Male Accessory Gland Secretions Modulate Female Post-Mating Behavior in the Moth Spodoptera litura

The study of male insects’ accessory gland (MAG) secretions will promote our understanding of reproductive strategies and their evolution, and will facilitate the development of new approaches for pest control. Here, we carried out a series of experiments to determine the functions of MAG secretions on modulating female post-mating behavior in the moth Spodoptera litura. Results showed that females injected with MAG secretions called and mated significantly less than controls in the night after treatment, which were independent of mechanical stimulation during mating and the presence of sperm. However, a successful mating resulted in a longer loss in sexual receptivity (lasting to the second night after mating). This study also demonstrated that MAG secretions not only triggered oviposition but also promoted egg development, which also were not dependent on mechanical stimulation during mating and the presence of sperm. MAG secretions also showed negative effect on female longevity, which may be because MAG secretions stimulate females to allocate more resources to egg development and oviposition, leaving fewer resources for survival. Results of this study also suggest that oviposition behaviors incur energy costs. The hypothesis that virgin females may conduct oosorption to prolong longevity is not supported.     

The Female Post-Mating Response Requires Genes Expressed in the Secondary Cells of the Male Accessory Gland in Drosophila melanogaster

Seminal proteins from the Drosophila male accessory gland induce post-mating responses (PMR) in females. The PMR comprise behavioral and physiological changes that include increased egg laying, decreased receptivity to courting males, and changes in the storage and use of sperm. Many of these changes are induced by a “sex peptide” (SP) and are maintained by SP’s binding to, and slow release from, sperm. The accessory gland contains two secretory cell types with distinct morphological and developmental characteristics. Products of these “main” and “secondary” cells work interdependently to induce and maintain the PMR. To identify individual genes needed for the morphology and function of secondary cells, we studied iab-6^cocu males, whose secondary cells have abnormal morphology and fail to provide products to maintain the PMR. By RNA-seq, we identified 77 genes that are downregulated by a factor of >5× in iab-6^cocu males. By functional assays and microscopy, we tested 20 candidate genes and found that at least 9 are required for normal storage and release of SP in mated females. Knockdown of each of these 9 genes consequently leads to a reduction in egg laying and an increase in receptivity over time, confirming a role for the secondary cells in maintaining the long-term PMR. Interestingly, only 1 of the 9 genes, CG3349, encodes a previously reported seminal fluid protein (Sfp), suggesting that secondary cells may perform essential functions beyond the production and modification of known Sfps. At least 3 of the 9 genes also regulate the size and/or abundance of secondary cell vacuoles, suggesting that the vacuoles’ contents may be important for the machinery used to maintain the PMR.     

A Novel Assay Reveals Hygrotactic Behavior in Drosophila

Humidity is one of the most important factors that determines the geographical distribution and survival of terrestrial animals. The ability to detect variation in humidity is conserved across many species. Here, we established a novel behavioral assay that revealed the thirsty Drosophila exhibits strong hygrotactic behavior, and it can locate water by detecting humidity gradient. In addition, exposure to high levels of moisture was sufficient to elicit proboscis extension reflex behavior in thirsty flies. Furthermore, we found that the third antennal segment was necessary for hygrotactic behavior in thirsty flies, while arista was required for the avoidance of moist air in hydrated flies. These results indicated that two types of hygroreceptor cells exist in Drosophila: one located in the third antennal segment that mediates hygrotactic behavior in thirst status, and the other located in arista which is responsible for the aversive behavior toward moist air in hydration status. Using a neural silencing screen, we demonstrated that synaptic output from the mushroom body α/β surface and posterior neurons was required for both hygrotactic behavior and moisture-aversive behavior.     

Sensory Integration Regulating Male Courtship Behavior in Drosophila

The courtship behavior of Drosophila melanogaster serves as an excellent model system to study how complex innate behaviors are controlled by the nervous system. To understand how the underlying neural network controls this behavior, it is not sufficient to unravel its architecture, but also crucial to decipher its logic. By systematic analysis of how variations in sensory inputs alter the courtship behavior of a naïve male in the single-choice courtship paradigm, we derive a model describing the logic of the network that integrates the various sensory stimuli and elicits this complex innate behavior. This approach and the model derived from it distinguish (i) between initiation and maintenance of courtship, (ii) between courtship in daylight and in the dark, where the male uses a scanning strategy to retrieve the decamping female, and (iii) between courtship towards receptive virgin females and mature males. The last distinction demonstrates that sexual orientation of the courting male, in the absence of discriminatory visual cues, depends on the integration of gustatory and behavioral feedback inputs, but not on olfactory signals from the courted animal. The model will complement studies on the connectivity and intrinsic properties of the neurons forming the circuitry that regulates male courtship behavior.     

The Circadian Clock Protein Timeless Regulates Phagocytosis of Bacteria in Drosophila

Survival of bacterial infection is the result of complex host-pathogen interactions. An often-overlooked aspect of these interactions is the circadian state of the host. Previously, we demonstrated that Drosophila mutants lacking the circadian regulatory proteins Timeless (Tim) and Period (Per) are sensitive to infection by S. pneumoniae. Sensitivity to infection can be mediated either by changes in resistance (control of microbial load) or tolerance (endurance of the pathogenic effects of infection). Here we show that Tim regulates resistance against both S. pneumoniae and S. marcescens. We set out to characterize and identify the underlying mechanism of resistance that is circadian-regulated. Using S. pneumoniae, we found that resistance oscillates daily in adult wild-type flies and that these oscillations are absent in Tim mutants. Drosophila have at least three main resistance mechanisms to kill high levels of bacteria in their hemolymph: melanization, antimicrobial peptides, and phagocytosis. We found that melanization is not circadian-regulated. We further found that basal levels of AMP gene expression exhibit time-of-day oscillations but that these are Tim-independent; moreover, infection-induced AMP gene expression is not circadian-regulated. We then show that phagocytosis is circadian-regulated. Wild-type flies exhibit up-regulated phagocytic activity at night; Tim mutants have normal phagocytic activity during the day but lack this night-time peak. Tim appears to regulate an upstream event in phagocytosis, such as bacterial recognition or activation of phagocytic hemocytes. Interestingly, inhibition of phagocytosis in wild type flies results in survival kinetics similar to Tim mutants after infection with S. pneumoniae. Taken together, these results suggest that loss of circadian oscillation of a specific immune function (phagocytosis) can have significant effects on long-term survival of infection.     

A Motor Function for the DEAD-Box RNA Helicase,Gemin3, in Drosophila

The survival motor neuron (SMN) protein, the determining factor for spinal muscular atrophy (SMA), is complexed with a group of proteins in human cells. Gemin3 is the only RNA helicase in the SMN complex. Here, we report the identification of Drosophila melanogaster Gemin3 and investigate its function in vivo. Like in vertebrates, Gemin3 physically interacts with SMN in Drosophila. Loss of function of gemin3 results in lethality at larval and/or prepupal stages. Before they die, gemin3 mutant larvae exhibit declined mobility and expanded neuromuscular junctions. Expression of a dominant-negative transgene and knockdown of Gemin3 in mesoderm cause lethality. A less severe Gemin3 disruption in developing muscles leads to flightless adults and flight muscle degeneration. Our findings suggest that Drosophila Gemin3 is required for larval development and motor function.     

The Effect of Stress on Motor Function in Drosophila

Exposure to unpredictable and uncontrollable conditions causes animals to perceive stress and change their behavior. It is unclear how the perception of stress modifies the motor components of behavior and which molecular pathways affect the behavioral change. In order to understand how stress affects motor function, we developed an experimental platform that quantifies walking motions in Drosophila. We found that stress induction using electrical shock results in backwards motions of the forelegs at the end of walking strides. These leg retrogressions persisted during repeated stimulation, although they habituated substantially. The motions also continued for several strides after the end of the shock, indicating that stress induces a behavioral aftereffect. Such aftereffect could also be induced by restricting the motion of the flies via wing suspension. Further, the long-term effects could be amplified by combining either immobilization or electric shock with additional stressors. Thus, retrogression is a lingering form of response to a broad range of stressful conditions, which cause the fly to search for a foothold when it faces extreme and unexpected challenges. Mutants in the cAMP signaling pathway enhanced the stress response, indicating that this pathway regulates the behavioral response to stress. Our findings identify the effect of stress on a specific motor component of behavior and define the role of cAMP signaling in this stress response.     

A New Chamber for Studying the Behavior of Drosophila

Methods available for quickly and objectively quantifying the behavioral phenotypes of the fruit fly, Drosophila melanogaster, lag behind in sophistication the tools developed for manipulating their genotypes. We have developed a simple, easy-to-replicate, general-purpose experimental chamber for studying the ground-based behaviors of fruit flies. The major innovative feature of our design is that it restricts flies to a shallow volume of space, forcing all behavioral interactions to take place within a monolayer of individuals. The design lessens the frequency that flies occlude or obscure each other, limits the variability in their appearance, and promotes a greater number of flies to move throughout the center of the chamber, thereby increasing the frequency of their interactions. The new chamber design improves the quality of data collected by digital video and was conceived and designed to complement automated machine vision methodologies for studying behavior. Novel and improved methodologies for better quantifying the complex behavioral phenotypes of Drosophila will facilitate studies related to human disease and fundamental questions of behavioral neuroscience.     

The Gemin Associates of Survival Motor Neuron Are Required for Motor Function in Drosophila

Membership of the survival motor neuron (SMN) complex extends to nine factors, including the SMN protein, the product of the spinal muscular atrophy (SMA) disease gene, Gemins 2–8 and Unrip. The best-characterised function of this macromolecular machine is the assembly of the Sm-class of uridine-rich small nuclear ribonucleoprotein (snRNP) particles and each SMN complex member has a key role during this process. So far, however, only little is known about the function of the individual Gemin components in vivo. Here, we make use of the Drosophila model organism to uncover loss-of-function phenotypes of Gemin2, Gemin3 and Gemin5, which together with SMN form the minimalistic fly SMN complex. We show that ectopic overexpression of the dead helicase Gem3^ΔN mutant or knockdown of Gemin3 result in similar motor phenotypes, when restricted to muscle, and in combination cause lethality, hence suggesting that Gem3^ΔN overexpression mimics a loss-of-function. Based on the localisation pattern of Gem3^ΔN, we predict that the nucleus is the primary site of the antimorphic or dominant-negative mechanism of Gem3^ΔN-mediated interference. Interestingly, phenotypes induced by human SMN overexpression in Drosophila exhibit similarities to those induced by overexpression of Gem3^ΔN. Through enhanced knockdown we also uncover a requirement of Gemin2, Gemin3 and Gemin5 for viability and motor behaviour, including locomotion as well as flight, in muscle. Notably, in the case of Gemin3 and Gemin5, such function also depends on adequate levels of the respective protein in neurons. Overall, these findings lead us to speculate that absence of any one member is sufficient to arrest the SMN-Gemins complex function in a nucleocentric pathway, which is critical for motor function in vivo.