K. Ravi Ram

Seminal influences: Drosophila Acps and the molecular interplay between males and females during reproduction

Successful reproduction requires contributions from both the male and the female. In Drosophila, contributions from the male include accessory gland proteins (Acps) that are components of the seminal fluid. Upon their transfer to the female, Acps affect the females physiology and behavior. Although primary sequences of Acp genes exhibit variation among species and genera, the conservation of protein biochemical classes in the seminal fluid suggests a conservation of functions. Bioinformatics coupled with molecular and genetic tools available for Drosophila melanogaster has expanded the functional analysis of Acps in recent years to the genomic/proteomic scale. Molecular interplay between Acps and the female enhances her egg production, reduces her receptivity to remating, alters her immune response and feeding behavior, facilitates storage and utilization of sperm in the female and affects her longevity. Here, we provide an overview of the D. melanogaster Acps and integrate the results from several studies that bring the current number of known D. melanogaster Acps to 112. We then discuss several examples of how the females physiological processes and behaviors are mediated by interactions between Acps and the female. Understanding how Acps elicit particular female responses will provide insights into reproductive biology and chemical communication, tools for analyzing models of sexual cooperation and/or sexual conflict, and information potentially useful for strategies for managing insect pests.

Sustained Post-Mating Response in Drosophila melanogaster Requires Multiple Seminal Fluid Proteins

Successful reproduction is critical to pass genes to the next generation. Seminal proteins contribute to important reproductive processes that lead to fertilization in species ranging from insects to mammals. In Drosophila, the males accessory gland is a source of seminal fluid proteins that affect the reproductive output of males and females by altering female post-mating behavior and physiology. Protein classes found in the seminal fluid of Drosophila are similar to those of other organisms, including mammals. By using RNA interference (RNAi) to knock down levels of individual accessory gland proteins (Acps), we investigated the role of 25 Acps in mediating three post-mating female responses: egg production, receptivity to remating and storage of sperm. We detected roles for five Acps in these post-mating responses. CG33943 is required for full stimulation of egg production on the first day after mating. Four other Acps (CG1652, CG1656, CG17575, and CG9997) appear to modulate the long-term response, which is the maintenance of post-mating behavior and physiological changes. The long-term post-mating response requires presence of sperm in storage and, until now, had been known to require only a single Acp. Here, we discovered several novel Acps together are required which together are required for sustained egg production, reduction in receptivity to remating of the mated female and for promotion of stored sperm release from the seminal receptacle. Our results also show that members of conserved protein classes found in seminal plasma from insects to mammals are essential for important reproductive processes.

Sex Peptide is Required for the Efficient Release of Stored Sperm in Mated Drosophila Females

The Drosophila seminal fluid protein (SFP) sex peptide (SP) elicits numerous post-mating responses, including increased egg laying and decreased sexual receptivity, in the mated female. Unlike other SFPs, which are detectable in mated females for only a few hours post mating, SP is maintained—and its effects are sustained—for several days. The persistence of SP in the mated females reproductive tract is thought to be a consequence of its binding to, and gradual release from, sperm in storage, which maintains SPs ability to act within the female reproductive tract. Recent studies have shown that several other SFPs, acting in a network, are needed for SPs localization to sperm and are necessary for the efficient release of sperm from storage. This result suggested an additional new role for SP modulating the release of sperm from storage. We tested for this possibility by examining sperm storage parameters in mated females that did not receive SP. We found that while sperm accumulation into storage was unaffected, sperm depletion from storage sites was significantly decreased (or impaired) in the absence of SP. Mates of males expressing a modified SP that is unable to be released from sperm showed a similar phenotype, indicating that release of sperm-bound SP is a necessary component of normal sperm depletion. Additionally, SP null males were more successful in a sperm competitive environment when they were first to mate, which is likely a consequence of higher retention of their sperm due to defective sperm release. Our findings illustrate a direct role for SP in the release of sperm from storage.

A role for Acp29AB, a predicted seminal fluid lectin, in female sperm storage in Drosophila melanogaster

Females of many animal species store sperm for taxon-specific periods of time, ranging from a few hours to years. Female sperm storage has important reproductive and evolutionary consequences, yet relatively little is known of its molecular basis. Here, we report the isolation of a loss-of-function mutation of the Drosophila melanogaster Acp29AB gene, which encodes a seminal fluid protein that is transferred from males to females during mating. Using this mutant, we show that Acp29AB is required for the normal maintenance of sperm in storage. Consistent with this role, Acp29AB localizes to female sperm storage organs following mating, although it does not appear to associate tightly with sperm. Acp29AB is a predicted lectin, suggesting that sugar–protein interactions may be important for D. melanogaster sperm storage, much as they are in many mammals. Previous association studies have found an effect of Acp29AB genotype on a males sperm competitive ability; our findings suggest that effects on sperm storage may underlie these differences in sperm competition. Moreover, Acp29ABs effects on sperm storage and sperm competition may explain previously documented evidence for positive selection on the Acp29AB locus.

The Drosophila melanogaster seminal fluid protease "seminase" regulates proteolytic and post-mating reproductive processes.

Proteases and protease inhibitors have been identified in the ejaculates of animal taxa ranging from invertebrates to mammals and form a major protein class among Drosophila melanogaster seminal fluid proteins (SFPs). Other than a single protease cascade in mammals that regulates seminal clot liquefaction, no proteolytic cascades (i.e. pathways with at least two proteases acting in sequence) have been identified in seminal fluids. In Drosophila, SFPs are transferred to females during mating and, together with sperm, are necessary for the many post-mating responses elicited in females. Though several SFPs are proteolytically cleaved either during or after mating, virtually nothing is known about the proteases involved in these cleavage events or the physiological consequences of proteolytic activity in the seminal fluid on the female. Here, we present evidence that a protease cascade acts in the seminal fluid of Drosophila during and after mating. Using RNAi to knock down expression of the SFP CG10586, a predicted serine protease, we show that it acts upstream of the SFP CG11864, a predicted astacin protease, to process SFPs involved in ovulation and sperm entry into storage. We also show that knockdown of CG10586 leads to lower levels of egg laying, higher rates of sexual receptivity to subsequent males, and abnormal sperm usage patterns, processes that are independent of CG11864. The long-term phenotypes of females mated to CG10586 knockdown males are similar to those of females that fail to store sex peptide, an important elicitor of long-term post-mating responses, and indicate a role for CG10586 in regulating sex peptide. These results point to an important role for proteolysis among insect SFPs and suggest that protease cascades may be a mechanism for precise temporal regulation of multiple post-mating responses in females.

Gellan gum blended PEI nanocomposites as gene delivery agents: Evidences from in vitro and in vivo studies

Branched Polyethylenimine, 25 kDa (PEI), was blended with gellan gum, an anionic heteropolysaccharide, for partial neutralization of its excess positive charge to form gellan gum-polyethylenimine (GP) nanocomposites (NCs). Subsequently, we manipulated the amount of gellan gum for obtaining a series of NCs and characterized them for their size, charge and morphology. Among all the NCs, one member, named GP3, showed the best transfection efficiency in tested cell lines in comparison with the rest of the series, PEI, Lipofectamine and other commercial transfection agents and also exhibited minimum cytotoxicity. It was found to transfect primary cells of mouse skin with better efficiency than PEI and Lipofectamine and was able to protect the plasmid DNA from nucleases and serum proteins present in the blood. GP3 exhibited efficient intracellular delivery of plasmid as revealed by confocal studies while its intracellular presence was also confirmed by the knockdown of GFP expression (using GFP specific siRNA) and JNKII by quantifying proteins in cell lysates and by western blotting and hybridization, respectively. In vivo cytotoxicity studies in Drosophila showed lack of induction of stress response in the exposed organisms. Further, exposed organisms did not show any developmental delay or mortality and no morphological defects were observed in the emerged flies. In vivo gene expression studies in Balb/c mice revealed maximum expression of luciferase enzyme in spleen. The study suggests that GP3 may act as an efficient non-viral gene carrier with diverse biomedical applications.

Effects of co-exposure of benzene, toluene and xylene to Drosophila melanogaster: alteration in hsp70, hsp60, hsp83, hsp26, ROS generation and oxidative stress markers.

Benzene, toluene and xylene are monocyclic aromatic hydrocarbon compounds, used both as individual compound and as mixtures, in industry as well as household. Previous studies involving exposures to these compounds, individually, have shown that benzene was more toxic compared to toluene or xylene. Here, we tested a working hypothesis that toluene and/or xylene in a mixture containing benzene affect benzene induced toxicity in a non-target organism, Drosophila melanogaster. We exposed D. melanogaster larvae transgenic for hsp70, hsp83 or hsp26 and wild type (Oregon R strain) larvae to 25.0-100.0mM benzene, 25.0-100.0mM toluene and 25.0-100mM xylene, individually or in mixtures. Subsequently, we examined the expression of stress genes (encoding heat shock proteins, hsps), generation of reactive oxygen species (ROS), induction of anti-oxidant stress markers and emergence of flies under treatment as well as control conditions. We observed that all these endpoints were significantly altered in all the treatment groups compared to their respective controls. However, the magnitude of toxicity of a benzene-toluene (BT) or benzene-xylene (BX) or benzene-toluene-xylene (BTX) mixture was significantly lower in the organism than that of individual chemical. Our results also show the modulation of toluene toxicity by xylene. Present study suggests antagonistic effect of xylene and toluene on benzene toxicity and additive/synergistic effect of xylene on toluene induced toxicity. Thus, expression of stress genes may be used as an assay for detection of early cellular toxicity. Further, our study supports the use of Drosophila as an alternative animal model for first tier screening of adverse effects of chemical mixtures.

Hazardous effect of tannery solid waste leachates on development and reproduction in Drosophila melanogaster: 70 kDa heat shock protein as a marker of cellular damage

Rapid industrialization has increased the burden of chemicals in the environment. These chemicals may be harmful to development and reproduction of any organism. We therefore analyzed the adverse effects of leachates from a tannery solid waste on development and reproduction using Drosophila. We show a significant delay in mean emergence of flies observed at the higher concentrations of the leachates, indicating their effect on the organisms development. Significant leachate-induced effect on reproduction of the organism was also observed. Sub-organismal analyses revealed Hsp70 expression and tissue damage in a sex-specific manner. Refractoriness of Hsp70 expression in accessory glands of male flies and ovaries of females was concurrent with tissue damage. Genes encoding certain seminal proteins (Acp70A and Acp36DE) from accessory glands were significantly down-regulated at higher concentrations of the leachates. The study suggests that (i) sub-organismal adverse responses are reflected at organismal level, (ii) tannery waste leachates cause adverse effects on the expression of genes encoding seminal proteins that facilitate normal reproduction and (iii) Hsp70 may be used as a marker of cellular damage for reproductive organs.

Environmental chemical mediated male reproductive toxicity: Drosophila melanogaster as an alternate animal model

Industrialization and indiscriminate use of agrochemicals have increased the human health risk. Recent epidemiological studies raised a concern for male reproduction given their observations of reduced sperm counts and altered semen quality. Interestingly, environmental factors that include various metals, pesticides and their metabolites have been causally linked to such adversities by their presence in the semen at levels that correlate to infertility. The epidemiological observations were further supported by studies in animal models involving various chemicals. Therefore, in this review, we focused on male reproductive toxicity and the adverse effects of different environmental chemicals on male reproduction. However, it is beyond the scope of this review to provide a detailed appraisal of all of the environmental chemicals that have been associated with reproductive toxicity in animals. Here, we provided the evidence for reproductive adversities of some commonly encountered chemicals (pesticides/metals) in the environment. In view of the recent thrust for an alternate to animal models in research, we subsequently discussed the contributions of Drosophila melanogaster as an alternate animal model for quick screening of toxicants for their reproductive toxicity potential. Finally, we emphasized the genetic and molecular tools offered by Drosophila for understanding the mechanisms underlying the male reproductive toxicity.

Transcriptome analysis provides insights for understanding the adverse effects of endosulfan in Drosophila melanogaster

Indiscriminate use of agrochemicals worldwide, particularly, persistent organic pollutants (POPs), is of concern. Endosulfan, a POP, is used by various developing/developed nations and is known to adversely affect the development and the hormonal profiles of humans and animals. However, little is known about the molecular players/pathways underlying the adverse effects of endosulfan. We therefore analyzed the global gene expression changes and subsequent adverse effects of endosulfan using Drosophila. We used Drosophila melanogaster keeping in view of its well annotated genome and the wealth of genetic/molecular reagents available for this model organism. We exposed third instar larvae of D. melanogaster to endosulfan (2.0 μg mL(-1)) for 24 h and using microarray, we identified differential expression of 256 genes in exposed organisms compared to controls. These genes are associated with cellular processes such as development, stress and immune response and metabolism. Microarray results were validated through quantitative PCR and biochemical assay on a subset of genes/proteins. Taking cues from microarray data, we analyzed the effect of endosulfan on development, emergence and survival of the organism. In exposed organisms, we observed deformities in hind-legs, reminiscent of those observed in higher organisms exposed to endosulfan. In addition, we observed delayed and/or reduced emergence in exposed organisms when compared to their respective controls. Together, our studies not only highlight the adverse effects of endosulfan on the organism but also provide an insight into the possible genetic perturbations underlying these effects, which might have potential implications to higher organisms.