch showing a low protein level below the control. This observation was similar to that in 4th instar. The male larvae treated to 36°C for 6h in contrast showed a sudden hike in the protein band intensity at 68-97 kDa almost in tune with the CSR2 male batch. Through the 1h recovery period the protein levels dropped below the control; the 40°C high temperature treatment showing a more drastic downshift. The dip in protein content kept on increasing further through the 24h. Once again the 40°C causing more depression. Further at 48 h they showed the same trend in contrast to that of CSR2 wherein the protein levels increased above the control. The 5th instar Nistari female worms exposed to 36°C 1h treatment did not show much variation from the control whereas the 40°C 1h treated worms showed a slight hike above the control. At 1h, both treatments showed further hike, the 40°C treated ones showing more. Further by 24h the former continued the increase above the control in contrast to the 40°C treated batch which showed a dip in protein concentration, still keeping above the control. But both showed decrease in the protein levels - the 36°C 1h reaching a level closer to control but above it. Whereas the 40°C treated ones dropped below the control. This trend also was in keeping with the trend shown by the 5th instar female larvae of CSR2 with the exception that the 36°C 1h treated worms of Nistari never showed a downshift in concentration below the control levels and also the 40°C 1h treated batch showed no dip below the control till 24h. With 36°C 6h treatment the larvae showed protein levels more or less same to that of control then showing continuous downshift till the 48h, the 40°C temperature treated ones showing a more pronounced downshift than the 36°C temperature treated ones. During 5th instar both the races showed appearance of new bands. CSR2 showed new bands at 29-43 kDa region in lanes 2 and 3 (Fig.-14). Nistari worms showed new bands at 29-43 kDa region in male larvae treated to 40°C 1h and at 29 kDa in male larvae treated with 36°C 1h after a recovery period of 1h and in female larvae treated with 40°C 1h after a recovery period of 1h (Fig.15) lanes 4, 8, and 11.

42

4.1.4. Variations in the total protein content The graphs (1-8) indicate the variations in the total protein content in the two races of 5th instar silkworms under various treatments in comparison with that of control worms. These were compared with the observations from the various gels to draw final conclusions (Scaglia et.al., 2003). CSR2: The graphs (1-4) indicate that the male worms treated with 36°C for 1h showed only a slight variation (up shift) in total protein content from the control levels till 24h. By 48 h it fell slightly below the control. Under 36°C for 6h they showed a slight initial hike but remained equal to control for the remaining period. Under 40°C for 1h the initial up shift was high above the control levels, then immediately fell slightly below the control within 1h and by 24h became equal to control then at 48 h it fell below the control. Under 40°C for 6h the protein levels fell below control immediately and continued without change till 24h and later rose above control by 48h. The female worms treated with 36°C for 1h showed a slight up shift from the control levels till 48h, the hike improving steadily. Under 40°C for 1h the initial up shift was above the control levels was delayed till 1h and by 24h became equal to control. Then at 48 h it fell slightly below the control. Under 40°C for 6h the protein levels fell below control immediately and continued without change till 48h. Striking similarities between the responses of male and female worms were observed except in the case of 40°C 1h exposure. In this case alone an immediate up shift in protein above the control was observed and that was by the male. Another important observation was that the lowering in protein levels caused by the 6h treatments was more pronounced in female. Nistari: The graphs (5-8) indicate that the male worms treated with 36°C for 1h showed a sudden down shift in total protein content from the control levels till 24h. By 48 h it became equal to the control. Under 36°C for 6h the initial downshift was minimal but became pronounced by 1h and remained below control levels till 48h. 40°C for 1h caused an initial down shift below the control levels then gradually improved by 24h to become equal to control. Under 40°C for 6h the protein levels fell below control by 1h and continued without much change till 48h. The female worms treated with 36°C for 1h did not show much deviation from the control till 1h but fell below the control levels by 48h. Under 36°C for 6h 43

there was a slight up shift, which persisted till 24h to reach control levels by 48h. Under 40°C for 1h there was an immediate up shift in protein content which persisted till 48h. Under 40°C for 6h there was a slight down shift initially, then it gradually rose till 24h and at 48h dropped below control levels. Though the gut juice samples were subjected to native PAGE analysis no much changes in protein profile could be observed (Fig.22 &23). 4.2. Discussion During the 3rd instar the control larvae were due to moult on the 3½-4th day. The 48h recovery period in the treated batches fell on the 4th day. A general downshift in protein content in all the treatments was observed at 24h, whereas the controls kept steady till 48h. It may be that all the treated larvae might have advanced towards moulting earlier than the control worms. It is reported that the storage protein content in the haemolymph in silkworm larvae increase at the feeding period and decrease during the moulting period (Nagata and Kobayashi, 1990).

It is also reported that the storage proteins function as a reservoir for

amino acids, which are utilized for tissue formation during adult development (Levenbook, 1985). Nagata and Kobayashi (1990) have observed that the cyclic fluctuation of the storage proteins repeats at each instar and storage proteins disappear from the haemolymph at each of the larval moults. They explained that it is mainly due to the degradation of these proteins. In the present case the high temperature appears to stimulate early moulting. After successful moulting, the protein levels were observed to either return to normal position or hike above the control in case of longer duration treatments. It may be that the longer duration (6h treatments) not only induced early moulting, but also caused early synthesis of new proteins in the larval body fluid after completion of moult. The short duration treatment (1h) also stimulated early moulting but gradually the pace slowed down to become equal to control. Thus the effects produced by the short duration treatments and long duration treatments were differential in the two races. In CSR2 the short duration exposure induced early moulting but the treated larvae became normal (equal to control) at 48h after treatment. The long duration treatments induced early moulting at 24h. Subsequently by 48h the protein synthesis was higher than that of the control. The treated Nistari worms also behaved in a similar fashion but all the treatments were taken more or less similarly such that by 48h there was a high level of protein content in all the treatments well above the control. The above observations imply that the 3rd instar larvae of both races were more affected by the long duration treatments rather than the temperature levels. 44

That is to say the worms were less affected by the 40°C 1h treatment than the 36°C treatment. The threshold for damage to 3rd instar may be marked by the duration of temperature. During 4th instar the control larvae were due to moult on the 4th day. 48h recovery period fell on the 4th day. The protein quantity at the 68-97 kDa region of treated worms started falling at 24h recovery itself, whereas the controls kept steady till 48h. The change in protein levels between the control and treatments should be seen in relation to moulting as well as the effect of temperature. Similar to the observations made in the 3rd instar these results can be interpreted. The treated CSR2 larvae showed the inability to recover from the dropping concentrations, even after moulting. This inability was more pronounced in 40°C treated larvae. This suggests that the 40°C treatment in spite of the duration of exposure did not hinder but enhanced the moulting process, but CSR2 larvae could not recover fully and regain protein synthesis in full swing after ecdysis. The 1h treatments did not cause much damage to the general transcription mechanism of the Nistari worms. After 36°C and 40°C 1h treatment they moulted early but recovered after moulting. 36°C 1h treatment had almost nil effect on the Nistari worms. But the 6h treatments affected Nistari worms in a more or less similar fashion as that of CSR2. This implies that both CSR2 and Nistari worms are more susceptible to temperature treatment in 4th instar than in 3rd instar. The Nistari worms exhibited tolerance with respect to high temperature in a duration specific manner. The severity of variations exhibited by the 5th instar male worms with respect to control was less for the 1h treatments as compared to 3rd and 4th instars. Only the 6h treatments varied drastically - may be the worms posses some mechanism

to

counter

the

shorter

duration

treatments.

The

differential

susceptibility to high temp is more clear in females, especially for the 6h duration. The initial hike in protein content immediately after the treatments always indicated a sign of response to temperature.

Lindquist

(1980) reported

that the heat

shocked Drosophilla tissues produced more quantity of protein initially and then at a declined rate. She proposed that this was due to the shift in the gene transcription mechanism to produce a small number of characteristic heat shock proteins. The most visible increase in the haemolymph protein profiles as visualized in the gels were in the 68-97 kDa region.

It is known that one of the most

important families of heat shock protein - Hsp 70 - is found expressed in a wide variety of insects including lepidopterans such as Calpodes ethilus, Manduca Sexta 45

and Bombyx mori (Fittinghoff and Riddiford, 1990). The consistent pattern of increase in protein in this particular range subsequent to exposure to high temperature indicates a possibility of appearance of certain new proteins in this region which is not found in the normal, unstressed individuals. A comparison between the kinetics of the proteins of 68-97 kDa region as observed in the gels with the differences observed in the total protein quantity during 5th instar in infected and control worms supports this view. In general the CSR2 (except 3rd instar) appeared to be more sensitive to the shift in temperature rather than the duration of treatment. Whereas the Nistari worms appeared to be not much sensitive to the temperature change alone but seemed to be affected more by the duration of temperature treatments. The 3rd instar CSR2 worms also showed the same trend. This implies that CSR2 being a temperature susceptible race is sensitized by hike in temperature even for shorter duration (1h). Nistari being a less susceptible race appeared to be not much sensitive for a short-term temperature hike but succumbed when exposure period increased. More protein content always lead to more damage and took more time for recovery. In general an apparent hike in 68-97 kDa protein region was observed as discussed above in various treatments in comparison with the control. Available literature strongly suggest that insects in general the order lepidoptera in particular and silkworm specifically is capable of expressing members of Hsp70 family under heat shock (Lindquist, 1980; Joy and Gopinathan, 1995). The increase of protein in the 69-97kDa region was not accompanied by visible increase or decrease of other protein bands in the gel. This strongly suggests that the increase in this particular region is due to synthesis of fresh proteins. This view is supported by the findings of Lindquist in 1980. She has pointed out that Hsp70 was produced in much larger amount than any other protein subsequent to heat shock in Drosophilla. Though silkworm need not necessarily follow the Hsp70 gene expression pattern, there are reports that the silkworm share similar patterns of Hsp expression with other insects (Landaise, et.al., 2001). When the changes in 69-97kDa protein bands were compared with total protein levels estimated in 5th instar larvae, it was observed that in many instances the increase in protein levels at this region was accompanied with a sudden drop in the total protein concentration. (CSR2 male 36°C 6h at 48h recovery, 40°C 1h at 1h & 48h recovery, 40°C 6h at 0h recovery; CSR2 female – 36°C 6h at 0h recovery, 40°C 6h at 0h recovery; Nistari male - 36°C 1h at 1h, 24h and 48h recovery, 36°C 6h at 0h recovery and 40°C 1h at 1h recovery; Nistari female - 36°C 1h at 1h, 24h & 48h recovery, 36°C 6h at 24h 46

recovery and 40°C 6h at 0h recovery). This strongly indicates that the increase observed at 68-97kDa region was not as part of the general increase in protein levels, but due to significant increase in this particular region. Available literature strongly suggest this view. Lindquist in 1980 reported that the vigour and speed of induction of heat shock proteins are greatly enhanced by the fact that synthesis of the normal complement of proteins and mRNAs is sharply and rapidly curtailed. Fittinghoff and Riddiford (1990) established heat induced shut down of normal protein synthesis in Manduca Sexta, a lepidopteran insect which is phylogenetically very close to silkworm. Protein level changes in the above discussed manner was observed in all the three instars with striking differences between the two races. An initial sudden hike in protein levels at 69-97 kDa region was observed in case of CSR2 3rd instar except in the case of 36°C 1h treatment, in 4th instar only in the case of 40°C 6h treatment and in 5th instar only in the case of 6h treatments. In case of Nistari the above phenomenon was noticed only in the case of 5th instar in males for the 6h treatments and in female in case of 36°C 6h and 40°C 1h treatments. This phenomenon has been observed by previous workers also (Lindquist, 1980). She has pointed out that when Drosophilla cells were shifted to higher temperatures and increase in normal cellular protein synthesis could be detected. She has also pointed out that, completed transcripts of heat shock mRNAs are produced within 4 min. of temperature elevation; within 8-12 min. these messages have been processed, transported to the cytoplasm and translated into protein. The current observations and above established facts support the prevailing understanding on the differential thermo tolerance of the two races and it suggests that the reason for the comparative thermo tolerance of the Nistari race over the CSR2 race is due to the induction of certain new proteins in the 68-97kDa region. It is expected that the new protein belongs to the Hsp70 family. Similar observations were made by previous researchers in case of the thermo tolerant race CSR18 in comparison with CSR2 (Chitra et.al., 2004; Nagaraja, 1991). For confirmation of this result, immuno blotting techniques are to be employed, which is beyond the scope of this study. Apart from the above certain new bands as mentioned under the results and as indicated in figures 9, 14, 15, 18 and 21 were observed. In 3rd instar appearance of new proteins subsequent to heat shock were observed in the Nistari race at 68-97 and 97-205 regions (Fig.6, lanes 3-7 & 8-12). Nistari known for its thermo tolerance might have the capacity to express certain proteins of molecular weight 90kDa under high temperature immediately after the 47

heat shock and proteins of 97-205kDa after 6h. The new bands appeared here tends to be of the 90kDa Hsp family. Bombyx mori is proved to be possessing the genes encoding Hsp90. Landaise et.al., (2001) completely sequenced the Hsp90 cDNA from the lepidopteran insects Bombyx mori and Spodoptera frugiperda and found a high consistency with known phylogeny at both high and low taxonomic levels between the two. They performed transcription analysis of hsp90 gene and found that the induction of the gene occurs only at 42°C (14°C above physiological growth conditions) gene of S. frugiperda. In 4th instar CSR2 worms new bands at 97-205kDa were observed under 36 & 40°C 1h 24h recovery & 48h recovery as well as 36 & 40°C 6h 24h recovery & 48h recovery. This was compared with the Nistari race for the same treatments (Fig.18) and found that Nistari did not show any fresh bands at this region. The high molecular weight Hsps are supposed to have role in protein folding. The consistent expression of these proteins indicates that their expression is specific to the less tolerant CSR2 race and are expressed under heat shock. Whereas the Nistari race which is comparatively tolerant might constitutively possess some other heat shock proteins and thus there was no fresh expression. The constitutive presence of heat shock proteins has been reported by Krebs and Feder in 1998 in Drosophilla. In the 5th instar male and female worms of both the races treated to 36°C 1h expressed a new protein of molecular weight between 29 & 43kDa region after 24h recovery. The dramatic appearance of these bands in both the races suggest that it is not a racial character. Since it was found in both the races under heat shock, and not found without heat shock. It also implies that this protein is specific to appear the time period between 1h recovery and 24h recovery, but not 48h recovery. Since these bands appeared only during 5th instar, it is to be suspected that it renders some sort of protection against the worms of both races which are particularly susceptible during the 5th instar. Since it is not found in other instars, it is expected that this is an age specific protein. Chances are that this is a heat shock protein. Similar observation was made by Nagaraja (1991); who observed a 36kDa protein in the haemolymph protein profile of C.nichi larvae. Similar proteins of the same size were observed in a parallel study done on BmNPV inoculated larvae at the Molecular Biology Lab, CSR&TI, Mysore recently (Unpublished). This provides a strong ground to believe that these new proteins are synthesized in response to viral infection also. It is to be suspected that it renders some sort of protection against the worms of both races which are particularly susceptible during the 4th and 5th instar. Since it is not found in other instars, it is expected that this is a late age specific protein. 48

It was observed that out of the 100 treated larvae from each treatment reared upto cocooning, only the 40°C 6h treated CSR2 worms showed severe mortality. Almost all the worms in other treatments in both the races successfully spun cocoons and pupated. This indicate that larvae could overcome the stress of high temperature at least 40°C 1h and 36°C 6h. The mechanism which help the same may lie in the newly appeared proteins bands or excessive incorporation into already existing ones. In the current experiment, we have come across new protein bands at low, medium and high molecular weight regions in the heat shocked larvae of both races at different stages. Probably Hsps: 100, 90, 70 families and 34-35kDa. The available literature strongly indicates the possibility that these new proteins can be heat shock proteins. However, this has to be confirmed by employing immuno blotting techniques or by sequence homology comparisons after electro elution and sequencing of the protein bands under question. However, the results indicate that there is tremendous scope for further studies in this direction. The mechanism of Temperature tolerance with respect to different races, sexes and varying levels of temperature will make a very interesting and useful subject which is also very important to sustain tropical bivoltine sericulture industry.

49

Chapter 5 SUMMARY •

Heat-shock specific protein synthesis was observed in both races.

•

3rd instar larvae exhibited changes in protein levels dependent on the

duration of the heat shock. Threshold for damage to the general transcription mechanism in 3rd instar may be marked by the duration of the exposure. •

CSR2 (4th & 5th instars) was more affected by the hike in temperature rather than its duration. Its proteins suffered damage even by temperature hike for a short duration.

•

Nistari was less affected by the instantaneous hike in temperature, rather than its duration.

•

The female larval haemolymph proteins were found to be more affected by heat shock, than that of male.

•

40°C treatment caused more protein damage in 4th instar than in 3rd instar in both races.

•

Exposure to high temperature in surviving animals lead to a sudden hike in protein levels at 68-97kDa region in both races in the order Nistari > CSR2 male > CSR2 female, indicating a stress related, race and sex specific transcription strategy.

•

New protein bands appeared in the molecular weight range of 97-205, 6897 and 29-43 kDa possibly representing Hsp: 100, 90, 70 families and 3435kDa.


50

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61

1

2

3

4

5

6

7

8

9

10

11

12

13

205

97

68

43

29

Fig.4: Haemolymph Protein Profile - CSR2 3rd Instar

1

2

3

4

5

6

7

8

9

10

11

12

13

205

97

68

43

29

Fig.5: Haemolymph Protein Profile – CSR2 3rd Instar

62

Fig.4 Lanes

Race

Temperature

Exposure

Recovery

1 2 3 4 5 6 7 8 9 10 11 12 13

MARKER

CSR 2

CONTROL 36°C 40°C 36°C 40°C 36°C 40°C 36°C 40°C 36°C 40°C CONTROL

1 H. 1 H. 1 H. 1 H. 6 H. 6 H. 1 H. 1 H. 6 H. 6 H. -

1 H. 1 H. 6 H. 6 H. 1 H. 1 H. -

Lanes

Race

Temperature

Exposure

Recovery

1 2 3 4 5 6 7 8 9 10 11 12 13

MARKER

36°C 40°C CONTROL 36°C 40°C CONTROL 36°C 40°C CONTROL 36°C 40°C CONTROL

1 H. 1 H. 6 H. 6 H. 1 H. 1 H. 6 H. 6 H. -

24 H. 24 H. 24 H. 24 H. 48 H. 48 H. 48 H. 48 H. -

Fig.5

CSR 2

63

1

2

3

4

5

6

7

8

9

10

11

12





13

205



97















68

43

29

Fig.6: Haemolymph Protein Profile – Nistari 3rd Instar 1

2

3

4

5

6

7

8

9

10

11

12

13

205 

97





68

43

29 

Fig.7: Haemolymph Protein Profile – Nistari 3rd Instar

64

Fig: 6 Lanes

Race

Temperature

Exposure

Recovery

1 2 3 4 5 6 7 8 9 10 11 12 13

MARKER

Nistari

CONTROL 36°C 40°C 36°C 40°C 36°C 40°C 36°C 40°C 36°C 40°C CONTROL

1 H. 1 H. 1 H. 1 H. 6 H. 6 H. 1 H. 1 H. 6 H. 6 H. -

1 H. 1 H. 6 H. 6 H. 1 H. 1 H. -

Lanes

Race

Temperature

Exposure

Recovery

1 2 3 4 5 6 7 8 9 10 11 12 13

MARKER

36°C 40°C CONTROL 36°C 40°C CONTROL 36°C 40°C CONTROL 36°C 40°C CONTROL

1 H. 1 H. 6 H. 6 H. 1 H. 1 H. 6 H. 6 H. -

24 H. 24 H. 24 H. 24 H. 48 H. 48 H. 48 H. 48 H. -

Fig.7

Nistari

65

1

2

3

4

5

6

7

8

9

10

11

12

13

205

97

68

43

29

Fig.8: Haemolymph Protein Profile – CSR2 4th Instar

1

2

3

4

5

6

7

8

9

10

11

12

13

205 















97

68

43

29

Fig.9: Haemolymph Protein Profile – CSR2 4th Instar

66

Fig.8 Lanes

Race

Temperature

Exposure

Recovery

1 2 3 4 5 6 7 8 9 10 11 12 13

MARKER

CSR 2

36°C 40°C CONTROL 36°C 40°C CONTROL 36°C 40°C CONTROL 36°C 40°C CONTROL

1 H. 1 H. 1 H. 1 H. 6 H. 6 H. 6 H. 6 H. -

1 H. 1 H. 1 H. 1 H. -

Lanes

Race

Temperature

Exposure

Recovery

1 2 3 4 5 6 7 8 9 10 11 12 13

MARKER

36°C 40°C CONTROL 36°C 40°C CONTROL 36°C 40°C CONTROL 36°C 40°C CONTROL

1 H. 1 H. 6 H. 6 H. 1 H. 1 H. 6 H. 6 H. -

24 H. 24 H. 24 H. 24 H. 48 H. 48 H. 48 H. 48 H. -

Fig.9

CSR 2

67

1

2

3

4

5

6

7

8

9

10

11

12

13

205

97

68

43

29

Fig.10: Haemolymph Protein Profile – Nistari 4th Instar 1

2

3

4

5

6

7

8

9

10

11

12

13

205

97

68

43

Fig.11: Haemolymph Protein Profile – Nistari 4th Instar

68

Fig.10 Lanes

Race

Temperature

Exposure

Recovery

1 2 3 4 5 6 7 8 9 10 11 12 13

MARKER

Nistari

36°C 40°C CONTROL 36°C 40°C CONTROL 36°C 40°C CONTROL 36°C 40°C CONTROL

1 H. 1 H. 1 H. 1 H. 6 H. 6 H. 6 H. 6 H. -

1 H. 1 H. 1 H. 1 H. -

Lanes

Race

Temperature

Exposure

Recovery

1 2 3 4 5 6 7 8 9 10 11 12 13

MARKER

36°C 40°C CONTROL 36°C 40°C CONTROL 36°C 40°C CONTROL 36°C 40°C CONTROL

1 H. 1 H. 6 H. 6 H. 1 H. 1 H. 6 H. 6 H. -

24 H. 24 H. 24 H. 24 H. 48 H. 48 H. 48 H. 48 H. -

Fig.11

Nistari

69

1

2

3

4

5

6

7

8

9

10

11

12

13

205

97

68

43

29

Fig.12: Haemolymph Protein Profile CSR2 5th Instar

1

2

3

4

5

6

7

8

9

10

11

12

13

205

97

68

43

Fig.13: Haemolymph Protein Profile - CSR2 5th Instar

70

Fig.12

Lanes

Race

Sex

Temperature

Exposure

Recovery

1 2 3 4 5 6 7 8 9 10 11 12 13

MARKER

M F M F M F M F M F M F

36°C

1 H.

-

40°C

1 H.

-

CONTROL

-.

-

36°C

1 H.

1 H.

40°C

1 H.

1 H.

CONTROL

-

-

CSR 2

Fig.13 Lanes

Race

Sex

Temperature

Exposure

Recovery

1 2 3 4 5 6 7 8 9 10 11 12 13

MARKER

M F M F M F M F M F M F

CONTROL

-

-

36°C

6 H.

-

40°C

6 H.

-

CONTROL

-

-

36°C

6 H.

1 H.

40°C

6 H.

1 H.

CSR 2

71

1

2

3

4

5

6

7

8

9

10

11

12

13

205

97

68

43





29

Fig.14: Haemolymph Protein Profile CSR2 5th Instar

1

2

3

4

5

6

7

8

9

10

11

12

13

205

97

68

43



29





Fig.15: Haemolymph Protein Profile - Nistari 5th Instar

72

Fig.14 Samples Exposure

Lanes

Race

Sex

Temperature

1 2 3 4 5 6 7 8 9 10 11 12 13

MARKER

M F M F M F M F M F M F

36°C

1 H.

24 H.

40°C

1 H.

24 H.

CONTROL

-

-

36°C

1 H.

48 H.

40°C

1 H.

48 H.

CONTROL

-

-

CSR 2

Recovery

Fig.15 Lanes

Race

Temperature

Exposure

Recovery

1 2 3 4 5 6 7 8 9 10 11 12 13

MARKER

36°C

1 H.

-

40°C

1 H.

-

CONTROL

-

-

36°C

1 H.

1 H.

40°C

1 H.

1 H.

CONTROL

-

-

Nistari

73

1

2

3

4

5

6

7

8

9

10

11

12

13

205

97

68

43

Fig.16: Haemolymph Protein Profile Nistari 5th Instar

1

2

3

4

5

6

7

8

9

10

11

12

13

205

97

68

43

Fig.17: Haemolymph Protein Profile - Nistari 5th Instar

74

Fig.16

Lanes

Race

Sex

Temperature

Exposure

Recovery

1 2 3 4 5 6 7 8 9 10 11 12 13

MARKER

M F M F M F M F M F M F

CONTROL

-

-

36°C

6 H.

-

40°C

6 H.

-

CONTROL

-

-

36°C

6 H.

1 H.

40°C

6 H.

1 H.

Nistari

Fig.17

Samples Exposure

Lanes

Race

Sex

Temperature

1 2 3 4 5 6 7 8 9 10 11 12 13

MARKER

M F M F M F M F M F M F

36°C

1 H.

24 H.

40°C

1 H.

24 H.

CONTROL

-

-

36°C

1 H.

48 H.

40°C

1 H.

48 H.

CONTROL

-

-

Nistari

Recovery

75

1

2

3

4

5

6

7

8

9

10

11

12

13

205 















97

68

43

29

Fig.18: Haemolymph Protein Profile CSR2& Nistari 4th Instar

1

2

3

4

5

6

7

8

9

10

11

12

13

205

97

68

43

29

Fig.19: Haemolymph Protein Profile - CSR2 & Nistari 5th Instar

76

Fig.18

Lanes

Race

Temperature

Exposure

Recovery

1 2 3 4 5 6 7 8 9 10 11 12 13

MARKER CSR2 " " " " " Nistari " " " " "

36°C 40°C CONTROL 36°C 40°C CONTROL 36°C 40°C CONTROL 36°C 40°C CONTROL

1 H. 1 H. 1 H. 1 H. 1 H. 1 H. 1 H. 1 H. -

24 H. 24 H. 48 H. 48 H. 24 H. 24 H. 48 H. 48 H. -

Fig.19

Samples Exposure

Lanes

Race

Sex

Temperature

Recovery

1 2 3 4 5 6 7 8 9 10 11 12 13

MARKER CSR 2 " " " " " Nistari " " " " "

M F M F M F M F M F M F

CONTROL

-

-

36°C

6 H.

24 H.

40°C

6 H.

24 H.

36°C

6 H.

24 H.

40°C

6 H.

24 H.

CONTROL

-

-

77

1

2

3

4

5

6

7

8

9

10

11

12

13

205

97

68

43

29

Fig.20: Haemolymph Protein Profile CSR2 & Nistari 5th Instar

1

2

3

4

5

6

7

8

9

10

11

12

13

205

97

68

43









29

Fig.21: Haemolymph Protein Profile - CSR2 & Nistari 5th Instar

78

Fig.20 Samples Exposure

Lanes

Race

Sex

Temperature

Recovery

1 2 3 4 5 6 7 8 9 10 11 12 13

MARKER CSR 2 " " " " " Nistari " " " " "

M F M F M F M F M F M F

CONTROL

-

-

36°C

6 H.

48 H.

40°C

6 H.

48 H.

36°C

6 H.

48 H.

40°C

6 H.

48 H.

CONTROL

-

-

Lanes

Race

Sex

Temperature

1 2 3 4 5 6 7 8 9 10 11 12 13

MARKER CSR 2 " " " " " Nistari " " " " "

M F M F M F M F M F M F

36°C

1 H.

24 H.

40°C

1 H.

24 H.

CONTROL

-

-

36°C

1 H.

24 H.

40°C

1 H.

24 H.

CONTROL

-

-

Fig.21 Samples Exposure

Recovery

79

1

2

3

4

5

6

7

8

9

10

11

12

13

Fig.22: Gut Juice Protein Profile Nistari 5th Instar

1

2

3

4

5

6

7

8

9

10

11

12

13

Fig.23: Gut Juice Protein Profile Nistari 5th Instar

80

Fig.22

Samples Exposure

Lanes

Race

Sex

Temperature

1 2 3 4 5 6 7 8 9 10 11 12 13

MARKER

M F M F M F M F M F M F

36°C

1 H.

-

40°C

1 H.

-

CONTROL

-

-

36°C

1 H.

1 H.

40°C

1 H.

1 H.

CONTROL

-

-

Lanes

Race

Sex

Temperature

1 2 3 4 5 6 7 8 9 10 11 12 13

MARKER

M F M F M F M F M F M F

36°C

1 H.

24 H.

40°C

1 H.

24 H.

CONTROL

-

-

36°C

6 H.

24 H.

40°C

6 H.

24 H.

CONTROL

-

-

Nistari

Recovery

Fig.23

Nistari

Samples Exposure

Recovery

81