Influence of Sudden Thermal Stresses on Growth Rate and Enzymatic Identification of Bombyx mori L. Larvae

the Egyptian Society for Biological Sciences ,Department of Entomology ,Faculty of Sciences Ain Shams University .  molecular biology journal is one of the series issued twice by the Egyptian Physiology & Academic Journal of Biological Sciences, and is devoted to publication of original papers that elucidate important biological, chemical, or physical mechanisms of broad al significance. physiologic  www.eajbs.eg.net Provided for non-commercial research and education use.  Not for reproduction, distribution or commercial use.


INTRODUCTION
Silkworm is one of the most important animals which produce silk thread in the form of cocoon by consuming mulberry leaves during larval period.Growth and development of silkworm is greatly influenced by environmental factors such as temperature and relative humidity (Hussain et al., 2011).The development of silkworms also depends on its metabolic modulations and physiological adaptability, besides its genetic constitution (Chatterjee et al.,1993) and (Thiagarajan et al., 1993).The seasonal differences in the environmental components considerably affect the genotypic expression in the form of phenotypic output such as cocoon weight, shell weight and cocoon shell ratio (Nacheva and Junka, 1989).High temperature affects nearly all biological processes including the rates of biochemical and physiological reactions and ultimately affecting the quality and quantity of cocoon crops (Hazel,1995 andWillmer andJohnston, 2004).
The silkworms likely adapt to low temperature by different mechanisms involving synthesis of low molecular weight proteins, heat shock proteins and regulation of activities of certain key enzymes which plays crucial roles in insect metabolism (Singh et al., 2013).
The wide occurrence of phosphatases in insect tissues is thought to be associated with the (a) transport of metabolites, (b) metabolism of phospholipids, phosphoproteins, nucleotides and carbohydrates and (c) synthesis of proteins (Ray et al., 1984 andChaubey et al., 2010).
Among the various isozymes, esterases have been studied extensively since they are the group of enzymes involved in metabolic and defense functions and are found in both soluble and membrane bound forms.Their involvement in resistance/stress to various kinds of insecticides and thermal stresses (Eguchi and Yoshitake,1967) and also studied for their function in the digestion, nutrition and detoxification in insects (Kasim and Ahmed,1980), reproduction (Richmond et al., 1980) and as a tool for genetic relationships analysis among B. mori races and hybrids (Mahmoud et al., 2011).
Although temperature and humidity have been known to play important role in silk production, no much information are available on the effect of temperature stresses on silkworm physiology and commercial traits.The present study was undertaken to study the effect of different thermal stresses for different periods at the egg stage (late embryo stage or blue stage) and at the beginning of fourth larval stage (grown larval stage ) of B. mori in relation to its enzymatic activity and their subsequent impact on larval growth and effective rate of rearing.

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, according to Krishnaswamy (1978).Larvae were fed on leaves of Kokuzo-27 mulberry variety.

Temperature stresses Egg stage:
At the first day of raising temperature from cold storage at 5°C to 27°C, 100 eggs at late embryo stage (blue stage) within three replications were kept in egg hatching boxes under four temperature stress conditions (T2 to T5) along with control (T1).Control-treatment 1 (T1) -maintain at 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.After being exposed to thermal stress all treatments (T2~T5) were kept at 15 °C for two hours for recovery then placed at 27 °C till hatching.

Fourth larval stage (grown larval stage):
At the beginning of the fourth larval stage (grown larval stage), larvae were divided into 5 groups in three replicates, with100 larvae/ replicate and the same above temperature 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.(T7 ~T10) were maintained at 15 °C for two hours for recovery, then the rearing technique was continued normally at room temperature.
Insect performance was assessed based on larval growth (GR) rate and effective rate of rearing (ERR).
Growth was calculated was calculated according to (Thyagaraja et al., 1991): GR = (body weight of freshly ecdysed larvae/ body weight of newly hatched larvae) / larval duration by days.
The effective rate of rearing (ERR %) was recorded according to Chanu and Ibotombi (2011).ERR (%) = (no. of cocoon harvested / total no. of larvae reared) X 100 The data were recorded and subjected to analysis of variance according to Snedecor and Cochran (1981).

Enzymatic determination:
At the sixth day of the fifth larval instar, haemolymph samples were collected from each treatment and deepfreeze in eppendrof tubes until use for electrophoretic studies.

Phosphatases and Esterases electrophoresis:
Isozymes were separated in 10 % Native-polyacrylamide gel electrophoresis as described by Stegemann et al. (1985).Wendel and Weeden (1989) technique was used for phosphatases.Gel staining protocols of Scandalios (1964) were used for esterases.

Growth rate and effective rate of rearing:
Among the egg groups exposed to sudden thermal stresses, the highest performance was recorded for control and egg group exposed to 40°C for one hour as presented in Table (1).In a row, means followed by same letter (s) are not significantly different at 0.01 by LSD For grown larvae, the highest growth rate was observed for larvae exposed to 0 ºC for one hr.No significant difference was observed among all treatments, (Table 2).Thermal stresses did not significantly affect survival or effective rate of rearing.In a row, means followed by same letter (s) are not significantly different at 0.01 by LSD

B-Electrophoretic analysis of Phosphatases and Esterases: B-1 Acid phosphatase (ACPase): Egg stage
A common band exist at Rf (0.065) in all treatments except control (Fig. 1).The quantitative changes was calculated to be (1.684) in acid phosphatase pattern under exposure to 0 ºC for one and two hours, (Table 3).The enzyme activity increased and showed 1, 2, 2 and 3 new bands in treatments 2,3,4 and 5, respectively comparing to control.

Larval stage:
As represented in Table (4), No common bands were found between the tested samples.Sudden thermal stresses at the beginning of fourth larval stage increased the enzyme activity which resulted in the appearance of 2, 3, 1 and 1 new bands in treatments 7, 8, 9 and 10, respectively comparing to control.A common band exist in all treatments and control at Rf (0.062), which increased quantitatively in control and treatment 2 (egg group exposed to 0ºC for one hr) to 3.6 ~ 4.6 over the other treatments (Table 5 and Fig. 2).
Thermal stress at 0 ºC for two hrs and at 40 ºC for one hr induced the production of two new characteristic bands.While thermal stress at 40 ºC for 2 hrs reduced enzyme activity which resulted in the appearance of one characteristic band only.

Larval stage
As shown in Table ( 6), a common band exist in all treatments and control at Rf (0.062), which increased quantitatively in control and treatments 40 ºC for one and two hours groups to (~ 3.6) over the other treatments.Thermal stresses for one and two hrs at 0 ºC induced the production of 2 new characteristic bands.

B-3 alpha (α) esterase: Egg Stage
No common bands were observed between the tested treatments as shown in Table (7) and Fig

Larval stage
Electrophoretic analysis for the haemolymphal samples of different treatments revealed that, new characteristic bands were 2, 1, 3, 2, and 4 in treatments 1, 2, 3, 4 and 5 respectively as shown in Table (8

3-b Beta (β) esterases Egg stage
Egg groups exposed to 0°C for one and two hours showed three and four characteristic bands, respectively.Egg groups exposed to 40°C showed four characteristic new bands (Table 9 and Fig. 4).

Larval stage
As shown in Table (10).One, two, four and five new characteristic bands were observed in treatments 7, 8, 9 and 10, respectively.

DISCUSSION
All groups showed increased growth from the first day of exposure till the end of larval stage.The results revealed that, the highest performance was recorded for 40ºC for one hr and 0 ºC for one hr for egg and grown larvae, respectively.Effective rearing rate (ERR) indicates the survivability of silkworm during the rearing period showed no significant differences between all treatments.The survivability of silkworm larvae at heat stresses extremes related to the *Egg stage Lane 1: T1 control Lane 2: T2 -0°C for 1 hr Lane 3: T3 -0°C for 2 hrs Lane 4: T4 -40°C for 1 hr Lane 5: T5 -40°C for 2 hrs *Fourth larval stage 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 production of proteins serving as molecular chaperons assisting in refolding of denaturated proteins as suggested by Samad et al.( 2005).Exposure grown larvae to sudden thermal stresses for different periods achieve growth rate better than eggs treatments.Differential levels of tolerance to thermal exposure (other than their normal growth temperature) were reported to be dependent on the life stages of the silkworm (Joy and Gopinathan, 1995).Manjunatha, et al. (2013) displayed that, the mild heat stress between 35 and 40 °C for two hours facilitate the silkworm larvae to overcome and withstand the fluctuating natural environmental conditions and perform better.Obviously, this physical state is supported by expression of one or many proteins, heat shock proteins (HSPs) in a given generation with the activation of some genes located in their chromosomes (Hong et al., 2010 andSosalegowda et al., 2010).
Phosphatases exhibited a positive relationship to the cocoon quality of the silkworm.Therefore, it suggests that ALKPase may be used as a biochemical index to evaluate the health and economic characteristics of the silkworm, Bombyx mori L. as well as different stress and disease result in considerable decrease in the ALKPase activity (Miao, 2002).These findings disagree with the present results as the thermal stresses in egg and larval stages induced the production of new enzyme bands.
Esterases are very large class of enzymes, all of which can break an ester bond with the help of a water molecule.Most enzymes of this class hydrolyze endogenous substances and are important in intermediary metabolism (Sivakumram and Maya, 1991).Esterases activity can be regarded as an indicator for thermotolerance in the silkworm generations (Wu and Hou, 1993).
In the present study thermal stresses affect the pattern of the studied isozymes (phosphatases and esterases) in both egg and larval stages.The disappearance of some bands in the tested treatments may be attributed the effect of thermal doses which inhibits the gene expression and synthesis of these deleted proteins (qualitative effect).The difference in bands densities (quantitative effect) 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 (Taha, 2013).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 and biological processes (Hussein et al.,1993 andEl-Banna, 2009).These differences were due to better adaptation of one group over another (Mahadav et al., 2009).
Finally, our conclusions agree with Sinha and Sanyal (2013) which recommended the practical application of sudden temperature stresses systematically in sericulture to achieve better growth, performance and cocoon production.

Fig. 2 :
Fig. 2: Haemolymphal ALKPase activity for all tested treatments of B. mori exposed to sudden thermal stresses for different periods at egg and fourth larval stages T9 -40°C for 1 hr Lane 10: T10 -40°C for 2 hrs *Egg stage Lane 1: T1 control Lane 2: T2 -0°C for 1 hr Lane 3: T3 -0°C for 2 hrs Lane 4: T4 -40°C for 1 hr Lane 5: T5 -40°C for 2 hrs . (3).Sudden thermal stresses at egg stage induced the production of only one band in all treatments while the control sample showed three characteristic bands.

Fig. 4 :
Fig. 4: Haemolymphal β esterase activity for all tested treatments of B. mori exposed to sudden thermal stresses for different periods at egg and fourth larval stages.

Table 1 :
Larval growth rate (GR) & effective rate of rearing (ERR) for B. mori eggs exposed to sudden thermal stresses for different periods

Table 2 :
Larval growth rate (GR) & effective rate of rearing (ERR) of B. mori fourth larval groups exposed to sudden thermal stresses for different periods

Table 3 :
Haemolymphal ACPase activity with bands relative fragmentation (Rf) and amount percentage (Am%) at egg stage exposed to sudden thermal stresses for different periods.

Table 4 :
Haemolymphal ACPase activities with bands relative fragmentation (Rf) and amount percentage (Am%) at fourth larval stage exposed to sudden thermal stresses for different periods.

Table 5 :
Haemolymphal ALKPase activity with bands relative fragmentation (Rf) and amount percentage (Am%) at egg stage exposed to sudden thermal stresses for different periods.

Table 6 :
Haemolymphal ALKPase activity with bands relative fragmentation (Rf) and amount percentage (Am%) at fourth larval stage exposed to sudden thermal stresses for different periods.

Table 7 :
Haemolymphal α esterase activity with bands relative fragmentation (Rf) and amount percentage (Am%) at egg stage exposed to sudden thermal stresses for different periods.

Table 8 :
Haemolymphal alpha esterase activity with bands relative fragmentation (Rf) and amount percentage (Am%) at fourth larval stage exposed to sudden thermal stresses for different periods.

Table 9 :
Haemolymphal β esterase activity with bands relative fragmentation (Rf) and amount percentage (Am%) at egg stage exposed to sudden thermal stresses for different periods.

Table 10 :
Haemolymphal β esterase activity with bands relative fragmentation (Rf) and amount percentage (Am%) at fourth larval stage exposed to sudden thermal stresses for different periods.