A Reliable and Consistent Method to Quantify Percent Lethality and Life Span in Drosophila melanogaster.

Drosophila melanogaster is a classic model organism to study gene function as well as toxicological effects. To study gene function, the expression of a particular gene of interest is disrupted by using the widely explorable Drosophila genetic toolkit, whereas to study toxicological effects the flies are exposed to a particular toxicant through diet. These experiments often require the quantification of lethality from embryonic to adult stages, as well as the assessment of the life span in order to check the role of the gene/toxicant of interest in Drosophila. Here, we propose an experimental protocol that enables a consistent and rigorous assessment of lethality and life span of cadmium chloride (CdCl2)-exposed or genetically perturbed flies [downregulation and overexpression of the cytosolic Cu, Zn superoxide dismutase (SOD1) gene], consecutively. The protocol insists upon the requirement of one single experimental setup that is unique, distinctive, and cost-effective as it engages minimal laboratory equipment and resources. The described methods lead to the smooth observation of the embryos, their successive stagewise transition, and life span of the adult flies post eclosion. Additionally, these methods also facilitate the assessment of crawling and climbing behavioral parameters of the larvae and adults, respectively, and allow the calculation of lethal concentration (LC50) for the mentioned toxicant as well as median survival of the flies, which can be a determining factor in proceeding with further stages of experiments. Graphical abstract.

Parvin: A hub of intracellular signalling pathways regulating cellular behaviour and disease progression.

α-actinin superfamily houses the family of parvins, comprising α, ß and γ isoforms in the vertebrates and a single orthologue in the invertebrates. Parvin as an adaptor protein is a member of the ternary IPP-complex including Integrin Linked Kinase (ILK) and particularly-interesting-Cys-His-rich protein (PINCH). Each of the complex proteins showed a conserved lineage and was principally used by the evolutionarily primitive integrin-adhesome machinery to regulate cellular behaviour and signalling pathways. Parvin facilitated integrin mediated integration of the extracellular matrix with cytoskeletal framework culminating in regulation of cellular adhesion and spreading, cytoskeleton reorganisation and cell survival. Studies have established role of parvin in pregnancy, lactation, matrix degradation, blood vessel formation and in several diseases such as cancer, NAFLD and cardiac diseases etc. This review narrates the history of parvin discovery, its elaborate gene structure and conservation across phyla including cellular expression, localisation and interacting partners in vertebrates as well as invertebrates. The review further discusses how parvin acts as an epicentre of signalling pathways, its associated mutants and diseased conditions.

Dietary cadmium induced declined locomotory and reproductive fitness with altered homeostasis of essential elements in Drosophila melanogaster.

Cadmium (Cd) exerts detrimental effects on multiple biological processes of the living organisms along with epigenetic transgenerational effect. Drosophila melanogaster offers unique opportunity to evaluate Cd toxicity when studying important life traits in short duration of time by designing distinct behavioural assays. Present study utilized this model organism to assess Cd induced lethality, retarded growth, decreased life span and altered behaviour of the animals either at larval or adult stage. Our investigations revealed reduced locomotion and reproductive fitness of the animals upon Cd exposure. Transgenerational effect on locomotion was found to be behaviour specific as larval crawling was affected, but adult fly negative geotaxis was comparable to the control. Mechanistically, decreased antioxidant enzymes activity, superoxide dismutase (SOD) and catalase (CAT) together with altered homeostasis of essential elements (Fe, Zn and Mg) may be responsible for the observed effects. Altogether our work showed extensive range of Cd altered Drosophila behaviour which warrants need to control environmental Cd toxicity.

Dietary cadmium (Cd) reduces hemocyte level by induction of apoptosis in Drosophila melanogaster.

Drosophila melanogaster larvae ensure continuous proliferation and differentiation of hemocytes to maintain a fixed range of different blood cell types during its various stages of development. Variation in this number is often an indicator of animal well-being, its genotype or an effect of environmental perturbation, including exposure to heavy metals. The present study investigates the effect of Cd on larval hemocytes. Embryos were allowed to grow in metal media till third instar larvae and finally circulating hemocyte were collected. The number of major hemocytes, plasmatocytes and crystal cells was determined to be lowered in Cd exposed animals. Our results also showed modulation of antioxidant biology of Cd exposed hemocytes by changing the major antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT) activity, and decreasing reduced glutathione (GSH) levels in hemocytes suspended in the hemolymph. Acridine orange (AO) staining further revealed induction of apoptosis in hemocytes of metal treated larvae. Our results suggest a negative impact of Cd exposure on the hemocytes of the Drosophila larvae culminating in their lowered count by induction of apoptosis.

An insight on Drosophila myogenesis and its assessment techniques.

Movement assisted by muscles forms the basis of various behavioural traits seen in Drosophila. Myogenesis involves developmental processes like cellular specification, differentiation, migration, fusion, adherence to tendons and neuronal innervation in a series of coordinated event well defined in body space and time. Gene regulatory networks are switched on-off, fine tuning at the right developmental stage to assist each cellular event. Drosophila is a holometabolous organism that undergoes myogenesis waves at two developmental stages, and is ideal for comparative analysis of the role of genes and genetic pathways conserved across phyla. In this review we have summarized myogenic events from the embryo to adult focussing on the somatic muscle development during the early embryonic stage and then on indirect flight muscles (IFM) formation required for adult life, emphasizing on recent trends of analysing muscle mutants and advances in Drosophila muscle biology.

Chronic lead (Pb) exposure results in diminished hemocyte count and increased susceptibility to bacterial infection in Drosophila melanogaster.

Heavy metal Pb is a common toxic pollutant present in our environment adversely affecting health of the living organisms. Recent studies suggest positive correlation between heavy metal exposure and immune dysfunction and present work utilizes Drosophila to address this issue in relation to Pb exposure. In-vivo Pb toxicity was established by dietary intake where essential parameters like development and life span were found to be hampered and augmented upon metallothionein B (mtnB) downregulation hinting towards potential role of mtnB in Pb detoxification. Further response of Drosophila to B. subtilis bacterial infection was monitored by carrying out oral infections. Pb fed flies showed increased susceptibility to infection as compared to their controls. Since Drosophila hemocytes play dual role as immune cells, we checked for the total hemocyte count and found significant decrease in hemocyte numbers in Pb fed larvae. Both crystal cells and plasmatocytes, the two major hemocytes in third instar larval hemolymph were reduced. However we did not find any visible morphological changes in Giemsa stained hemocytes. Crystal cells are crucial for synthesis and release of phenoloxidase (PO), an enzyme required for melanin clot synthesis and deposition. PO activity assessed from total hemolymph protein isolates was found to be substantially decreased in Pb raised animals. Results were also confirmed by spot test and native gel activity assay of PO. Overall our results suggest immunotoxic effect of Pb through decrease in hemocyte count including crystal cell which in turn leads to decreased PO activity and increased susceptibility to B. subtilis.

A cis-regulatory mutation in troponin-I of Drosophila reveals the importance of proper stoichiometry of structural proteins during muscle assembly.

Rapid and high wing-beat frequencies achieved during insect flight are powered by the indirect flight muscles, the largest group of muscles present in the thorax. Any anomaly during the assembly and/or structural impairment of the indirect flight muscles gives rise to a flightless phenotype. Multiple mutagenesis screens in Drosophila melanogaster for defective flight behavior have led to the isolation and characterization of mutations that have been instrumental in the identification of many proteins and residues that are important for muscle assembly, function, and disease. In this article, we present a molecular-genetic characterization of a flightless mutation, flightless-H (fliH), originally designated as heldup-a (hdp-a). We show that fliH is a cis-regulatory mutation of the wings up A (wupA) gene, which codes for the troponin-I protein, one of the troponin complex proteins, involved in regulation of muscle contraction. The mutation leads to reduced levels of troponin-I transcript and protein. In addition to this, there is also coordinated reduction in transcript and protein levels of other structural protein isoforms that are part of the troponin complex. The altered transcript and protein stoichiometry ultimately culminates in unregulated acto-myosin interactions and a hypercontraction muscle phenotype. Our results shed new insights into the importance of maintaining the stoichiometry of structural proteins during muscle assembly for proper function with implications for the identification of mutations and disease phenotypes in other species, including humans.