Comprehensive analysis of differentially expressed proteins in the male and female Bombyx mori larval instars exposed to thermal stress.

Gender sensitivity to ambient heat, despite well known in insect species, how it manifests during young and late larval instars of Bombyx mori is still unclear. To uncover this cryptic feature, different instars male and female larvae were subjected to varied thermal stress separately and sex-stage specific expression of proteins was investigated. Interestingly, heat shock proteins (HSPs) 90 and 70 were expressed differently in all the instars and also between male and female larvae as confirmed by immunoblot assay. Besides up- and downregulation of few HSPs and other normal proteins, discreet expression of protein was noticed in the two-dimensional gel electrophoresis of male than female larvae which were identified as HSP70 by mass spectrometry. Furthermore, quantitative polymerase chain reaction results show 3.98- and 4.86-fold higher levels of Bmhsp70 and Bmhsp90 transcripts in male and female larvae, respectively, as a response to 40°C heat shock (HS) treatment. Conversely, in spite of the massive production of HSPs due to HS at 45°C, all the larvae were found dead, which is a strong proof of concept for autophagy. Comparatively, female larvae HS at 40°C succeed to spin cocoons with increased weight and silk contents than non-HS larvae. Comprehensively, in the present study, we have noticed a strong correlation for the first time that Bmhsp90 and Bmhsp70 genes expressions due to thermal stress are not only sex specific but also explicit preferential and coordinated action on survivability and biosynthetic potential of the silkworm, B. mori larvae during different instars.

Silkworm thermal biology: a review of heat shock response, heat shock proteins and heat acclimation in the domesticated silkworm, Bombyx mori.

Heat shock proteins (HSPs) are known to play ecological and evolutionary roles in this postgenomic era. Recent research suggests that HSPs are implicated in cardiovascular biology and disease development, proliferation and regulation of cancer cells, cell death via apoptosis, and several other key cellular functions. These activities have generated great interest amongst cell and molecular biologists, and these biologists are keen to unravel other hitherto unknown potential functions of this group of proteins. Consequently, the biological significance of HSPs has led to cloning and characterization of genes encoding HSPs in many organisms including the silkworm, Bombyx mori L. (Lepidoptera: Bombycidae). However, most of the past investigations in B. mori were confined to expression of HSPs in tissues and cell lines, whereas information on their specific functional roles in biological, physiological, and molecular processes is scarce. Naturally occurring or domesticated polyvoltines (known to be the tropical race) are more resistant to high temperatures and diseases than bi- or univoltines (temperate races). The mechanism of ecological or evolutionary modification of HSPs during the course of domestication of B. mori - particularly in relation to thermotolerance in geographically distinct races/strains - is still unclear. In addition, the heat shock response, thermal acclimation, and hardening have not been studied extensively in B. mori compared to other organisms. Towards this, recent investigations on differential expression of HSPs at various stages of development, considering the concept of the whole organism, open ample scope to evaluate their biological and commercial importance in B. mori which has not been addressed in any of the representative organisms studied so far. Comparatively, heat shock response among different silkworm races/strains of poly-, bi-, and univoltines varies significantly and thermotolerance increases as the larval development proceeds. Hence, this being the first review in this area, an attempt has been made to collate all available information on the heat shock response, HSPs expression, associated genes, amino acid sequences, and acquired/unacquired thermotolerance. The aim is to present this as a valuable resource for addressing the gap in knowledge and understanding evolutionary significance of HSPs between domesticated (B. mori) and non-domesticated insects. It is believed that the information presented here will also help researchers/breeders to design appropriate strategies for developing novel strains for the tropics.

Biomolecular changes and somatic mutations induced by UV laser irradiation at embryonic stage of Bombyx mori.

PURPOSE: To determine the impact of ultraviolet (UV) laser radiation on Bombyx mori embryos in terms of its effect on embryonic and larval haemolymph proteins and morphological traits. MATERIALS AND METHODS: The eggs of silkworm strain NB4D2 were exposed to third harmonic laser pulses at 355 nm from a Nd:YAG laser for different durations of 30, 60, 90, 120, 150, or 180 sec. Morphological changes induced by the UV laser were analysed at larval, pupal and adult stages. The eggs exposed to UV laser irradiation at different developmental stages were subjected to protein analysis by sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE). The haemolymph derived from irradiated and control larva was also analysed by SDS-PAGE. RESULTS: UV laser irradiation resulted in various structural polymorphisms. Asymmetrical fusion of segments was not confined to larva but persisted throughout pupal and adult stages. Development of extra caudal horn, unequal size and lack of antenna, retarded thoracic legs and variation in larval markings were observed. Comparatively, the effect of the UV laser on 8- and 16-h old embryo was greater than on the other stages. The changes in protein pattern were not distinct until the 5th day of embryogenesis as revealed by SDS-PAGE. A 178 kiloDalton (kDa) protein resolved into 198, 184 & 169 kDa polypeptides and 154 kDa new protein band along with other proteins of 110, 45, 41 & 38 kDa were noticed in irradiated eggs at the 6th day. Further, 33, 32, and 6.2 kDa new protein bands were observed in the haemolymph of 5th instar silkworm larvae derived from UV laser irradiated embryos. CONCLUSIONS: A comparative analysis of the present study revealed that UV laser not only induced continuous structural polymorphisms (somatic mutations), but also induced protein changes with the appearance of new protein bands in embryonic and haemolymph protein. The UV laser could be a potential tool for biochemical genetics and genome analysis in B. mori.