Leena Thorat

Insect trehalase: Physiological significance and potential applications

Trehalose, a non-reducing disaccharide, is widespread throughout the biological world. It is the major blood sugar in insects playing a crucial role as an instant source of energy and in dealing with abiotic stresses. The hydrolysis of trehalose is under the enzymatic control of trehalase. The enzyme trehalase is gaining interest in insect physiology as it regulates energy metabolism and glucose generation via trehalose catabolism. The two forms of insect trehalase namely, Tre-1 and Tre-2, are important in energy supply, growth, metamorphosis, stress recovery, chitin synthesis and insect flight. Insect trehalase has not been reviewed in depth and the information available is quite scattered. The present mini review discusses our recent understanding of the regulation, mechanism and biochemical characterization of insect trehalase with respect to its physiological role in vital life functions. We also highlight the molecular and biochemical properties of insect trehalase that makes it amenable to competitive inhibition by most glycosidase inhibitors. Due to its crucial role in carbon metabolism in insects, application of inhibitors against trehalose can form a promising area towards formulating strategies for insect pest control.

Trehalose as an indicator of desiccation stress in Drosophila melanogaster larvae: A potential marker of anhydrobiosis

In the current scenario of global climate change, desiccation is considered as one of the major environmental stressors for the biota exposed to altered levels of ambient temperature and humidity. Drosophila melanogaster, a cosmopolitan terrestrial insect has been chosen as a humidity-sensitive bioindicator model for the present study since its habitat undergoes frequent stochastic and/or seasonally aggravated dehydration regimes. We report here for the first time the occurrence of anhydrobiosis in D. melanogaster larvae by subjecting them to desiccation stress under laboratory conditions. Larvae desiccated for ten hours at <5% relative humidity could enter anhydrobiosis and could revive upon rehydration followed by resumption of active metabolism. As revealed by FTIR and HPLC analyzes, our findings strongly indicated the synthesis and accumulation of trehalose in the desiccating larvae. Biochemical measurements pointed out the desiccation-responsive trehalose metabolic pathway that was found to be coordinated in concert with the enzymes trehalose 6-phosphate synthase and trehalase. Further, an inhibitor-based experimental approach using deoxynojirimycin, a specific trehalase inhibitor, demonstrated the pivotal role of trehalose in larval anhydrobiosis of D. melanogaster. We therefore propose trehalose as a potential marker for the assessment of anhydrobiosis in Drosophila. The present findings thus add to the growing list of novel biochemical markers in specific bioindicator organisms for fulfilling the urgent need of environmental biomonitoring of climate change.

Downregulation of dTps1 in Drosophila melanogaster larvae confirms involvement of trehalose in redox regulation following desiccation.

As a survival strategy to environmental water deficits, desiccation-tolerant organisms are commonly known for their ability to recruit stress-protective biomolecules such as trehalose. We have previously reported the pivotal role of trehalose in larval desiccation tolerance in Drosophila melanogaster. Trehalose has emerged as a versatile molecule, serving mainly as energy source in insects and also being a stress protectant. While several recent reports have revealed the unconventional role of trehalose in scavenging reactive oxygen species in yeast and plants, this aspect has not received much attention in animals. We examined the status of desiccation-induced generation of reactive oxygen species in D. melanogaster larvae and the possible involvement of trehalose in ameliorating the harmful consequences thereof. Insect trehalose synthesis is governed by the enzyme trehalose 6-phosphate synthase 1 (TPS1). Using the ubiquitous da-GAL4-driven expression of the dTps1-RNAi transgene, we generated dTps1-downregulated Drosophila larvae possessing depleted levels of dTps1 transcripts. This resulted in the inability of the larvae for trehalose synthesis, thereby allowing us to elucidate the significance of trehalose in the regulation of desiccation-responsive redox homeostasis. Furthermore, the results from molecular genetics studies, biochemical assays, electron spin resonance analyses and a simple, non-invasive method of whole larval live imaging suggested that trehalose in collaboration with superoxide dismutase (SOD) is involved in the maintenance of redox state in D. melanogaster.

Effects of water hyacinth Eichhornia crassipes root extracts on midge Chironomus ramosus larvae: a preliminary note

The effects of crude root extracts of Eichhornia crassipes (Marts) Solms on Chironomus ramosus Chaudhuri eggs and larvae are studied under laboratory conditions. Egg masses of C. ramosus that are subjected to varying concentrations of exudates (final concentrations 0.25–2.5%) show 100% viability. However, larvae reared further at these concentrations reveal a dose–response relationship, with an LC50 value of 1.33% and an LC90 value of 2.05%. A striking feature is the occurrence of supernumerary salivary glands in larvae (three glands per larva) reared in the 1%, 1.25%, 1.5% and 1.75% extracts. This deviates from the normal two glands per larva and may be explained by altered growth as a result of the putative bioactive compounds present in the hyacinth extracts. In nature, hyacinth and midges co‐exist in many freshwater habitats, implying that hyacinth compounds present from natural leaching could be encountered by the larvae. These preliminary findings suggest that hyacinth can influence the larval development of midges, giving rise to long‐term ecological implications.

Tolerance to desiccation stress in Chironomus ramosus through plasticity in homeostatic control

Desiccating environments pose physiological challenges to organisms, especially those experiencing them on recurring basis. Several desiccation-tolerant organisms are known to withstand loss of body water in response to seasonal dehydration patterns through adaptations that facilitate their sustenance in the dry state followed by recovery upon return of favourable hydrating conditions. In the present study, we have chosen Chironomus ramosus , an aquatic midge species as a laboratory model system towards the exploration of its desiccation tolerance ability. We confirm that upon desiccation exposure at low relative humidity, larvae of C. ramosus exhibited rapid water loss and could revive upon rehydration. Moreover, the revived individuals underwent successful metamorphosis; albeit delayed. This heterochrony in the developmental calendar was experimentally verified by the investigation of ecdysone levels which suggested an altered pattern of ecdysis in response to the stress. These data suggested the organism’s plasticity in developmental homeostasis when confronted with dehydration stress. Furthermore, spectrofluorometric assays indicated the occurrence of thiol damage in the larvae. Investigation of Hsp70, the evolutionarily conserved stress responsive gene suggested that Hsp70 was indeed up-regulated during the rehydration period, thus facilitating recovery of the larvae from dehydration. In summary, several lines of evidence involving cellular, physiological and developmental adaptations revealed the ability of C. ramosus larvae to minimize the diverse categories of damage caused by desiccation stress through plasticity in their homeostatic mechanism.

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.

High-throughput mass spectrometry analysis revealed a role for glucosamine in potentiating recovery following desiccation stress in Chironomus

Desiccation tolerance is an essential survival trait, especially in tropical aquatic organisms that are vulnerable to severe challenges posed by hydroperiodicity patterns in their habitats, characterized by dehydration-rehydration cycles. Here, we report a novel role for glucosamine as a desiccation stress-responsive metabolite in the underexplored tropical aquatic midge, Chironomus ramosus. Using high- throughput liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QToF-MS) analysis, biochemical assays and gene expression studies, we confirmed that glucosamine was essential during the recovery phase in C. ramosus larvae. Additionally, we demonstrated that trehalose, a known stress-protectant was crucial during desiccation but did not offer any advantage to the larvae during recovery. Based on our findings, we emphasise on the collaborative interplay of glucosamine and trehalose in conferring overall resilience to desiccation stress and propose the involvement of the trehalose-chitin metabolic interface in insects as one of the stress-management strategies to potentiate recovery post desiccation through recruitment of glucosamine.

Desiccation stress induces developmental heterochrony in Drosophila melanogaster

Stressful environments are known to perturb developmental patterns in insects. In the purview of desiccation as a stressor, relatively little is known about the developmental consequences linked with desiccation tolerance. In this study, we have particularly focused on the exploration of the temporal profile of postembryonic development in response to desiccation exposure in Drosophila melanogaster and the associated trade-offs. We document a correlation between variations in 20-hydroxyecdysone levels and the altered timing of metamorphic events during the life cycle. Following desiccation, we observed an extension in the larval longevity whereas the duration of the pupal and adult stages was significantly shortened. Alternately, feeding of 20-hydroxyecdysone apparently led to the restoration of the normal temporal pattern of development in the desiccated group. In spite of the desiccation-responsive heterochronic shifts in development, the overall lifespan post recovery remained almost unaltered among the desiccated and undesiccated groups suggesting plasticity in developmental control. This observation reminisces ‘canalization-like’ phenomenon that buffers alterations in the overall lifespan. We thus identified a desiccation-responsive period in the lifespan of D. melanogaster during which variations in ecdysone levels are capable to alter the temporal course of development.