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The expression pattern of two major chaperones, the heat shock proteins (HSPs) HSP60 and HSP70 was studied in vitro in tissues of the housefly Musca domestica during larval and adult stages of development to identify their immunological relatives and understand their functional significance in normal cellular activities and during thermal stress. Fluorographs of labeled polypeptides and western blots demonstrated that both HSPs are expressed constitutively and heat-induced in all the larval and adult cell types examined. The pattern of whole tissue immunocytochemical staining using anti-HSP60 and anti-HSP70 antibodies corresponded well with the observations from western blots or fluorographs. In developing oocytes, both constitutive and heat inducible expression of HSP60 were regulated in an oocyte stage-specific manner. In unstressed ovaries the expression of these proteins was less pronounced in early stage oocytes (1 st -8 th ) than at later stages (9 th and onward). The heat shock, however, induced both HSP70 and HSP60 to a significantly high level in early stage oocytes (1 st -8 th ) as compared to their respective controls. Our findings indicate the involvement of the HSP60 and HSP70 proteins in the development, growth and differentiation of both somatic and germ line tissues. Furthermore, the enhanced co-expression of HSP70 and HSP60 upon heat shock in various larval and adult cell types suggests the possible role of HSP60 in thermoprotection.

DNA-based identification of species for phylogenetic analysis as well as forensic identification is widely being carried out with the help of polymerase chain reaction (PCR). In this study, a successful effort has been made to identify 5 species of Indian freshwater turtles, including 3 hard-shell turtles (Geoemydidae), i.e. Kachuga dhongoka, K. kachuga and Geoclemys hamiltoni, and 2 species of soft-shell turtles (Trionychidae), i.e. Aspideretes gangeticus and Lissemys punctata punctata, by using a well-optimized PCR-RFLP method. The analysis of nucleotide sequence variations in the PCR-amplified mitochondrial cyt-b genes (encoding cytochrome b) from the 5 species revealed its usefulness in the taxonomic differentiation of these species. On the basis of cyt-b sequence data and the PCR-RFLP pattern, a phylogeny was developed to resolve the genetic relationships between these species, living in the same habitat type. In comparison, the PCR-RFLP of mitochondrial 16S rDNA genes appeared less decisive in analysing phylogenetic relationships or even in species differentiation. Further, the molecular method (PCR-RFLP) developed here is simple, rapid, reliable and reproducible; hence it can be routinely applied for species identification, essential for conservation and management of endangered chelonian species.

The effect of two most commonly used and highly toxic organic pesticides, endosulphan (organochlorine) and monocrotophos (organophosphate), was studied in a blowfly, Lucilia cuprina, to test whether these pesticides induce the stress response and, if so, whether the intensity of the response, in terms of induction of heat shock proteins (HSPs), HSP60 and HSP70 in particular, is pesticide concentration dependent. The in vitro exposure of larval and adult tissues to varying concentrations of these pesticides (endosulphan: 1.0-4.0 ppm for larva and 0.05-0.50 ppm for adult; monocrotophos: 0.0005-0.0050 ppm for larva and 0.0001-0.0010 ppm for adult) revealed that both compounds were able to induce the expression of HSP60 and HSP70 proteins. Western blot analysis of these HSPs indicated that the induction of expression was tissue-specific. The trypan blue staining of pesticide-exposed tissues demonstrated monocrotophos to exert more severe effect than endosulphan, as the former compound induced both HSP60 and HSP70 significantly at a much lower concentration than that of the later. The pattern of expression of these HSPs, in general, appeared in direct correlation with the pesticide concentration. Gut tissues were found relatively more sensitive to pesticide toxicity than other tissues, as revealed by trypan blue staining, and hence, they might serve as primary targets for early detection of pesticide toxicity. The results indicated that either of these HSPs or both could serve as a potential biomarker toward assessment and monitoring of toxicity induced by these pesticides.

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