Impact of thermal stress on the haemolymphal proteins , biological and economical characters of the silkworm , bombyx mori L . Rehab

The purpose of this study is to determine the impact of different thermal stresses on univoltine Bombyx mori embryos and grown larva in the terms of larval haemolymph proteins , biological and economical characters. The experiment was designed to expose egg groups to different temperatures as follows; a group exposed to 0°C for one hour, a group exposed to 0°C for two hours, a group exposed to 40°C for one hour and a group exposed to 40°C for two hours, followed by 2 hours recovery at room temperature in addition to a control group, the same was applied to the grown larval groups. The larval haemolymph of tested groups were subjected to protein analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE). Data revealed that considerable alteration in temperature hamper the protein activities which affects the embryonic development and hatching of eggs. 40oC for two hours was lethal dose for embryo. The highest measured parameters were recorded for eggs exposed to 40oC for one hour. While for grown larvae, the highest results were recorded for larvae exposed to 0oC for 1 hour. Thermal stresses induced protein changes with the appearance of new bands. The epigenetic degree of similarity recorded 50% between the control egg group and the egg groups exposed to 0oC for 2 hrs and 40oC for 1 hrs. as well as, for grown larva, between control larval group and larval group exposed to 40oC for 2 hrs.


INTRODUCTION
The mulberry silkworm, Bombyx mori L. (Lepidoptera: Bombycidae) is a very heat sensitive organism.The differences in the environmental components considerably affect the genotypic expression in the form of phenotypic output of silkworm crop such as cocoon weight, shell weight and cocoon shell ratio (Rahmathulla, 2012).Therefore, intensive and careful domestication over centuries has apparently deprived this commercial insect of the opportunity to acquire thermo-tolerance.
Lack of thermo-tolerance may be attributed as one of the many factors responsible for poor performance of B mori strains at times as many quantitative characters decline sharply at higher/lower temperatures.Therefore, one of the key considerations in developing hybrids could be the need for thermo-tolerant strains (Kumari et al., 2011).The development and economic production of sericulture greatly depends on the metabolic modulations and physiological adaptability of silkworm, besides its genetic constitutions (Sinha and Sanyal 2013).The recent advances in silkworm breeding and those in stress-induced protein synthesis have opened up new avenues to evolve robust productive silkworm hybrids (Moghaddam et al., 2008).Such information my provide useful clues for the establishment of methods for prevention of viral diseases in the silkworm (Kobayashi and Chaeychomsri, 1993).
Insects have evolved different biological and physiological strategies to overcome drastic and adverse changes in their surrounding environment (Singh et al., 2010).It depends on the molecular mechanism of the cell involving the rapid synthesis of special proteins, the heat shock proteins (Park et al., 2008).The term "heat shock protein" was introduced by Tissieres et al. (1974) as these proteins increased in synthesis due to sudden increases/decreases in temperature.They act as 'molecular chaperones' to ensure better survival under stressful conditions, including thermo-stress (Mosser and Morimoto, 2004) and have been implicated in immunogenicity to cancers/infectious diseases (Srivastava, 2002).
Generally, the heat shock response depends on the magnitude of temperature and duration of exposure, and is relative to the environmental temperature at which the organism normally survives as suggested by Lagerspetz (2006).
The determination of the electrophoretic patterns of the protein bands mobility and number (qualitative changes) and the intensity of some bands (quantitative changes) probably, reflect some of physiological processes (Hussein et al.,1993).Quantitative protein profiling is an essential part of proteomics and requires new technologies that accurately, reproducibly, and comprehensively identify and quantify the proteins contained in biological samples (Smolka et al., 2002).Screening for thermotolerance in the silkworm, is an essential prerequisite for the development of thermo-tolerant strains.The current study, aims to study the effect of temperature stress at the egg and fourth larval stages of B. mori in relation to its biochemical alternations and their subsequent impact on commercial traits.

MATERIAL AND METHODS
Silkworm eggs were obtained from the Sericulture Research Department (SRD) of Plant Protection Research Institute (PPRI), Agricultural Research Center (ARC), Giza, Egypt.Rearing technique was done in the lab.under the hygro-thermic conditions 28 ± 1 °C and 75 ± 5% RH, following the standard methodology of rearing in SRD according to Krishnaswamy (1978).Larvae were fed on leaves of Kokuzo-27 mulberry variety.

A-Temperature stresses Egg stage
To study the impact of temperature stresses on egg hatching percentages, 100 eggs at late embryo stage (blue stage) in three replications were kept in egg hatching boxes in four temperature stress conditions (T2 to T5) along with control (T1) in the first day of raising temperature from cold storage at 5°C to 27°C .Control-treatment 1 (T1) -27°C till hatching.Treatment -2 (T2) -0 degree Celsius for one hour in an incubator.Treatment -3 (T3) -0 degree Celsius for two hours in an incubator.Treatment -4 (T4) -40 degree Celsius for one hour in an incubator.Treatment -5 (T5) -40 degree Celsius for two hours in an incubator.
All the treatment batches (T2~T5) after being exposed to thermal stress were kept at 15°C for two hours for recovery and finally placed at 27 °C till hatching.

Fourth larval stage
At the beginning of the fourth larval stage (grown larval stage), larvae were divided into 5 groups, with100 larvae/ replicate and the following treatments were imposed: Control -Treatment -6 (T6) -room temperature.Treatment -7 (T7) -0 degree Celsius for one hour in an incubator.
Treatment -8 (T8) -0 degree Celsius for two hours in an incubator.Treatment -9 (T9) -40 degree Celsius for one hour in an incubator.Treatment -10 (T10) -40 degree Celsius for two hours in an incubator Later all the treated larval boxes (T7 ~T10) were maintained at 15 °C for two hours for recovery, then the rearing technique was continued normally at room temperature.
Insect performance in all treatments was assessed based on pre-cocooning parameters such as hatching percentage, larval duration, and larval weight.Commercial characteristics such as cocoon weight, shell weight and shell percentage for female and male were also recorded and subjected to analysis of variance according to Snedecor and Cochran (1981).
At the sixth day of the fifth larval instar, haemolymph samples were collected from each treatment.

B-Sodium Dodecyl Sulphate (SDS)polyacrylamide gel electrophoresis
SDS-polyacrylamide gel electrophoresis was performed in 12% acrylamide slab gels following the system of Laemmli, (1970).10 microlitres of haemolymph sample was taken in an eppendrof tube and centrifuged at 5000 rpm for 10 min.Then a volume of 20 μl protein extract was added to 10 μl of treatment buffer (1M Tris-HCl pH6.8+ 10% (w/v) SDS + glycerol + 0.5M EDTA + Bromophenol blue + b-mercaptoethanol).Then 20 μl of each sample was loaded in the gel.After the electrophoresis run was completed, gels of SDS-protein was stained in Coomassie Brilliant Blue solution for 12-18 h, then rinsed in destaining solution until the dark background became colourless except the blue protein bands and photographed.The gels were scanned to calculate the relative mobility and concentration of identified bands using Gel-Pro Analyzer software (V.3).

C-Similarity Index:
The similarity index between all tested treatments were calculated according to Nei & Li (1979) using the formula S.I: Similarity Index N: the number of common bands Na and Nb: the number of bands in individuals (a) and (b).

Bombyx mori L. eggs
Egg groups exposed to 0 ºC for one and two hours and 40 ºC for one hr hatched normally, while exposure to 40º C for two hours resulted in death of embryos.The depressed eggs with fully formed dead larvae inside the eggs were found after high temperature stress as shown in Figure (1).-------------Na + Nb significantly compared to the other groups.While, exposed to 40 ºC for one hour increased hatching percentage, single larval and cocoon weights and single silk ratio for both male and female.Fourth larval stage exposed to 0ºC for one hour showed increment in single larval weight, single female and male cocoon weights and silk ratios .All larvae exposed to 0ºC and 40ºC for one and two hours resumed feeding and spun normal cocoons (Table 2).The same small liters in a column means there is no significance between means.The same small liters in a column means there is no significance between means.Relative fragmentation (RF), molecular weight(Mw) and amount percent (Am %)

B-Electrophoretic studies
Common band at R 10 appeared in (T 2, T 3 and T 4) were two times in amount percent comparing to control (T 1), the protein amount of the common band at R 17 in (T 4) declined ~ three times less than control (T 1).Also, the common band R 23, protein amount percent of control (T 1) and (T 5) were ~ two times than (T 2 and T 3) and four times than (T 4).The protein amount percent of the common band at R 24, of (T 2) showed the lowest density among the other samples.
The degree of similarity among the tested treatments was calculated and tabulated in Table (4).The Similarity was not less than 0.5, the highest similarity 0.8 was recorded between control (T 1) and (T 5).Relative fragmentation (RF), molecular weight (Mw) and amount percent (Am %) of protein bands Lane 6 : T6 control Lane 7 : T7 -0°C for 1 hr Lane 8 : T8 -0°C for 2 hrs Lane 9 : T9 -40°C for 1 hr Lane 10 : T10 -40°C for 2 hrs Sample 1 : T1 control Sample 2 : T2 -0°C for 1 hr Sample 3 : T3 -0°C for 2 hrs Sample 4 : T4 -40°C for 1 hr Sample 5 : T5 -40°C for 2 hrs Data presented in Table ( 5) and demonstrated in Figure (2) showed the protein pattern at fourth larval stage, six common bands no. 1, 7, 9, 25, 26 and 27were detected between the control (T 6) and the other treatments.The common band at R 7 of (T 8) showed the highest protein density among the other treatments.The common band at R 9, sample (T 9) showed the highest density ~ 2 times than (T 8) and (T 10).The protein density of band at R 25of (T 9) and (T 10) were 2 times than the protein of control (T 6).The protein densities of common band at R 26, T 7 and T 9 were ~ 2 times of control (T 6) and ~ 4 times of (T 8) and (T 10).The common protein band density at R 27 of (T 10) recorded the lowest density among the other tested samples.
Degree of similarity among the tested treatments are presented in Table ( 6).The similarity was not lowered than 0.5, the highest similarity was 0.7 and recorded between control (T 6) and treatments (T 7) and (T 8), as well as between (T 7) and (T 8).

DISCUSSION
Temperature plays an important role and decides the fate of mulberry silkworm, Bombyx mori L. during embryonic and postembryonic development (Kumar et al., 2012).Nowadays, the spread and success of silkworm rearing was mainly due to the introduction of F1 hybrids of native multivoltine as female parent (for resistance) and bivoltine as male parent (for high quality silk).Even now it is a challenging task to develop not only stress and disease-resistant strains, but also to provide high yielding silkworm strains with improved stress tolerance (Manjunatha et al., 2010).
The heat stress treatments could be employed to determine the level of thermo-tolerance based on mortality (Loeschcke and Sorensen, 2005).
Notably, thermotolerance increased as larval development proceeded, sequentially in the order of first instar > second instar > third instar > fourth instar > fifth instar (Vasudha et al., 2006).The highest mortality (21%) was observed in the first instar and 100% survival in the fifth instar larvae.Thermo-tolerance races of silkworms and other insects (Lohmann and Riddiford, 1992).
In the present study, thermotolerance varied from egg stage to larval stages as the embryos died at exposure to 40ºC for 2 hrs.and the hatching percentages were affected among the tested groups.Both thermal stresses at 0ºC for 2 hrs.and 40ºC for 1 hr at egg stage almost have the same effect on the pre-cocooning and post cocooning parameters.while in the grown larvae, Sample 6 : T6 control Sample 7 : T7 -0°C for 1 hr Sample 8 : T8 -0°C for 2 hrs Sample 9 : T9 -40°C for 1 hr Sample 10 : T10 -40°C for 2 hrs the highest values were recorded for the group exposed to 0ºC for 1 hr.The increased cocoon weights and cocoon shell weights over control, reflects the positive correlation between heat stresses response and silk protein content in the cocoon.
It is well known that both prokaryotic and eukaryotic cells respond to unfavorable environmental conditions by increased/decreased synthesis of stress proteins.It is a universal phenomenon in bacteria to human, but certain features of the response do vary from organism to organism (Craig, 1985).
Studying protein profile of an insect is of considerable importance for understanding different physiological processes.
The separation and characterization of the individual proteins facilitates the study of the chemical nature and physiological function of each protein (Mohamed, 1990).
Data in the present study showed the disappearance of some protein bands or appearance of new ones with different exposure time.The disappearance in certain protein bands of tested treatments may be attributed the effect of thermal doses which inhibits the synthesis and expression process of these deleted proteins (qualitative effect).In addition, even the remained band after treatment usually differs in the amount of protein, this may be explained that, thermal stresses could not inhibit the synthesis of this protein type, but affect its quantitative amount.
Considerable changes in protein fractions were demonstrated electrophoretically.The changes in the protein mobility and number of bands (qualitative determination) and the intensity of some bands (quantitative determination) probably, reflect some of physiological processes (Hussein et al.,1993).In the present study the quantitative increase or decrease in common protein bands among the tested treatments may be due to the presence of different numbers of iso-genes responsible for the production of this protein type or due to the prolongation of the genes related to this protein in their action compared to the other treatments.
The inhibition of some protein bands can explain some other experimental results such as reduction in weight, slow development, tissue degeneration and preventing adult emergence (Schloter, 1985).
The protein pattern differences may act as a tool to identify (estimate) the similarity and genetic distance between the control and treated samples as concluded by Rao et al. (1999) andAyeed et al. (2001).
The present results revealed that, protein pattern monitors number of bands as a difference between the control and other treatments.This difference is translated as similarity degree , which recorded a low value 50% between the control egg group and the egg groups exposed to 0ºC for 2 hrs.and 40ºC for 1 hrs (which recorded the best economical characters).Also, for grown larval treatments, the similarity degree was 50% between control larval group and larval group exposed to 40ºC for 2 hrs.The data from the present study enhance knowledge for the development of thermo tolerant silkworm breeds/ hybrids and their effective commercial utilization in the sericulture industry.

Fig. 1 :
Fig. 1: egg group exposed to 40 C for two hours Fourth larval groups which exposed to 0ºC for one and two hours were seen in a stillness case for about 15 minutes and then returned to normal condition.Nothing happened in larvae exposed to 40 ºC for one and two hours.Data represented in Table (1) showed that, egg group exposed to 0 ºC for two hours decreased incubation period Fractionated protein Data presented in Tables (3) and demonstrated in Figure (2) showed egg stage protein pattern of Bombyx mori, in all treatments the bands molecular weight ranged from 24.853 to 124.354 KDa.Six common bands no. 1, 8, 10, 17, 23 and 24 were detected in the control (T 1) and the different treatments.Percentages of protein bands amount were compared quantitatively among different treatments to clarify the quantitative changes.

Table 1 :
Bombyx mori L. pre and post-cocooning parameters of eggs exposed to different temperatures for different times

Table 2 :
Pre and post-cocooning parameters of Bombyx mori L. fourth larval stage exposed to different temperatures for different times

Table 3 :
larval haemolymph protein pattern of Bombyx mori exposed to different temperatures for different times during egg stage.

Table 4 :
Epigenetic degree of similarity between the tested treatments of Bombyx mori exposed to different temperatures for different times during egg stage.

Table 5 :
Larval haemolymph protein pattern of Bombyx mori grown larvae exposed to different temperatures for different times

Table 6 :
Epigenetic degree of similarity between the tested treatments of Bombyx mori grown larvae exposed to different temperatures for different times