HARSH IMPACT OF TEMPERATURE ON PROTEOMIC PROFILE OF THE SILKWORM BOMBYX MORI L.

Transcription

1 Journal of Cell and Tissue Research Vol. 16(3) (2016) (Available online at www. Tcrjournals.com) ISSN: ; E-ISSN: Original Article HARSH IMPACT OF TEMPERATURE ON PROTEOMIC PROFILE OF THE SILKWORM BOMBYX MORI L. BHAT, M. A., 1? BUHROO, Z. I. 2 AND MANJUNATHA, H. B. 1 1 Proteomics and Genomics Laboratory, DOS in Sericulture Science, University of Mysore, Manasagangothri, Mysuru, Karnataka, India Temperate Sericulture Research Institute, Mirgund, Faculty Of Agriculture, Sher-e- Kashmir University of Agricultural Sciences and Technology of Kashmir, Srinagar, India E.mail: mbhat.muzafar@gmail.com, Cell Received: November 1, 2016; Accepted: November 22, 2016 Abstract: The present study investigates the proteomic profile in bivoltine ( ) and polyvoltine (Daizo) silkworm strains of Bombyx mori L. Following the concept of whole organism, silkworm larvae were subjected to thermal stress at 40 0 C for 2h followed by 2h recovery from first to fifth instar. Heat shock proteins were analyzed by SDS-PAGE and it was found that the appearance of 97 and 70kDa protein were assessed in all instars in all silkworm strains. Organism responses to thermal stress can be detected at the proteomic level where the specific proteins are linked to the physiological changes. These results propose that heat shock protein based breeding strategy should be used for inducing robustness in silkworm strains. Heat shock proteins act as molecular markers for evaluation and evolution of thermo tolerant silkworm strains. Key words: Bombyx mori, Temperature effect. INTRODUCTION The mechanisms of fundamental thermal tolerance are innermost to forecast the impact of climate change at the individual level [1]. One of the most important hypothesis projected to explain the mechanism of thermal tolerance of ectotherms is the theory of oxygen and capacity-limited thermal tolerance [2], which argues that thermal tolerance may be constrained by oxygen limitation and the mismatch between oxygen supply and demand at thermal extremes (i.e. at temperatures outside thermal optima). The insects are the most abundant animal group on earth and the scientific interest to understand their response to environmental stresses has increased considerably because of their medical, economical and ecological values. The impact of these stresses, depending on its severity, varies from increased synthesis of heat shock proteins for the survival to the physiological and developmental insults. The heat shock proteins act as molecular chaperones and protect the cells by controlling the appropriate folding and translocation of polypeptides to different cellular compartments. There are many families of heat shock proteins (hsps) based on their molecular weight and homology, and include Hsp100, Hsp90, Hsp70, Hsp60, Hsp40 and (small heat shock proteins) shsps [3-5]. Heat shock proteins (HSPs) are universally proteins found in plant and animal cells. They originally were described in relation to heat shock [6] but are now known to be induced by a wide variety of stresses, including expo sure to cold, UV light, wound healing, tissue remodeling, or biotic stresses [7]. Thus, the term heat shock protein is a misnomer since many stresses other than heat 5929

2 J. Cell Tissue Research induce expression of hsp genes. The biological response to fluctuated/elevated temperature under natural environment is measured as heat shock response (HSR) and this phenomenon has also extensively been studied by subjecting various organisms to induced thermal stress under laboratory conditions. The response exhibited by an organism is crisis coping type of reaction that facilitates physiological adaptability and survivability, which vary not only among organisms but also species and strains. This response has also influenced by varied biomolecules of which a set of proteins synthesized during the critical period are identified as heat shock proteins (HSPs) that commonly occur from bacteria to man but their functional domain differs. The communiqué between HSR and expression of HSPs in relation to biological properties and function although has been studied extensively still gaining prime attention due to climate change [8] that affecting bio-world and lepidopterons in particular which comprises most of the agricultural pests including a beneficial insect the silkworm, Bombyx mori L. Although, both prokaryotic and eukaryotic cells respond to unfavorable environmental conditions by increased synthesis of HSPs and have conserved sequences, but certain features of the response do vary from organism to organism. In recent years, the processes of heat shock responses and the role of heat shock proteins (HSPs) have not been confined merely to molecular chaperons [9], but spread over to determine their ecological and evolutionary role in the post genomic era [10] and as a biomarker in biomedical research. Proteomics is a large-scale study of gene expression, which ultimately provides direct measurement of protein expression levels and insights into the activity of all relevant proteins. The proteome of B. mori is highly dynamic and complex with respect to the environmental changes while it grows in the laboratory and field under controlled and fluctuated conditions respectively. Due to economic importance it has been exploited through continuous domestication. B. mori. lost its tolerance to environmental insults unlike B. mandarina [11]. As a consequence, conventionally, breeders have been using female parent from multivoltine (known for thermotolerance) male parent from bivoltine for production of F1 hybrid. Productive bivoltine breeds (conventional breeding) can t be reared during hot climate and/or fluctuated environment, because insects have a narrow range of tolerance to elevated temperature unlike humans, plants, hence they struggle to cope up with the fluctuated environmental conditions. Especially, tropical countries like India, the temperature and humidity vary from season to season and region to region, while fluctuation occur between dawn to dusk, sericulturists experience a great risk in rearing of silkworms [5]. The fluctuated environment lead to improper growth of embryo, poor hatching, weak larvae, inferior cocoons and ultimately production of low grade silk. Even, few hours of elevated temperature 40 0 C and above in the rearing house causes considerable damage in biological and commercial traits of silkworm B. mori L. [12,13]. In view of the above, the present study was proposed with an aim of understanding specific expression of HSPs in order to assess, whether silkworms differ in the constitutive level of protein expression in response to changing temperature, and its role in biological and commercial traits in the selected strains of Bombyx mori, which as per available literature has not been documented to date with reference to other organism. The resultant candidate HSPs can be used as markers for evaluation and breeding of better temperature tolerant silkworm strains under tropical environmental conditions in view of present global warming scenario. MATERIALS AND METHODS Polyvoltine (Daizo) and bivoltine (NB4D2) strains were selected for the present investigation and procured from the germplasm, Department of Studies in Sericulture Science, University of Mysore, Manasagangothri, Mysuru. The silkworm eggs were incubated under optimum temperature 25±1 C and 75±5% relative humidity, followed by black boxing, until hatching in the laboratory. The larvae were reared on mulberry leaves up to spinning following standard rearing procedure [14]. Induction of Heat Shock: Larvae of polyvoltine (Daizo) and bivoltine (NB4D2) silkworm strains in each instar were placed separately in thin-walled 5930

3 Bhatt et al. Fig. 1B Figure 1: Heat Shock protein profile derived from different instar larvae of Bombyx mori heat shocked at 40 0 C. Fig. 1A = Protein profile of first instar, Fig. 1B = Protein profile of second instar, Fig. 1C = Protein profile of third instar, Fig. 1D = Protein profile of fourth instar. Fig. 1E= Protein profile of fifth instar. In first, second, third and fourth instars: 1= Molecular Marker, 2 = Control, 3= heat shocked, 4= Daizo Control, 5= Daizo heat shocked, In fourth instar: 1= Control, 2= heat shocked, 3= Daizo Control, 4= Daizo heat shocked Fig. 1C 5931

4 J. Cell Tissue Research Fig. 1D Fig. 1E Table 1: Densitometric analysis of proteome of larvae different silkworm strains of Bombyx mori. SD: Standard deviation ±, Distance: It is the distance (In pixels) that each peak starts from the binging of the line scan,width: The size of each peak, Height: The size of each peak, Area: It is the integrated area under each peak. This number reflects the intensity of each peak %: It is the percentage each peak contributes to the total density measured on the graph, Rf Value: It measure the distance from the start line to the solvent front and to the front of each spot. Larval Instars I II III IV V Strains Peak Distance Width Height Area % Rf Mean No. SD Daizo Daizo Daizo Daizo Daizo

5 Bhatt et al. petri dishes/test tubes for thermal stress at 40 C with 75% ± 5% relative humidity in the water bath for 2h. The heat induced larvae were transferred to room temperature for recovery period of 2h [13]. Protein Extraction: After recovery period of 2h, HS and control larvae were homogenized separately in extraction buffer (containing Tris HCl - ph 6.8, Dithiothretol (DTT) and henylmethanesulphonylfluoride (PMSF) at 4ºC. Electrophoretic separation of proteins extracted from the silk glands was performed following the procedure established [13]. Precisely, the running gel (12%) was prepared with a mixture of 4 ml of solution a (14.6 g Acrylamide Bisacrylamide + 50 ml double distilled water) with 2.5 ml of b (18.15 g Tris ml double distilled water, ph 8.8) and 3.38 ml distilled water. The mixture was degassed and 0.1 ml of solution c (10 g Sodium dodecyl sulphate + 50 ml distilled water), 20 μl of TEMED and 6.0 mg of ammonium per sulphate were added. The homogenate was centrifuged at 4000 rpm for 20 mins at 4 C and the supernatant collected was used for SDS-PAGE (Weber and Osborn 1969). The components were mixed carefully was poured between the assembled glass plates and allowed to polymerize at room temperature. Consequently, stacking gel (5%) was prepared mixing solutions a (0.83 ml) and d (1.25 ml - 3 g Tris + 50 ml double distilled water, ph 6.8) with distilled water (3.0 ml). The degaussed mixture was added to 0.05 ml of solution c, 10 μl TEMED and 2 mg ammonium per sulphate and poured above the polymerized running gel. The polymerization of the gel was achieved at room temperature. Samples were prepared dissolving protein in solution d (0.1ml) containing SDS (4%), β- mercaptoethanol (10%) and glycerol (20%). To this, bromophenol blue was added and heated in boiling water bath for 5 mins. ~20 μl of cool samples were loaded into the wells immersed in solution e (0.3 g Tris g glycine g SDS ml distilled water). Electrophoresis was performed at constant voltage (100V) until the tracking dye reaches the lower end of the gel. Protein molecular markers were run along with samples. Thereafter, the gel was stained with Coomassie brilliant blue and de-stained with destaining solution for clarity of the protein bands. Densitometric Analysis: Gels were analyzed comparing the banding patterns and their molecular mass against protein molecular markers run parallel along with samples using gel image analysis software-alfa installed in gel documentation unit. RESULTS AND DISCUSSION Expression of 97, 70 and 35kDa hsps were clearly observed in first instar, while as 97 and 70kDa were obvious in second, third, fourth and fifth instars larvae of NB4D2 and Daizo (Figs. 1A-1E). To support densitiometric analysis, NB4D2 revealed (35) and Daizo (36) bands in first instar larvae. In fifth instar NB4D2 revealed (42) and Daizo (44) protein bands in fifth instar larvae. RF value in first instar of NB4D2 observed was ( %) and Daizo ( %) bands in first instar larvae. In fifth instar NB4D2 revealed ( %) and Daizo ( %) in fifth instar larvae (Table 1). The present study clearly reveals the response of silkworm under thermal stress and the development of the expression of hsp97 and 70kDa. Heat shock at 40ᴼC for 2h was found to be mild. The current investigation is exceptional from that of other published research reports in four aspects i.e. whole body of the silkworm larvae was considered instead of tissue under the concept of whole organism; all instars (I to V instars) larvae were subjected to HS rather limited to V instar alone; Keeping this shared impact, in the present study, all the instar larvae were taken into account to measure their response to a temperature stress, expression of HSPs along with their biological and biosynthetic events because fifth instar alone scientifically insufficient to determine the tolerance level of a silkworm breed/strain ignoring the physiological and biochemical contribution of earlier instars [15]. However, previous studies explicit that the tolerance level of young silkworms is low compared to late age silkworm and most commonly the whole larvae have been exposed to hot events over a period of 22 to 30 days that occur either for short or long duration in a day or days [8]. During this period of exposure the cuticle and the underlying epidermis as external tissue act as an initial defense organ [13,16] followed by internal tissues like fat body and haemolymph [14,16] greatly contribute to overcome the environmental insults. In this milieu, larvae of polyvoltine and bivoltine from first to fifth instars were selected separately to examine 5933

6 J. Cell Tissue Research the cumulative response against and evaluate the rate of expression of genes coding for proteins (HSPs) that play a pivotal role in tolerance to harsh environmental conditions. For the first time we have established the total protein map for whole proteomic profile of larvae from I to V instars (Fig, 2), wherein SDS-PAGE although revealed an almost similar protein banding pattern as reported in different strains of B. mori [18] but the densitometric analysis explicit more protein bands poly-voltine (Daizo) larvae in all instars (Table 1). Thus, potentiality of these proteins shall be taken into account while screening better parents for the development of productive silkworm breeds/strains of B. mori. CONCLUSIONS The present study revealed that expression of 97, 70 and 35kDa hsps were clearly observed in NB4D2 and Daizo during first instar larval period, while as 97 and 70kDa hsps were obvious during their second, third, fourth and fifth instar larval periods respectively. Induction and variable expression of hsps in the current study opens up avenues in screening silkworm gemplasm for breeding temperature tolerant silkworm strains for sustainable tropical sericulture industry. 5934

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