INTRODUCTION

Due to global warming, impact of temperature on living organisms has been core of scientific investigation in recent past. In insect, all species has its own choice of temperatures for its usual growth and very high temperature slows down the growth, may leads to developmental malfunction, such as larval ecdysis and adult emergence (Chapman, 2002). Being a poikilothermic organism, temperature decides the fate of insect, during embryonic and postembryonic development. It is well understood that, temperature affects the insect life-cycle plasticity, physiological thermal traits, immune responses and gene expression (Neven, 2000; Kiuchi et al., 2008; Arias et al., 2011; Beckett, 2011; Catalan et al., 2011). It also affects the metabolic rate and oxidative stress status (Contreras and Bradley, 2010; Lalouette et al., 2011). Woods and Bonnecaze (2006) studied the impact of temperature, water and oxygen on insect eggs at a transition between diffusion and reaction limitation. Even oxygen consumption profiles of insect cells get affected due to temperature (Gotoh et al., 2004). In a number of insect species, the growth rate is improved and the developmental period and immune cells (hemocytes) are abridged as the temperature raises (Wigglesworth, 1972; Kiuchi et al., 2008; Pandey et al., 2010).

In recent past, several physiological studied have been undertaken in different aspects of tropical tasar silkworm, Antheraea mylitta (Kumar et al., 2011; Pandey et al., 2010, 2011, 2012). Although, embryonic development of insects in relation to various stresses have been studied by several researchers (Khattak et al., 2001; Duraimurugan and Regupathy, 2005; Temerak, 2006; Ali and Rizvi, 2007; Seidavi et al., 2008; Ahmad et al., 2008; Sujatha and Joseph, 2011; Bajaj and Sharma, 2011) but impact of temperature in relation to embryonic development of tasar silkworm eggs (seed) is not studied much which is backbone of tasar silk industry.

The tropical tasar silkworm, A. mylitta is produces tasar silk of commercial importance at industry level and it gives direct supplementary livelihood to poor people of the society. The basic reproductive unit in the tasar silk industry is the silkworm eggs and temperature plays very crucial role during embryonic development. Embryonic development starts within few hours of egg lying and it needs proper incubation and care. It reported that, as the development of the embryo proceeds, both lipids and carbohydrates gets utilized, resulting in increase in the concentration of citrate, pyruvate and lactate (Pant et al., 1979). Due to global warming elevation in temperature with less humidity was recorded during 1st crop grainage which not only affect the egg hatching but also adversely affects the seed sector of tasar industry which is very essential input of sericulture. Although, embryology of several sericigenous insects have been studied by researchers (Tazima, 1978; Jolly et al., 1974; Kumar et al., 1998). But biochemical and morphological changes occurs in tasar silkworm eggs under temperature stress conditions are not understood well. Hence, the present study has been conducted to investigate impact of temperature stress on embryonic development and biochemical profile of the A. mylitta eggs.

MATERIALS AND METHODS

Insect culture: Stock of tropical tasar silkworm, A. mylitta Drury (Daba ecorace) was maintained in outdoor condition and larvae were fed on fresh leaves of Asan, Terminalia tomentosa W&A. Disease free laying was obtained from our laboratory grainage and utilized for different set of experiments (Kumar et al., 2011). The 72 h old eggs were used for hatching percentage experiment. For embryonic development study 24 h old eggs were used. Present research work was carried out during the year 2010-2011 in Silkworm Physiology Laboratory, Central Tasar Research and Training Institute, Ranchi, India. Our rearing farm and laboratory are located at Latitude-23°.21’N, Longitude- 85°.20’E and an altitude of 652 m MSL, in the state of Jharkhand, India (Kumar et al., 2011).

Embryonic development: For proper embryonic development, tasar silkworm eggs were incubated after standardisation, at optimum temperature and relative humidity i.e., 26-28°C and 75-80%, respectively. Different age groups of samples were collected daily for embryonic development and biochemical study.

Temperature stresses: To study the impact of temperature stresses on hatching of tasar silkworm eggs, 200 eggs in five replications were kept in egg hatching boxes having transparent top to permit light and perforated sides for aeration (5.5x4x2 cm) at four temperature stress conditions (T1 to T4) along with control (T5). For treatment-1 (T1) temperature 37°C and 40% RH in Vista Biocell environmental chamber incubator (This condition was observed during first crop grainage), for treatment-2 (T2) temperature 27°C and 60% RH in Vista Biocell environmental chamber followed by one higher temperature stress at 37°C daily, for treatment-3 (T3) temperature at 25°C and 60-70% RH at room temperature and treatment-4 (T4) cyclic temperature 38 and 18°C and 50-85% RH under outdoor direct sun light covered with polythene sheet. In treatment-5 (T5) respective control were maintained and considered as controls. After completion of treatment the eggs samples of experimental and control groups were kept at -80°C for biochemical analysis. Hatching percentage was recorded after 1st, 2nd and 3rd day separately by using following formula:

Eggs samples collection: Eggs samples of different treatments were taken in Eppendorf tube and crushed and homogenized in 1 mL, 50 mm phosphate buffer (pH 6.9). These samples were centrifuged at 5000 rpm for 10 min and supernatant was collected in to pre-chilled Eppendorf tube and utilized for present study.

Estimation of protein and carbohydrate in eggs: Samples were collected daily for biochemical study. Eggs samples of different temperature stress conditions and control were analyzed for protein and carbohydrate after third day of egg hatching. The protein estimation was carried according to Bradford’s micro protein assay (Bradford, 1976) with slight modifications. Concentration of the protein sample was determined from a standard curve drawn using bovine serum albumin and OD was taken at 595 nm. Colorimetric determination of carbohydrate was performed by using phenol-sulfuric acid based reaction absorbance at 490 nm to see colored aromatic complex formed between phenol and the carbohydrate (Fournier, 2001). UV 5704-SS spectrophotometer was used in the present experiment.

Microscopic observation: For proper embryonic development, eggs were incubated at optimum temperature and relative humidity i.e., 26-28°C and 75-80%, respectively. Different temperature stress was given to eggs and samples were collected daily for microscopic observation. To see the impact of temperature stress on embryonic development, eggs samples of different age groups of control and experimental were dissected out by opening the eggs shell and subjected to microscopic morphological observation by using stereo zoom microscope. Comparative morphological evaluation of pictures was done and temperature stress impact on embryonic development was analysed.

Data analysis: The data was subjected to the statistical analysis by using ANOVA. The Microsoft Excel 2003 software was used for data analysis assistance.

RESULTS

Temporal changes in protein and carbohydrate profile of A. mylitta eggs: Throughout embryonic development of egg, the concentration of protein was recorded maximum (442.86±4.120 mg g^-1) on day 1 and its level decreases on advancement of age of eggs or progression of embryonic development up to 123.380±9.630 mg g^-1. The utilization of protein was observed highest on day 10 (35.50%) followed by day 2 (24.74%), day 3 (17.71%) and day 8 (11.41%). The utilization of protein was observed lowest on day 5 (1.80%) followed by day 7 (1.36%) and day 1 (0.00%). Regression equation shows Y = -26.332x+397.87 and regression trend shows R² = 0.8476.

During embryonic development of egg, the concentration of carbohydrate was recorded maximum (21.240 mg g^-1) on day 1 and its level decreases on advancement of age of eggs or progression of embryonic development up to 8.110±0.880 mg g^-1. The utilization of carbohydrate was observed highest on day 3 (24.73%) followed by day 2 (23.27%), day 5 (16.06%) and day 4 (12.45%). Whereas, the utilization of carbohydrate was observed lowest on day 7 (2.93%) followed by day 8 (1.81%), day 6 (0.35%) and day 9 (0.16%). (y = -26.332x+397.87 and R² = 0.8476 (Table 1).

Impact of temperature stress on protein concentration: The concentration of total protein in eggs kept in different temperature stress conditions reveals that differences were highly significant among all treatments (p<0.1). Total protein concentration was higher in T1 (230.62±5.65 mg g^-1) followed by 197.10±3.40 mg g^-1 in T4 whereas, lowest ranged from 163.31±4.85 to 171.47±5.30 mg g^-1 in T2, T3 and T5. It was evidence that higher concentration of protein in T1 temperature stress condition due to unfavourable condition for embryonic development and block the embryogenesis and not utilization of protein (Fig. 1). For incubation of tasar eggs, 26-28°C temperature and 75-80% RH is found appropriate to acquire proper embryonic development as well as uniform and timely hatching.

Impact of temperature stress carbohydrate concentration: Total carbohydrate concentration in eggs of different temperature stress conditions is presented in Fig. 2. The level of carbohydrate was observed significantly higher in T1 (p<0.1) than other conditions. The maximum concentration of carbohydrate in eggs kept in temperature stress condition of T1 (103.51±11.27 mg g^-1) followed by T4 (72.22±5.12 mg g^-1) and in T2, T3 and T5 it’s ranged between 48.67±0.83 to 55.94±4.24 mg g^-1 (Fig. 2).

Table 1:	Temporal changes in protein and carbohydrate profile of A. mylitta eggs during embryonic development

Fig. 1:	Protein profile of A. mylitta eggs kept in different temperature stress conditions. Mean followed by different alphabets are significantly different at p<0.1

Fig. 2:	Carbohydrate profile of A. mylitta eggs kept in different temperature stress conditions. Mean followed by different alphabets are significantly different at p<0.1

Temporal changes during embryonic development of normal eggs: The eggs of A. mylitta contain yolk and cleavage takes place in superficial nature. Chronological variation during embryonic development of normal eggs was found from 24 h to till eggs hatching (Fig. 3a-h). Within 6 to 10 h of egg deposition after repeated division, cleavage energids originate as aggregated cytoplasm of the cytoplasmic reticulum. During 10-16 h as a result of increased cell division in ventral region the ventral plate is formed and by 22 h trough shaped embryonic primodium floats in yolk and the protocephalon and protocorn are separated by transverse furrows. Throughout 22-28 h, the germ band becomes slender and elongated and by 28-34 h along the mid portion of the germ band on upper side a long narrow depression called the primitive groove or streak or median plate is formed. By 24-36 h the development of embryo was well differentiated into head and trunk region. After 48 h, segmentation of the body is clearly visible and in 72 h embryo showed three distinct regions of the body i.e., head, thorax and abdomen were well differentiated. After 96 h the embryo became C-shaped. Although, mouthparts are yet not developed completely but they were at the advanced stage of development. The thoracic region is clearly divisible into three thoracic segments. Interestingly after 120 h organogenesis in embryo takes place. The antennae bear segments and head region was detachable into three segments. The head capsule formation completed in after 144 h and mouthparts got matured. Three segmented antennae with antennal setae, the mandibles and labrum are well developed.

Fig. 3(a-h):	Temporal morphological changes during embryonic development of A. mylitta eggs, (a) 24 h old embryo, (b) 48 h old embryo, (c) 72 h old embryo, (d) 96 h old embryo, (e) 120 h old embryo, (f) 144 h old embryo, (g) 168 h old embryo and (h) 192 h old embryo

After 168 h old embryo well pigmented integument were found and coloration of head capsule takes place. After 192 h embryo was fully formed larva, which is ready to cut off the egg shell or to hatch out.

Impact of temperature stress on embryonic development: For incubation of tasar eggs, 26-28°C temperature and 75-80% RH is found appropriate to acquire proper embryonic development. Marked variation in temperature and humidity negatively affects the embryonic development of tasar silkworm eggs (Fig. 4a-f). Data reveals considerable alteration in temperature used to hamper the protein and carbohydrate profile which leads to affect the embryonic development hatching of eggs. The depressed eggs with fully formed dead larvae inside the eggs were found after high temperature stress (Fig. 4a, b). But higher temperature with low humidity during embryonic development of eggs resulted in death of embryo during early age (Fig. 4c-f).

Impact of temperature stress on hatching percentage: When stress was given to tasar silkworm eggs, it showed great deal of variation in hatching percentage (Table 2). On the 1st day-maximum hatching percentage was recorded in T5 (63.50) followed by T2 (42.50) and T3 (27.67). Highly significance different in hatching percentage was observed between T2 (42.50) and T3 (27.67); between T2 (42.50) and T5 (63.50); between T3 (27.67) and T5 (63.50). On the 2nd day-maximum hatching percentage was observed in T3 (42.00) followed by T2 (30.67); T4 (38.33) and T5 (29.50) shows less hatching percentage. Significant differense in hatching percentage was also observed between T2 (30.67) and T3 (42.00); between T2 (30.67) and T4 (38.33); between T3 (42.00) and T5 (29.50); between T4 (38.33) and T5 (29.50). No significant difference was observed between T2 (30.67) and T5 (29.50).

In day 3, maximum hatching percentage was recorded in T4 (17.50) followed by T3 (14.00), T2 (9.33) and T5 (0.833) and T5 shows less hatching percentage (0.833). No significant difference in hatching percentage was observed between T2 (9.33) and T3 (14.00), between T3 (14.00) and T4 (17.50) but highly significance different was observed between T2 (9.33) and T5 (0.833), between T3 (14.00) and T5 (0.833) and between T4 (17.50) and T5 (0.833).

Fig. 4(a-f):	Impact of temperature stress on embryonic development of A. mylitta eggs (a-b) Fresh and depressed eggs, (c-f) Larvae died inside egg shell due to higher temperature stress

Table 2:	Impact of temperature stress on A. mylitta egg hatching
Note: T1: Temperature at 37°C and 40% RH in incubator (This condition was observed during first crop grainage), T2: Temperature at 27°C ant 60% RH in incubator followed by one higher temperature stress at 37°C daily, T3: Temperature at 25°C and 60-70% RH at room, T4: Max. temperature. 38°C and mini. temperature. 18°C and 50-85% RH under outdoor direct sun light covered with polythene sheet, T5: Control: Mean followed by different letters with in column are significantly different at p<0.1

In total, maximum hatching percentage was recorded in T5 (93.83) followed by T2 (82.50) and T3 (83.67) whereas, T4 shows less hatching percentage (55.83). No significant difference in hatching percentage was observed between T2 (82.50) and T3 (83.67), between T2 (82.50) and T5 (93.83), between T3 (83.67) and T5 (93.83), but highly significant difference was observed between T2 (82.50) and T4 (55.83), between T3 (83.67) and T4 (55.83). It is observed that high temperature and less humidity are not good for larval hatching (Table 2).