Interactive parallel sex pheromone circuits that promote and suppress courtship behaviors in the cockroach.

Many animals use multicomponent sex pheromones for mating, but the specific function and neural processing of each pheromone component remain unclear. The cockroach Periplaneta americana is a model for studying sex pheromone communication, and an adult female emits major and minor sex pheromone components, periplanone-B and -A (PB and PA), respectively. Attraction and courtship behaviors (wing-raising and abdominal extension) are strongly expressed when adult males are exposed to PB but weakly expressed when they are exposed to PA. When major PB is presented together with minor PA, behaviors elicited by PB were impaired, indicating that PA can both promote and suppress courtship behaviors depending on the pheromonal context. In this study, we identified the receptor genes for PA and PB and investigated the effects of knocking down each receptor gene on the activities of PA- and PB-responsive sensory neurons (PA- and PB-SNs), and their postsynaptic interneurons, and as well as effects on courtship behaviors in males. We found that PB strongly and PA weakly activate PB-SNs and their postsynaptic neurons, and activation of the PB-processing pathway is critical for the expression of courtship behaviors. PA also activates PA-SNs and the PA-processing pathway. When PA and PB are simultaneously presented, the PB-processing pathway undergoes inhibitory control by the PA-processing pathway, which weakens the expression of courtship behaviors. Our data indicate that physiological interactions between the PA- and PB-processing pathways positively and negatively mediate the attraction and courtship behaviors elicited by sex pheromones.

A Closer Look at Histamine in Drosophila.

The present work intends to provide a closer look at histamine in Drosophila. This choice is motivated firstly because Drosophila has proven over the years to be a very simple, but powerful, model organism abundantly assisting scientists in explaining not only normal functions, but also derangements that occur in higher organisms, not excluding humans. Secondly, because histamine has been demonstrated to be a pleiotropic master molecule in pharmacology and immunology, with increasingly recognized roles also in the nervous system. Indeed, it interacts with various neurotransmitters and controls functions such as learning, memory, circadian rhythm, satiety, energy balance, nociception, and motor circuits, not excluding several pathological conditions. In view of this, our review is focused on the knowledge that the use of Drosophila has added to the already vast histaminergic field. In particular, we have described histamine's actions on photoreceptors sustaining the visual system and synchronizing circadian rhythms, but also on temperature preference, courtship behavior, and mechanosensory transmission. In addition, we have highlighted the pathophysiological consequences of mutations on genes involved in histamine metabolism and signaling. By promoting critical discussion and further research, our aim is to emphasize and renew the importance of histaminergic research in biomedicine through the exploitation of Drosophila, hopefully extending the scientific debate to the academic, industry, and general public audiences.

Neural thyroid hormone metabolism integrates seasonal changes in environmental temperature with the neuroendocrine reproductive axis.

We asked if environmental temperature alters thyroid hormone metabolism within the hypothalamus, thereby providing a neuroendocrine mechanism by which temperature could be integrated with photoperiod to regulate seasonal rhythms. We used immunohistochemistry to assess the effects of low-temperature winter dormancy at 4 °C or 12 °C on thyroid-stimulating hormone (TSH) within the infundibulum of the pituitary as well as deiodinase 2 (Dio2) and 3 (Dio3) within the hypothalamus of red-sided garter snakes (Thamnophis sirtalis). Both the duration and, in males, magnitude of low-temperature dormancy altered deiodinase immunoreactivity within the hypothalamus, increasing the area of Dio2-immunoreactivity in males and females and decreasing the number of Dio3-immunoreactive cells in males after 8-16 weeks. Reciprocal changes in Dio2/3 favor the accumulation of triiodothyronine within the hypothalamus. Whether TSH mediates these effects requires further study, as significant changes in TSH-immunoreactive cell number were not observed. Temporal changes in deiodinase immunoreactivity coincided with an increase in the proportion of males exhibiting courtship behavior as well as changes in the temporal pattern of courtship behavior after emergence. Our findings mirror those of previous studies, in which males require low-temperature exposure for at least 8 weeks before significant changes in gonadotropin-releasing hormone immunoreactivity and sex steroid hormones are observed. Collectively, these data provide evidence that the neuroendocrine pathway regulating the reproductive axis via thyroid hormone metabolism is capable of transducing temperature information. Because all vertebrates can potentially use temperature as a supplementary cue, these results are broadly applicable to understanding how environment-organism interactions mediate seasonally adaptive responses.

An Improved Chromosome-Level Genome Assembly of the Firefly Pyrocoelia pectoralis.

The endemic and endangered Chinese firefly Pyrocoelia pectoralis is a sexually dimorphic, nocturnal species. A previous attempt by this team to assemble a draft genome of P. pectoralis using PacBio and Illumina HiSeq X Ten platforms was limited in its usefulness by high redundancy and contamination. This prompted us to conduct an improved chromosome-level genome assembly of P. pectoralis. Ten chromosomes were further assembled based on Hi-C data to a 532.25 Mb final size with a 52.87 Mb scaffold N50. The total repeat lengths in the genome of P. pectoralis amount to 227.69 Mb; 42.78%. In total, 12,789 genes could be functionally annotated using at least one public database. Phylogenetic inference indicated that P. pectoralis and P. pyralis diverged ~51.41 million years ago. Gene family expansion and contraction analysis of 12 species were performed, and 546 expanded and 2660 contracted gene families were identified in P. pectoralis. We generated a high-quality draft of the P. pectoralis genome. This genome assembly should help promote research on the species' sexual dimorphism and its unique courtship behavior, which involves a combination of pheromonal and bioluminescent signals. It also can serve as a resource for accelerating genome-assisted improvements in the conservation of this species.

n-octyl acrylate is a candidate sex pheromone component involved in courtship in parasitoid wasp Microplitis mediator.

Sex pheromones are considered to play critical roles in partner communication of most parasitic Hymenoptera. However, the identification of sex pheromone components remains limited to a few families of parasitoid wasps. In this study, we functionally characterized a candidate sex pheromone component in Microplitis mediator (Hymenoptera: Braconidae), a solitary parasitoid of Noctuidae insects. We found that the body surface extract from female wasps could significantly stimulate courtship behavior of males. Gas chromatography-electroantennographic detection (GC-EAD) analysis revealed that a candidate semiochemical from extract triggered significant electrophysiological response of antennae of males. By performing gas chromatography-mass spectrometer (GC-MS) measurement, GC-EAD active compound was identified as n-octyl acrylate, a candidate sex pheromone component in female M. mediator. In electroantennogram (EAG) tests, antennae of male wasps showed significantly higher electrophysiological responses to n-octyl acrylate than those of females. Y-tube olfactometer assays indicated that male wasps significantly chose n-octyl acrylate compared with the control. Furthermore, male wasps showed a remarkable preference for n-octyl acrylate in a simulated field condition behavioral trial; simultaneously, n-octyl acrylate standard could also trigger significant courtship behavior in males. We propose that n-octyl acrylate, as a candidate vital sex pheromone component, could be utilized to design behavioral regulators of M. mediator to implement the protection and utilization of natural enemies.

Transcriptional profiling of Drosophila male-specific P1 (pC1) neurons.

In Drosophila melanogaster, the P1 (pC1) cluster of male-specific neurons both integrates sensory cues and drives or modulates behavioral programs such as courtship, in addition to contributing to a social arousal state. The behavioral function of these neurons is linked to the genes they express, which underpin their capacity for synaptic signaling, neuromodulation, and physiology. Yet, P1 (pC1) neurons have not been fully characterized at the transcriptome level. Moreover, it is unknown how the molecular landscape of P1 (pC1) neurons acutely changes after flies engage in social behaviors, where baseline P1 (pC1) neural activity is expected to increase. To address these two gaps, we use single cell-type RNA sequencing to profile and compare the transcriptomes of P1 (pC1) neurons harvested from socially paired versus solitary male flies. Compared to control transcriptome datasets, we find that P1 (pC1) neurons are enriched in 2,665 genes, including those encoding receptors, neuropeptides, and cell-adhesion molecules (dprs/DIPs). Furthermore, courtship is characterized by changes in ~300 genes, including those previously implicated in regulating behavior (e.g. DopEcR, Octß3R, Fife, kairos, rad). Finally, we identify a suite of genes that link conspecific courtship with the innate immune system. Together, these data serve as a molecular map for future studies of an important set of higher-order and sexually-dimorphic neurons.

Knock-out of vasotocin reduces reproductive success in female zebrafish, Danio rerio.

The vertebrate nonapeptide vasotocin/vasopressin is evolutionarily highly conserved and acts as neuromodulator and endocrine/paracrine signaling molecule. Circumstantial and mechanistic evidence from pharmacological manipulations of the vasotocin system in several teleost fishes suggest sex- and species-specific reproductive roles of vasotocin. While effects of vasotocin on teleost reproductive physiology involve both courtship behaviors and the regulation of the hypothalamic-pituitary-gonadal (HPG) axes, comprehensive studies investigating behavioral and physiological reproductive consequences of genetic ablation of vasotocin in a genetically tractable fish model, such as the zebrafish, are currently lacking. Here, we report the generation of homozygous CRISPR/Cas9-based vasotocin gene knock-out zebrafish. Breeding pairs of vasotocin knock-out fish produce significantly fewer fertilized eggs per clutch compared to wildtype fish, an effect coincident with reduced female quivering courtship behavior. Crossbreeding experiments reveal that this reproductive phenotype is entirely female-dependent, as vasotocin-deficient males reproduce normally when paired with female wild-type fish. Histological analyses of vasotocin knock-out ovaries revealed an overall reduction in oocytes and differential distribution of oocyte maturation stages, demonstrating that the reproductive phenotype is linked to oocyte maturation and release. Ovarian hormone quantification and gene expression analysis in mutant fish indicated reduced synthesis of Prostaglandin F2α, a hormone involved in ovarian maturation, egg release and regulation of female courtship behavior in some cyprinids. However, acute injection of vasotocin did not rescue the female mutant reproductive phenotype, suggesting a contribution of organizational effects of vasotocin. Together, this study provides further support for emerging roles of vasotocin in female teleost reproduction in an important teleost model species.

Visual feedback neurons fine-tune Drosophila male courtship via GABA-mediated inhibition.

Many species of animals use vision to regulate their social behaviors. However, the molecular and circuit mechanisms underlying visually guided social interactions remain largely unknown. Here, we show that the Drosophila ortholog of the human GABAA-receptor-associated protein (GABARAP) is required in a class of visual feedback neurons, lamina tangential (Lat) cells, to fine-tune male courtship. GABARAP is a ubiquitin-like protein that maintains cell-surface levels of GABAA receptors. We demonstrate that knocking down GABARAP or GABAAreceptors in Lat neurons or hyperactivating them induces male courtship toward other males. Inhibiting Lat neurons, on the other hand, delays copulation by impairing the ability of males to follow females. Remarkably, the fly GABARAP protein and its human ortholog share a strong sequence identity, and the fly GABARAP function in Lat neurons can be rescued by its human ortholog. Using in vivo two-photon imaging and optogenetics, we reveal that Lat neurons are functionally connected to neural circuits that mediate visually guided courtship pursuits in males. Our work identifies a novel physiological function for GABARAP in regulating visually guided courtship pursuits in Drosophila males. Reduced GABAA signaling has been linked to social deficits observed in the autism spectrum and bipolar disorders. The functional similarity between the human and the fly GABARAP raises the possibility of a conserved role for this gene in regulating social behaviors across insects and mammals.

Cell assembly analysis of neural circuits for innate behavior in Drosophila melanogaster using an immediate early gene stripe/egr-1.

Innate behavior, such as courtship behavior, is controlled by a genetically defined set of neurons. To date, it remains challenging to visualize and artificially control the neural population that is active during innate behavior in a whole-brain scale. Immediate early genes (IEGs), whose expression is induced by neural activity, can serve as powerful tools to map neural activity in the animal brain. We screened for IEGs in vinegar fly Drosophila melanogaster and identified stripe/egr-1 as a potent neural activity marker. Focusing on male courtship as a model of innate behavior, we demonstrate that stripe-GAL4-mediated reporter expression can label fruitless (fru)-expressing neurons involved in courtship in an activity (experience)-dependent manner. Optogenetic reactivation of the labeled neurons elicited sexual behavior in males, whereas silencing of the labeled neurons suppressed courtship and copulation. Further, by combining stripe-GAL4-mediated reporter expression and detection of endogenous Stripe expression, we established methods that can label neurons activated under different contexts in separate time windows in the same animal. The cell assembly analysis of fru neural population in males revealed that distinct groups of neurons are activated during interactions with a female or another male. These methods will contribute to building a deeper understanding of neural circuit mechanisms underlying innate insect behavior.

The Effect of Chromosomes on Courtship Behavior in Sibling Species of the Drosophila virilis Group.

Prezygotic isolation mechanisms, particularly courtship behavior, play a significant role in the formation of reproductive barriers. The action of these mechanisms leads to the coexistence of numerous closely related insect species with specific adaptations in a shared or adjacent territory. The genetic basis of these mechanisms has been studied using closely related Drosophila species, such as the D. virilis group. However, the investigation of individual courtship behavior elements has been limited until recently, and the effect of genotype on the species-specific features of courtship as a whole has not been thoroughly examined. It should be noted that courtship behavior is not a typical quantitative trait that can be easily measured or quantified in both females and males, similar to traits like wing length or bristle number. Each courtship element involves the participation of both female and male partners, making the genetic analysis of this behavior complex. As a result, the traditional approach of genetic analysis for quantitative traits, which involves variance decomposition in a set of crosses, including parental species, F1 and F2 hybrids, and backcrosses of F1 to parental species, is not suitable for analyzing courtship behavior. To address this, we employed a modified design by introducing what we refer to as 'reference partners' during the testing of hybrid individuals from F1, F2, and backcrosses. These reference partners represented one of the parental species. This approach allowed us to categorize all possible test combinations into four groups based on the reference partner's sex (female or male) and their constant genotype towards one of the parental species (D. virilis or D. americana). The genotype of the second partner in the within-group test combinations varied from completely conspecific to completely heterospecific, based on the parental chromosomal sets. To assess the contribution of partner genotypes to the variability of courtship-element parameters, we employed structural equation modeling (SEM) instead of the traditional analysis of variance (ANOVA). SEM enabled us to estimate the regression of the proportion of chromosomes of a specific species type on the value of each courtship-element parameter in partners with varying genotypes across different test combinations. The aim of the current study was to analyze the involvement of sex chromosomes and autosomes in the formation of courtship structure in D. virilis and D. americana. The genetic analysis was complemented by video recording and formalization of courtship-ritual elements. D. virilis was found to be more sensitive to mate stimuli compared to D. americana. The majority of species-specific parameters, such as latency and duration of courtship elements (e.g., male and female song, following, licking, and circling), were shown to be influenced by the D. virilis genotype. However, not all of these parameters significantly impact copulation success, with the male song, licking, and following being the most significant. In females, the female song was found to have a significant relationship only with copulation duration. The influence of the female genotype on the species-specific parameters of courtship elements is primarily related to autosomes, while the male genotype is associated with the X chromosomes. The study suggests that sexual selection primarily occurs through acoustic and chemoreceptor channels.

Loss of gdnfa disrupts spermiogenesis and male courtship behavior in zebrafish.

Spermatogenesis is essential for establishment and maintenance of reproduction in male vertebrates. Spermatogenesis, which is mainly regulated by the combined action of hormones, growth factors, and epigenetic factors, is highly conserved. Glial cell line-derived neurotrophic factor (GDNF) is a member of the transforming growth factor-ß superfamily. In this study, global gdnfa knockout and Tg (gdnfa: mcherry) transgenic zebrafish lines were generated. Loss of gdnfa resulted in disorganized testes, decreased gonadosomatic index, and low percentage of mature spermatozoa. In the Tg (gdnfa: mcherry) zebrafish line, we found that gdnfa was expressed in Leydig cells. The mutation in gdnfa significantly decreased Leydig cell marker gene expression and androgen secretion in Leydig cells. In addition, courtship behavior was disrupted in the male mutants. We present in vivo data showing that global knockout of gdnfa disrupts spermiogenesis and male courtship behavior in zebrafish. The first viable vertebrate model with a global gdnfa knockout may be valuable for studying the role of GDNF in animal reproduction.

Ornaments indicate parasite load only if they are dynamic or parasites are contagious.

Choosing to mate with an infected partner has several potential fitness costs, including disease transmission and infection-induced reductions in fecundity and parental care. By instead choosing a mate with no, or few, parasites, animals avoid these costs and may also obtain resistance genes for offspring. Within a population, then, the quality of sexually selected ornaments on which mate choice is based should correlate negatively with the number of parasites with which a host is infected ("parasite load"). However, the hundreds of tests of this prediction yield positive, negative, or no correlation between parasite load and ornament quality. Here, we use phylogenetically controlled meta-analysis of 424 correlations from 142 studies on a wide range of host and parasite taxa to evaluate explanations for this ambiguity. We found that ornament quality is weakly negatively correlated with parasite load overall, but the relationship is more strongly negative among ornaments that can dynamically change in quality, such as behavioral displays and skin pigmentation, and thus can accurately reflect current parasite load. The relationship was also more strongly negative among parasites that can transmit during sex. Thus, the direct benefit of avoiding parasite transmission may be a key driver of parasite-mediated sexual selection. No other moderators, including methodological details and whether males exhibit parental care, explained the substantial heterogeneity in our data set. We hope to stimulate research that more inclusively considers the many and varied ways in which parasites, sexual selection, and epidemiology intersect.

Sexual dysfunction precedes motor defects, dopaminergic neuronal degeneration, and impaired dopamine metabolism: Insights from Drosophila model of Parkinson's disease.

Sexual dysfunction (SD) is one of the most common non-motor symptoms of Parkinson's disease (PD) and remains the most neglected, under-reported, and under-recognized aspect of PD. Studies have shown that Dopamine (DA) in the hypothalamus plays a role in regulating sexual behavior. But the detailed mechanism of SD in PD is not known. Drosophila melanogaster shares several genes and signaling pathways with humans which makes it an ideal model for the study of a neurodegenerative disorder such as PD. Courtship behavior of Drosophila is one such behavior that is closely related to human sexual behavior and so plays an important role in understanding sexual behavior in diseased conditions as well. In the present study, a sporadic SD model of PD using Drosophila was developed and SD phenotype was observed based on abnormalities in courtship behavior markers. The Drosophila SD model was developed in such a way that at the window of neurotoxin paraquat (PQ) treatment [PQ is considered a crucial risk factor for PD due to its structural similarity with 1-methyl-4-phenyl pyridinium (MPP+), the active form of PD-inducing agent, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)], it does not exhibit mobility defects but shows SD. The whole brain tyrosine hydroxylase immunostaining showed no observable dopaminergic (DAergic) degeneration (number of DA neurons and fluorescence intensity of fluorescently labeled secondary antibodies that target anti-TH primary antibody) of the SD model. Similarly, there was no significant depletion of brain DA and its metabolite levels (HVA and DOPAC) as determined using HPLC-ECD (High-Performance Liquid Chromatography using Electrochemical Detector). The present study illustrates that the traits associated with courtship and sexual activity provide sensitive markers at the earlier stage of PD onset. This PQ-induced SD fly model throws an opportunity to decipher the molecular basis of SD under PD conditions and to screen nutraceuticals/potential therapeutic molecules to rescue SD phenotype and further to DAergic neuroprotection.

Olfactory receptor OR52N2 for PGE2 in mediation of guppy courtship behaviors.

Prostaglandins (PGs) are a type of physiologically active unsaturated fatty acids. As an important sex pheromone, PGs play a vital role in regulating the reproductive behaviors of species by mediating nerve and endocrine responses. In this study, guppy (Poecilia reticulate) was used as the model specie to detect the function of PGE2 in inducing the onset of courtship behaviors. Our results showed that adding PGE2 into the water environment could activate the courtship behavior of male guppy, indicating that the peripheral olfactory system mediated the PGE2 function. Thereafter, the open reading frame (ORF) of olfactory receptor or52n2 was cloned, which was 936 bp in length, coding 311 amino acids. As a typical G protein-coupled receptor, OR52N2 had a conservative seven α-helix transmembrane domains. To confirm the regulatory relationship between OR52N2 and PGE2, dual-luciferase reporter assay was employed to verify the activation of downstream CREB signaling pathways. Results showed that PGE2 significantly enhanced CRE promoter activity in or52n2 ORF transient transfected HEK-293 T cells. Finally, localization of or52n2 mRNA were observed in ciliated receptor cells of the olfactory epithelium using in situ hybridization. Our results provide a novel insight into sex pheromone signaling transduction in reproductive behavior.

Bridging conservation across the ex situ-in situ spectrum: Insights into the reproductive ecology of the threatened narrow-headed gartersnake (Thamnophis rufipunctatus).

Zoo-based (ex situ) conservation breeding programs provide invaluable opportunities to uncover enigmatic behaviors and traits of focal species under managed care, which can support research and conservation management efforts. A suite of factors and a limited range have yielded population declines in the threatened narrow-headed gartersnake (Thamnophis rufipunctatus). Better understanding its cryptic ecology and life history (e.g., reproductive ecology) offers conservation benefits. We analyzed data on courtship behavior, parity and litter size, offspring size, and neonatal growth from an ex situ T. rufipunctatus population at the Phoenix Zoo from 2009 to 2018. Courtship behavior and parturition phenology are likely linked with the North American monsoon season, yet the courtship window may be wider than realized. We document the first instances of interannual iteroparity and multigenerational rearing of successful breeders at the ex situ level. Litter sizes varied but were relative to maternal body mass, suggesting that fecundity may be driven by intrinsic condition (e.g., age and size) of breeding females. Mean offspring body masses were equivalent between sexes, and neonate growth trends were quadratic during their first 9 months. Sexual dimorphism became apparent around 4-5 months age. Much of these data are novel for T. rufipunctatus and provide insight into their reproductive ecology. Phenology of reproductive ecology and body size metrics can guide field surveillance, age estimations, and population ecology monitoring, as well as inform ex situ adaptive management practices. Strategies spanning the ex situ-in situ spectrum are applicable to other imperiled taxa to better inform conservation management decisions.

Eat Better to Look Better: The Relationship between Food Availability, UV Brightness of the Major Claw, and Mating Success of the Fiddler Crab Austruca lactea.

Many factors affect male fiddler crab courting and female choice during underground mating, including claw-waving patterns, claw-waving frequency, hood structures, burrow quality, and visible and ultraviolet signals of the major claw. Under food-limited conditions, fiddler crabs decrease their investments in reproduction-e.g., lower their claw-waving frequency. However, the effect of food availability on the visible or ultraviolet signals important for courtship success is unknown, not only for fiddler crabs, but for crustaceans in general. This is the first study to explore the influence of food availability on the early stages of female choice in Austruca lactea. In the present study, we tested (1) the effect of food availability on the UV brightness of A. lactea's major claw and burrow quality and (2) the correlation between A. lactea courtship success and the UV brightness of its major claw. Our results showed that higher food availability increased the UV brightness of the major claw and enhanced the male's burrow quality. The UV component to the signal is important for courtship success. The males with a UV component of signal on their major claw had a higher courtship success. This is an indication that food availability may affect the attractiveness of male fiddler crabs.

The masculinization steroid milieu caused by fluorene-9-bisphenol disrupts sex-typical courtship behavior in female zebrafish (Danio rerio).

In vertebrates, the behavior of congenital sex differences between males and females is highly dependent on steroid signals and hormonal milieu. The presence of endocrine disrupting chemicals (EDCs) in the environment generally plays a similar role to sex hormones, so its interference with aquatic organism population stability can not be ignored and is worth studying. Fluorene-9-bisphenol (BHPF) has been clarified as an endocrine disruptor on organisms by several studies but its mechanism in perturbation of courtship behavior of female zebrafish is not clear. Here, we proposed an automated multi-zebrafish tracking method quantifying the courtship process and reported that zebrafish females exposed to BHPF, are not receptive to males but rather court females, and lose normal ovarian function with an altered sex steroid milieu. Our results showed that BHPF damaged 17ß-estradiol synthesis by down-regulation of sox3 and cyp19a1a, linking apoptosis with ovary development and female fecundity. The down-regulated expression of estrogen signaling through an estrogen receptor, esr2b, caused the induction of masculinization of female courtship behavior and sexual preference in zebrafish females after BHPF treatment. This process might be mediated by inhibiting the transcription of a neuropeptide B (npb) in the brain. Our study reveals that the estrogen signaling pathway may play an important role in classical courtship behavior and sexual preference of zebrafish. This study provided evidence that anti-estrogenic chemical exposure caused adverse effects on the regulation of the brain-gonad-estrogen axis of aquatic organisms, which should be of concern and highlighted the importance of controlling environmental contamination.

HGT is widespread in insects and contributes to male courtship in lepidopterans.

Horizontal gene transfer (HGT) is an important evolutionary force shaping prokaryotic and eukaryotic genomes. HGT-acquired genes have been sporadically reported in insects, a lineage containing >50% of animals. We systematically examined HGT in 218 high-quality genomes of diverse insects and found that they acquired 1,410 genes exhibiting diverse functions, including many not previously reported, via 741 distinct transfers from non-metazoan donors. Lepidopterans had the highest average number of HGT-acquired genes. HGT-acquired genes containing introns exhibited substantially higher expression levels than genes lacking introns, suggesting that intron gains were likely involved in HGT adaptation. Lastly, we used the CRISPR-Cas9 system to edit the prevalent unreported gene LOC105383139, which was transferred into the last common ancestor of moths and butterflies. In diamondback moths, males lacking LOC105383139 courted females significantly less. We conclude that HGT has been a major contributor to insect adaptation.

Role of Melatonin in Temperature-Induced Activation of the Neuroendocrine Reproductive Axis in Garter Snakes.

An animal's ability to respond optimally to changing environmental conditions is paramount to successfully reproducing and thus maximizing fitness. Studies on photoperiod-induced changes in neural thyroid hormone metabolism have conclusively linked environmental cues to the neuroendocrine reproductive axis of birds and mammals. Whether this conserved mechanism also transduces changes in environmental temperature, however, has not been fully addressed. We investigated whether the hormone melatonin mediates the effects of low-temperature dormancy on thyroid hormone metabolism within the hypothalamus of red-sided garter snakes (Thamnophis sirtalis parietalis). To address this question, we used immunohistochemistry to assess changes in thyroid-stimulating hormone (TSH) in the infundibulum of the pituitary and deiodinase 3 (Dio3) and gonadotropin-releasing hormone (GnRH) in the hypothalamus. We also asked if changes in TSH, Dio3, and/or GnRH immunoreactivity are associated with changes in male courtship behavior. In contrast to our predictions, 6 weeks of dormancy at 4°C significantly decreased the number of TSH-labeled cells in the infundibulum. It is possible that the observed decrease in TSH is related to the release of snakes from temperature refractoriness, but this idea needs further testing. Treatment of snakes with the melatonin precursor 5-hydroxytryptophan during dormancy at 4°C both reversed the temperature-induced change in TSH immunoreactivity and disrupted the temporal pattern of male courtship behavior. These results suggest that TSH cells within the infundibulum are both modulated by temperature and sensitive to changes in melatonin. As predicted, male snakes hibernated at an elevated temperature of 12°C for 6 weeks and treated with vehicle showed no change in TSH-, Dio3-, or GnRH-immunoreactive cell number. Treatment of snakes with the melatonin receptor antagonist luzindole was not sufficient in rescuing the effects of dormancy at 12°C on TSH immunoreactivity or courtship behavior. However, luzindole-treated snakes showed a significant increase in GnRH-immunoreactive cell number, suggesting that melatonin exerts an inhibitory effect on GnRH in garter snakes. In summary, our results provide critical insights into the mechanisms that mediate the effects of temperature on reproductive physiology and behavior.

Lateralization of Courtship Traits Impacts Pentatomid Male Mating Success-Evidence from Field Observations.

Lateralization has been documented in many insect species, but limited information on courtship and mating lateralization in wild conditions is available. We conducted field investigation on the courtship and mating behavior of the neem bug, Halys dentatus, a polyphagous insect mainly infesting Azadirachta indica, with particular attention to lateralization of mating displays. We investigated the presence of population-level behavioral asymmetries during H. dentatus sexual interactions and their influence on male mating success. Two lateralized traits were found: left or right-biased male approaches to the female and left or right-biased male turning displays. Males approaching females from their left side were mainly right-biased in the 180° turning display, and males that approached females from their right side were mainly left-biased. Right-biased males by turning 180° to carry out end-to-end genital contact, performed a lower number of copulation attempts, thus starting copula earlier than left-biased males. Mating success was higher when males approached the left side of females during sexual interactions. A higher number of successful mating interactions was observed in right-biased males when turning 180°. Our results add useful knowledge on the reproductive behavior of H. dentatus in the field, with potential applications for identifying useful benchmarks to monitor the quality of individuals mass-reared for pest control purposes over time.