Targeted gene deletion and phenotypic analysis of the Drosophila melanogaster seminal fluid protease inhibitor Acp62F

Internally fertilizing organisms transfer a complex assortment of seminal fluid proteins, a substantial fraction of which are proteolysis regulators. In mammals, some seminal protease inhibitors have been implicated in male infertility and these same molecular classes of protease inhibitors are also found in Drosophila seminal fluid. Here, we tested the reproductive functions of the Drosophila melanogaster seminal fluid protease inhibitor Acp62F by generating a precise deletion of the Acp62F gene. We did not detect a nonredundant function for Acp62F in modulating the egg laying, fertility, remating frequency, or life span of mated females. However, loss of Acp62F did alter a male's defensive sperm competitive ability, consistent with the localization of Acp62F to sperm storage organs. In addition, the processing of at least one seminal protein, the ovulation hormone ovulin, is slower in the absence of Acp62F

Multiplatform Metabolomics to Understand the Imidacloprid-Induced Toxicity in <i>Drosophila</i>

Neonicotinoids, the class of insecticides used for crop protection, are subjected to vigilance due to their pernicious impacts. Imidacloprid (IMD) is one of the most representative insecticides of the neonicotinoid family, which has shown unfriendly consequences for non-target species. Metabolomics, a multidisciplinary approach, is being used in toxicological research to understand the metabolic responses to toxicant exposure by utilizing modern analytical techniques. Yet, no solitary analytical technique can cover the broad metabolite spectrum, but a multi-technique metabolomics platform can aid in analyzing the majority of the metabolites. In the present study, an effort has been made to identify the differential metabolites in Drosophila after exposure to IMD at 2.5 and 25 ng/mL using liquid chromatography-high-resolution mass spectrometry (LC-HRMS), gas chromatography-MS (GC-MS), and NMR-based untargeted metabolomics. Multivariate pattern recognition analysis helped in identifying/recognizing 19 (LC-HRMS), 7 (GC-MS), and 13 (NMR) differential metabolites mainly belonging to the category of amino acids, sugars, fatty acids, and organic acids. The pathway analysis of differential metabolites predominantly showed impact on aminoacyl-tRNA biosynthesis, amino acid metabolism, and glycerophospholipid metabolism. Among these, arginine and proline metabolism was observed to be the common metabolic pathway perturbed in Drosophila due to IMD exposure. The multiplatform metabolomics based on LC-HRMS, GC-MS, and NMR analysis with an advanced level of statistical analysis can provide insights into potential perturbations in the metabolome of IMD-exposed Drosophila

Evolution in the Fast Lane: Rapidly Evolving Sex-Related Genes in Drosophila

A large portion of the annotated genes in Drosophila melanogaster show sex-biased expression, indicating that sex and reproduction-related genes (SRR genes) represent an appreciable component of the genome. Previous studies, in which subsets of genes were compared among few Drosophila species, have found that SRR genes exhibit unusual evolutionary patterns. Here, we have used the newly released genome sequences from 12 Drosophila species, coupled to a larger set of SRR genes, to comprehensively test the generality of these patterns. Among 2505 SRR genes examined, including ESTs with biased expression in reproductive tissues and genes characterized as involved in gametogenesis, we find that a relatively high proportion of SRR genes have experienced accelerated divergence throughout the genus Drosophila. Several testis-specific genes, male seminal fluid proteins (SFPs), and spermatogenesis genes show lineage-specific bursts of accelerated evolution and positive selection. SFP genes also show evidence of lineage-specific gene loss and/or gain. These results bring us closer to understanding the details of the evolutionary dynamics of SRR genes with respect to species divergence

Functional male accessory glands and fertility in Drosophila require novel ecdysone receptor

<div><p>In many insects, the accessory gland, a secretory tissue of the male reproductive system, is essential for male fertility. Male accessory gland is the major source of proteinaceous secretions, collectively called as seminal proteins (or accessory gland proteins), which upon transfer, manipulate the physiology and behavior of mated females. Insect hormones such as ecdysteroids and juvenoids play a key role in accessory gland development and protein synthesis but little is known about underlying molecular players and their mechanism of action. Therefore, in the present study, we examined the roles of hormone-dependent transcription factors (Nuclear Receptors), in accessory gland development, function and male fertility of a genetically tractable insect model, <i>Drosophila melanogaster</i>. First, we carried out an RNAi screen involving 19 hormone receptors, individually and specifically, in a male reproductive tissue (accessory gland) for their requirement in Drosophila male fertility. Subsequently, by using independent RNAi/ dominant negative forms, we show that Ecdysone Receptor (EcR) is essential for male fertility due to its requirement in the normal development of accessory glands in Drosophila: EcR depleted glands fail to make seminal proteins and have dying cells. Further, our data point to a novel ecdysone receptor that does not include Ultraspiracle but is probably comprised of EcR isoforms in Drosophila male accessory glands. Our data suggest that this novel ecdysone receptor might act downstream of homeodomain transcription factor paired (prd) in the male accessory gland. Overall, the study suggests novel ecdysone receptor as an important player in the hormonal regulation of seminal protein production and insect male fertility.</p></div

Western blots showing the levels of EcR and USP in knockdown males compared to control males.

<p>The EcR panel represents EcR levels in accessory glands from EcR control (+ lane, EcR), EcR knockdown (-lane, EcR), USP control (+ lane, USP) and USP knockdown (- lane, USP). Similarly, the USP panel represents the USP levels observed in accessory glands from above groups. Blots probed with α-tubulin antibodies (α-tubulin panels) served as controls for protein loading. Knockdowns were specific to the targeted hormone receptor. Further, the deficiency of EcR did not affect USP levels and vice-versa.</p

The effect of EcR knockdown on the cellular organization of the accessory glands.

<p>Immunofluorescence panels shown here are the overlay images of accessory glands immunostained with α-Spectrin antibody (marking cell membrane, Green color) and labeled with nuclear stain DAPI (blue color). In EcR control glands, several polygonally shaped binucleate cells (the main cells) and a few large and spherical binucleate cells (the secondary cells, marked with arrow) interspersed between the polygonally shaped cells were observed at 400X (Panel A) and at a higher magnification of 630X (Panel A′). However, in EcR-deficient glands, cell membranes are highly disrupted (Panel B at 400X) and the nuclear distribution is distinctly different from that of control (Panel B′ at a total magnification of 630X). To assess the effect of depletion of EcR or USP, in accessory glands,western blots were probed for the secondary cell markers, Abd-B and ANCE. The glands lacked Abd-B (Panel C,—lane under EcR in Abd-B blot) while EcR control (Panel C, + lane under EcR), USP control (Panel C, + lane under USP) or USP knockdown (Panel C, -lane under USP) had detectable levels of Abd-B. In addition, EcR knockdown glands did not contain ANCE (Panel D, -lane under EcR in ANCE blot) while the same could be detected in controls, suggesting the disruption of secondary cells due to depletion of EcR.</p

Morphology and secondary cells markers of accessory glands in males over-expressing dominant negative EcR isoforms.

<p>The morphology of accessory glands from (A) control or males over expressing (B) EcR-A, (C) EcR-B1, or (D) EcR-B2 was observed under light microscopy. Morphology of accessory glands from males over expressing EcR-B1, or EcR-B2 is comparable to their controls. However, accessory glands from EcR-A appear slightly reduced in comparison to their controls but still not as extremely reduced as those in EcR-miRNA based knockdown males. (B) Western blots of accessory gland protein extracts depicting levels of Abd-B (Abd-B panel), ANCE (ANCE panel) proteins and cleaved Caspase 3 immunoreactivity (cleaved Caspase 3 panel) in males over expressing EcR-A, EcR-B1 and EcR-B2. Blots were probed with β-actin antibodies (β-actin panels) served as controls for protein loading.</p