Molecular cloning and in silico studies of physiologically significant trehalase from Drosophila melanogaster.

Trehalase, a physiologically important glycosidase is known for its crucial role in insect glycometabolism and stress recovery. The present study describes the molecular cloning of a gene fragment, encoding the catalytically active trehalase from Drosophila melanogaster (DmTre) and its heterologous expression in Escherichia coli. The 1275bp gene was overexpressed in two different vectors viz., pET28a and pCOLD TF and investigated for variable soluble expression, purification and activity of the recombinant enzyme with optimum pH and temperature of enzyme as 6 and 55°C, respectively. The sequence was characterized in silico by subjecting it to homology search, multiple sequence alignment and phylogenetic tree construction revealing its identity to other trehalases which belong to glycoside hydrolase family 37. The deduced amino acid sequence and modeled 3D structure of DmTre possessed all features of trehalase superfamily, including signature motifs and catalytic domain. The active site pocket of recombinant DmTre was compared with the crystal structure of E. coli trehalase identifying Glu424 and Asp226 as the putative catalytic residues. Additionally, enzyme-substrate docking suggests possible involvement of other residues in the catalysis along with Asp226. The present study holds significance in understanding the structural aspects of Drosophila trehalase in spite of unavailabilty of eukaryotic trehalase crystal structure.

Increased level of superoxide dismutase (SOD) activity in larvae of Chironomus ramosus (Diptera: Chironomidae) subjected to ionizing radiation.

A battery of enzymes from the eukaryotic antioxidant defense system was measured in salivary gland and in whole body extract of fourth instar larvae of Chironomus ramosus with an objective of finding any clue for the dipteran insect's capacity to tolerate heavy doses of ionizing radiation. Levels of activity of antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), and glutathione peroxidase (GSH-Px) were quantified in 30 days old larvae exposed to LD(20) dose of gamma radiation. Compared to controls, activity of Cu,Zn-SOD increased 3 to 4 fold and catalase 2 fold in response to ionizing radiation stress, while activities of GR and GSH-Px enzymes were decreased. Among the other SOD isoenzymes, our results showed comparable levels of Mn-SOD and Cu,Zn-SOD activity in control and irradiated groups of larvae. The increase in levels of the Cu,Zn-SOD isoenzyme was also confirmed by Western blot and zymography supported by densitometric quantification. No evidence of Fe-SOD was found in C. ramosus larvae. These findings could help to explain the persistence of natural populations of Chironomus in radioactively contaminated regions.

Assessment of Cell Viability in Intact Glandular Tissue in Chironomus ramosus using Dye-exclusion and Colorimetric Assays.

Conventionally, dye-exclusion test for determining cell viability has been restricted only for cells in suspension in tissue culture. In this paper, salivary gland of Chironomus has been proposed as a simple tissue model system where dye-exclusion test can be reliably employed for the intact gland. We have compared suitability of commonly used vital dyes and nigrosin was found suitable for the salivary gland cells. Biochemical tests using tetrazolium salts are also commonly used for determining quantitative indices of cell viability in metabolically active cells. Ours is the first attempt to extend the same technique for the whole tissue. We standardized the conditions and prepared a protocol for MTT-based colorimetric assay suitable for the salivary gland of Chironomus. A strong correlation (r(2) = 0.9893) was obtained where increasing O.D. correlated linearly with the number of live glands. We concluded that nigrosin dye-exclusion and MTT metabolic inclusion assays are suitable methods for the viability test of metabolically active intact salivary gland of Chironomus which can serve as a potential model for the assessment of cytotoxicity in future.

Extracellular electrical activity from the photoreceptors of midge.

The ontogeny of photosensitivity has been studied in a holometabolous insect, the midge Chironomus ramosus. The life cycle of midges shifts from an aquatic environment to a non-aquatic environment. Extracellular electrical activity of photoreceptor organs was recorded at larval and adult stages. We found an increase in photosensitivity as the larva metamorphosed to the adult stage. This is the first report of changes in photosensitivity during the development of any insect described in an ecological context.

Chromosomal polymorphism is associated with nematode parasitism in a natural population of a tropical midge.

A natural population of a tropical midge, Chironomus ramosus (Diptera: Chironomidae), was found to be polymorphic for a paracentric inversion (IV: 18C-19D). Based on the characteristic banding pattern of the fourth chromosome in the larval salivary gland polytene nuclei, individuals were classified as either structural homozygotes or heterozygotes. Isofemale lines were obtained and subsequently standard (S/S) and inversion (I/I) homozygotes were characterised by careful progeny testing in the laboratory. While exploring various biotic and abiotic factors that might be responsible for the maintenance of inversion polymorphism, we detected nematode (Family: Mermithidae) infections among the larval population. A detailed study indicated that the inversion polymorphism in the natural population of C. ramosus was apparently being maintained as a result of the selective pressure exerted by the nematode parasite. The corresponding pattern of increase and decrease in genotype frequencies and the relative fitness values indicated a selective advantage of inversion heterozygotes (S/I) over both homozygous types (S/S and I/I). Both empirical and experimental data suggest the strong heterotic nature of adaptation in this C. ramosus population towards nematode infection. This is the first report of its kind where inversion polymorphism has been shown to be associated with nematode parasitism.

Search for a Drosophila-93D-like locus in Chironomus and Anopheles.

The results of experiments to explore the possible existence of a heat shock locus in Chironomus and Anopheles which may be comparable to the 93D heat shock locus of Drosophila, are presented. None of the heat shock loci in C. striatipennis were inducible by benzamide, colchicine, vitamin B6, thiamphenicol or a homogenate of heat shocked glands, all of which are known to selectively induce the 93D-like loci in the genus Drosophila. Benzamide also failed to induce any locus in A. stephensi. The effect of all these treatments on general transcription in Chironomus and Anopheles polytene nuclei were comparable to those known in polytene cells of Drosophila. It thus appears that a heat shock locus homologous to 93D of D. melanogaster is absent in Chironomus and Anopheles so far as inducibility of a puff by specific agents is concerned. The existence of a possible 'functional counterpart' of the 93D locus in Chironomus and Anopheles genomes cannot be eliminated.