Yael Heifetz

Mating-responsive genes in reproductive tissues of female Drosophila melanogaster

Male-derived accessory gland proteins that are transferred to females during mating have profound effects on female reproductive physiology including increased ovulation, mating inhibition, and effects on sperm utilization and storage. The extreme rates of evolution seen in accessory gland proteins may be driven by sperm competition and sexual conflict, processes that may ultimately drive complex interactions between female- and male-derived molecules and sperm. However, little is known of how gene expression in female reproductive tissues changes in response to the presence of male molecules and sperm. To characterize this response, we conducted parallel genomic and proteomic analyses of gene expression in the reproductive tract of 3-day-old unmated and mated female Drosophila melanogaster. Using DNA microarrays, we identified 539 transcripts that are differentially expressed in unmated vs. mated females and revealed a striking peak in differential expression at 6 h postmating and a marked shift from primarily down-regulated to primarily up-regulated transcripts within 3 h after mating. Combining two-dimensional gel electrophoresis and liquid chromatography mass spectrometry analyses, we identified 84 differentially expressed proteins at 3 h postmating, including proteins that appeared to undergo posttranslational modification. Together, our observations define transcriptional and translational response to mating within the female reproductive tract and suggest a bimodal model of postmating gene expression initially correlated with mating and the final stages of female reproductive tract maturation and later with the declining presence of male reproductive molecules and with sperm maintenance and utilization.

Factors affecting behavioral phase transition in the desert locust,Schistocerca gregaria (Forskål) (Orthoptera: Acrididae).

The behavior of the desert locust,Schistocerca gregaria (Forskål) (Orthoptera: Acrididae), is adjusted rapidly to population density and is a phase characteristic. We used discriminant analysis to quantify the extent of phase transition from the solitary to the gregarious phase and accurately classify the phase status on the basis of two decisive behavioral parameters: nymphal activity and social interaction. Fecal extracts. examined by olfactometry, attract solitarious nymphs but do not contribute to behavioral phase transition. Neither do visual stimuli alone. Short-range olfaction of airborne volatiles slightly affects behavioral phase transition. Antennectomy abolishes density response. Cuticular lipid extract, containing presumptive contact pheromones, does not attract nymphs, but does significantly affect behavioral phase transition.

Mating induces an immune response and developmental switch in the Drosophila oviduct

Mating triggers physiological and behavioral changes in females. To understand how females effect these changes, we used microarray, proteomic, and comparative analyses to characterize gene expression in oviducts of mated and unmated Drosophila females. The transition from non-egg laying to egg laying elicits a distinct molecular profile in the oviduct. Immune-related transcripts and proteins involved in muscle and polarized epithelial function increase, whereas cell growth and differentiation-related genes are down-regulated. Our combined results indicate that mating triggers molecular and biochemical changes that mediate progression from a “poised” state to a mature, functional stage.

Mating-increases trypsin in female Drosophila hemolymph

Male-derived accessory gland proteins (Acps) are transferred to the female reproductive tract during mating and affect female reproductive maturation and behavior. Some Acps subsequently enter the female hemolymph. We hypothesized that humoral proteases are the primary effectors of Acp bioactivity by processing (activating) and/or degrading them. To test this hypothesis we examined the fate of one Acp, Drosophila melanogaster Sex Peptide (Acp70A, DrmSP), which possesses several putative serine-protease cleavage sites, in hemolymph of unmated and mated females. In D. melanogaster, DrmSP induces post-mating non-receptivity and enhances oogenesis. To determine if serine proteases regulate the duration of DrmSP activity in mated females, we performed kinetic analysis of cleavage of a synthetic N-terminal truncated DrmSP(8-36) (T-SP) with hemolymph of unmated versus mated females. We found that T-SP is cleaved more rapidly and completely in mated female hemolymph. Using LC-MS/MS analyses, we identified its primary cleavage sites, indicating that trypsin was the major endopeptidase regulating T-SP in hemolymph. This was verified in vitro by utilizing specific chromogenic serine-protease substrates and inhibitors. We propose that post-mating cleavage of DrmSP in the female hemolymph regulates the duration of the rapidly induced post-mating responses in D. melanogaster and that this is a specific example of Acp bioactivity regulated by hemolymph serine proteases.

Juvenile hormone and locust phase

Juvenile hormone (JH) is directly and indirectly involved in the determination of many phase characteristics of locusts. Temporal differences in JH titer and the biosynthetic capacity of excised corpora allata of solitary nymphs are correlated to some extent with the effects on them of treatment with JH or JH analogs. Juvenile hormone modulates cuticular melanization and the rate of reproductive maturation—specifically regulating vitellogenesis at the transcriptional level, and nonspecifically stimulating the translational capacity of the locust fat body. Juvenile hormone does not appear to be involved in behavioral phase transition of locusts. Long-term treatment of crowded nymphs with the JH analog methoprene does not lessen their gregarious behavior, and does not reduce hemolymph lipids or carbohydrates. Reducing endogenous JH levels of solitary nymphs by chemical allatectomy with precocene III does not induce gregarious behavior. However, methoprene does affect nymphal coloration. The relevance of these results to locust control is discussed. Arch. Insect Biochem. Physiol. 35:375–391, 1997.

Cuticular hydrocarbons control behavioural phase transition in Schistocerca gregaria nymphs and elicit biochemical responses in antennae

Abstract Cuticular lipid extracts have been shown to induce gregarious phase-related behaviour in Schistocerca gregaria solitarious nymphs. Analysis of various fractions of the cuticular lipids reveals that the behavioural response is specific to the hydrocarbon fraction. On exposure to cuticular hydocarbons, antennal preparations from crowded S. gregaria nymphs and adults generate a rapid and transient increase in the concentration of inositol trisphosphate (IP 3 ), whereas the level of cAMP is not affected. Antennal preparations from locust nymphs are more responsive to the hydrocarbon fraction than are preparations from adults. IP 3 generation is dose-dependent and species-specific. The notion that the cuticular hydrocarbons may interact with membrane receptors of antennal sensory cells is supported by the observation that hydrocarbon-induced IP 3 formation is significantly impaired in the presence of GDP-β-S, an inhibitor of trimeric G-protein function. These findings confirm the view that components of cuticular hydrocarbons function in locusts as contact pheromones, specifically inducing gregarious phase behaviour, and that they initially act via induction of IP 3

Tissue remodeling: a mating-induced differentiation program for the Drosophila oviduct

BackgroundIn both vertebrates and invertebrates, the oviduct is an epithelial tube surrounded by visceral muscles that serves as a conduit for gamete transport between the ovary and uterus. While Drosophila is a model system for tubular organ development, few studies have addressed the development of the flys oviduct. Recent studies in Drosophila have identified mating-responsive genes and proteins whose levels in the oviduct are altered by mating. Since many of these molecules (e.g. Muscle LIM protein 84B, Coracle, Neuroglian) have known roles in the differentiation of muscle and epithelia of other organs, mating may trigger similar differentiation events in the oviduct. This led us to hypothesize that mating mediates the last stages of oviduct differentiation in which organ-specific specializations arise.ResultsUsing electron- and confocal-microscopy we identified tissue-wide post-mating changes in the oviduct including differentiation of cellular junctions, remodeling of extracellular matrix, increased myofibril formation, and increased innervation. Analysis of once- and twice-mated females reveals that some mating-responsive proteins respond only to the first mating, while others respond to both matings.ConclusionWe uncovered ultrastructural changes in the mated oviduct that are consistent with the roles that mating-responsive proteins play in muscle and epithelial differentiation elsewhere. This suggests that mating triggers the late differentiation of the oviduct. Furthermore, we suggest that mating-responsive proteins that respond only to the first mating are involved in the final maturation of the oviduct while proteins that remain responsive to later matings are also involved in maintenance and ongoing function of the oviduct. Taken together, our results establish the oviduct as an attractive system to address mechanisms that regulate the late stages of differentiation and maintenance of a tubular organ.

Expression of a pheromone receptor in ovipositor sensilla of the female moth (Heliothis virescens).

Female moths release pheromones that influence various behavioral and physiological processes. The highly specific responses elicited by pheromones are mediated via specific chemosensory proteins, pheromone binding proteins and chemoreceptors, operating in the antennal sensory neurons. In Heliothis virescens, the response to the major pheromone component (Z)‐11‐hexadecenal (Z11‐16:Al) is mediated by the pheromone binding protein PBP2 and the receptor type HR13. PCR experiments revealed that transcripts for relevant chemosensory molecules are also present in the abdomen suggesting an additional role. In the female, mRNA for HR13 as well as for the related PBP2 was found in the ovipositor tip and in an immunohistochemical analysis with a specific antiserum it was possible to visualize the receptor protein in distinct sensilla types surrounding the ovipositor tip. The expression of HR13 implies a chemosensory responsiveness of these sensilla types to pheromones possibly provided by PBP2. Due to the close vicinity of sensillar HR13 cells and pheromone producing cells in the ovipositor we propose that the HR13 cells might mediate abdominal responses to the emitted pheromones.

Cuticular Surface Hydrocarbons of Desert Locust Nymphs, Schistocerca gregaria, and Their Effect on Phase Behavior

The quantity of cuticular hydrocarbons is higher in solitarious nymphs of the desert locust, Schistocerca gregaria, compared to gregarious nymphs, but the total hydrocarbon fraction of solitarious nymphs does not significantly divert behavioral transition of isolated nymphs to the gregarious phase, while gregarious hydrocarbon extracts do. This suggests that qualitative differences in composition are responsible for the biological effect. The profile of cuticular hydrocarbon components is similar in the two phases, but some peak ratios differ. Crowding of solitarious nymphs leads to rapid changes in the profile of the hydrocarbon fraction, suggesting that specific hydrocarbons are produced and secreted as a consequence. Isolating previously crowded nymphs has an opposite effect. The composition of cuticular hydrocarbons from the migratory locust, Locusta migratoria, which differs considerably from that of S. gregaria, does not induce the gregarious behavioral phase in solitarious nymphs of the latter.

Nature and Functions of Glands and Ducts in the Drosophila Reproductive Tract

Successful reproduction requires interactions between males and females at many levels: the organisms, their cells (the gametes), and their molecules. Among the latter, secreted products of male and female reproductive glands are especially important. These molecules are particularly well understood in Drosophila melanogaster, because of this insect’s excellent molecular genetic tools. Here, we discuss the biology of Drosophila reproductive glands, including their development, structure, and secreted products. These glands include important secretory centers, tissues that play roles in gamete maintenance and perhaps in modification, and organs that mediate dynamic transfer of gametes and molecules, and gamete support and/or discharge. Components of seminal fluid produced by male reproductive glands enter the female during mating. There, they interact with female proteins, neurons, and pathways to convert the female from a “poised” pre-mated state to an active post-mating state. This mated state is characterized by high levels of egg production, by sperm storage, and by post-mating behaviors related to re-mating, activity, and feeding. Female reproductive gland secretions include additional molecules important for sperm survival or egg transit. The interplay and coordination between male- and female-derived molecules is an area of intense study. Its conclusions are relevant to understanding reproduction in insects and, more broadly, in all animals, and as well as to questions about chemical communication, hormone biology and evolution.