Comparative aspects of the internal reproductive system of males in species of Melolonthinae, Dynastinae, and Rutelinae (Coleoptera: Scarabaeoidea) from Mexico

The anatomy of the internal male reproductive systems of 12 species of Melolonthinae (Phyllophaga, Chlaenobia, Macrodactylus, Isonychus), six species of Dynastinae (Cyclocephala), and three species of Rutelinae (Paranomala) (Coleoptera, Scarabaeoidea) of Mexico are described. A total of 350 male specimens representing 21 species were collected. From each species, the reproductive systems were obtained by micro-dissection, placed in a liquid fixative, stained, and drawn to scale. The internal genitalia of each species was described and compared among the species examined. The reproductive system of the Melolonthinae species is comprised of two testicles, each with six follicles, two deferent ducts, two accessory glands, two glandular ducts, an ejaculatory duct, and the aedeagus (not described for any of the species examined). The number of testicular follicles per testicle is as reported in different species of other Scarabaeoidea subfamilies. The length of the accessory glands and the ejaculatory duct varies in the species studied. The ejaculatory bulb is present in all of the species of Dynastinae and Rutelinae examined but in only three species of Melolonthinae.

3D imaging reveals potential anticipatory responses to increasing intraspecific competition in the Drosophila male reproductive system

Selection favour males that anticipate intraspecific competition levels and develop appropriate competitive traits. In holometabolous insects, larval density can act as an ecological cue of males future intraspecific competition levels while also limiting access to food allocated to sexually selected traits (e.g., testes). To date, however, most studies have been conducted in larval densities that may have little or no relevance to species ecology. Here, we manipulated Drosophila melanogaster larval density based on the natural history of the species and used 3D micro-CT imaging to show a strong modulation of testes volume based on larval density: males from high larval densities generally had larger testes. Conversely, males from high densities tended (albeit not statistically significantly) to have the smallest accessory glands and ejaculatory bulb. Overall, we used micro-CT to measure male reproductive morphology in an ecologically relevant design for which the findings broadly support predictions from sexual selection theory.

Seminal fluid protein divergence among populations exhibiting postmating prezygotic reproductive isolation

ABSTRACTDespite holding a central role for fertilisation success, reproductive traits often show elevated rates of evolution and diversification. The rapid evolution of seminal fluid proteins (Sfps) within populations is predicted to cause mis-signalling between the male ejaculate and female reproductive tract between populations resulting in postmating prezygotic (PMPZ) isolation. Crosses between populations of Drosophila montana show PMPZ isolation in the form of reduced fertilisation success in both noncompetitive and competitive contexts. Here we test whether male ejaculate proteins deriving from either the accessory glands or the ejaculatory bulb differ between populations using liquid chromatography tandem mass spectrometry. We find more than 150 differentially abundant proteins between populations which may contribute to PMPZ isolation. These proteins include a number of proteases and peptidases, and several orthologs of D. melanogaster Sfps, all known to mediate fertilisation success and which mimic PMPZ isolation phenotypes. Males of one population typically produced greater quantities of Sfps and the strongest PMPZ isolation occurs in this direction. The accessory glands and ejaculatory bulb have different functions and the ejaculatory bulb contributes more to population differences than the accessory glands. Proteins with a secretory signal, but not Sfps, evolve faster than non-secretory proteins although the conservative criteria used to define Sfps may have impaired the ability to identify rapidly evolving proteins. We take advantage of quantitative proteomics data from three Drosophila species to determine shared and unique functional enrichments of Sfps that could be subject to selection between taxa and subsequently mediate PMPZ isolation. Our study provides the first high throughput quantitative proteomic evidence showing divergence of reproductive proteins implicated in the emergence of PMPZ isolation between populations.IMPACT SUMMARYIdentifying traits that prevent successful interbreeding is key to understanding early stages of the formation of new species, or speciation. Reproductive isolation arising prior to and during fertilisation frequently involves differences in how the sexes interact. In internally fertilising taxa, such interactions are mediated between the female reproductive tract where fertilisation occurs and the receipt of the ejaculate necessary for fertilisation. Because ejaculate proteins are at least partially responsible for these interactions, differences in male ejaculate protein composition could negatively impact fertilisation success, generating reproductive isolation. While the biological classes of ejaculate proteins are shared across all animal taxa, proteins that are secreted by males tend to show rapid evolution in gene expression and genetic sequence. Thus, reproductive proteins are suggested as prime targets facilitating reproductive isolation that arises after mating but before fertilisation (PostMating PreZygotic or PMPZ isolation). Most research on PMPZ isolation has focussed on differences between species for which it is not possible to determine the causative and temporal order of early speciation processes. Here, we test whether populations that exhibit few genetic differences but show strong PMPZ isolation also exhibit variation in ejaculate composition using quantitative high throughput proteomic analyses. We find a number of proteins are differentially abundant between populations including several known to impact fertilisation success in other species. We show that secreted proteins are evolving at an elevated rate, implicating their potential role in PMPZ isolation. We test divergence in ejaculate composition between species, finding a core set of functions that were conserved across species which last shared a common ancestor more than 40 million years ago along with species-specific investment. This work highlights the divergent evolution of reproductive proteins which may contribute to barriers between populations within a species early during speciation, extendable to similar analyses in other taxa in the future.

Morphology and histology of the male reproductive system of Collaria oleosa (Distant, 1883) (Heteroptera: Miridae)

Collaria oleosa (Distant, 1883) is a phytophagous bug often observed causing injuries in wheat, barley, and oat crops, besides grass pastures. The male reproductive system of C. oleosa has been studied anatomically and histologically. It consists of a pair of testes, each one with two follicles, one pair of seminal vesicles, two pairs of external accessory glands, and an ejaculatory bulb. The testicular follicles exhibit cysts with spermatogonia, spermatocytes, spermatids, and spermatozoa bundles. The testes are connected to the seminal vesicles by the vas deferens. The seminal vesicles are connected to the ejaculatory bulb and are bound by the accessory glands. In mirids belonging to the Stenodemini tribe, the general morphology of the male reproductive system is similar. However, two follicles per testis may be characteristic of the genus Collaria. The anatomy and histology of the male reproductive system in C. oleosa provides new information and also contributes to the understanding of the systematics of Stenodemini as well as the Miridae family in general.

A new genus and species of the Dasybatotreminae Bychowsky, 1957 (Monogenea: Monocotylidae), parasitic on Hypanus dipterurus (Jordan & Gilbert) (Myliobatiformes: Dasyatidae) in the Southeastern Pacific Ocean off Peru

Peruanocotyle n. gen. is proposed to accommodate Peruanocotyle chisholmae n. sp. (Monogenea: Monocotylidae). The new species is a gill parasite of the diamond stingray, Hypanus dipterurus (Jordan & Gilbert) (Myliobatiformes: Dasyatidae), a demersal chondrichthyan collected off the coast of Callao, Peru. Peruanocotyle n. gen. is placed in the Dasybatotreminae Bychowsky, 1957 because its anterior attachment organ has multiple glandular openings. Peruanocotyle chisholmae n. gen., n. sp. is unique among the Dasybatotreminae, by having: a well-developed, broadly ovoid anterior attachment organ with numerous grooves and an anteromedial notch; three prominent anterior glands that are connected to numerous peripheral glands; an unusual whip-shaped male copulatory organ composed of four connate tubes; an ejaculatory duct with slightly sclerotized walls where the proximal end has criss-crossed creases and the distal portion is highly convoluted; an ejaculatory bulb with one bipartite internal seminal vesicle; a wheel-shaped haptor with one central loculus and eight peripheral loculi; anchors that are much shorter than the width of marginal membrane and have an accessory sclerotized piece present; four oval testes, arranged in two groups; an ovary that has six clavate, proximal lobules and a distal portion that is strongly coiled; intestinal ceca with large lateral and medial diverticula; an oral opening surrounded by numerous small dome-like papillae; and a single vagina with sclerotized walls. Dasybatotreminae is amended to accommodate the new genus, and the new species is fully described and illustrated herein. The present finding adds to the other three marine monocotylids previously reported from Peru, namely Anoplocotyloides chorrillensis Luque & Iannacone, 1991; Anoplocotyloides papillatus (Doran, 1953) Young, 1967 and Monocotyle luquei Chero, Cruces, Iannacone, Sanchez, Minaya, Sáez & Alvariño, 2016.

Pre-imaginal conditioning alters adult sex pheromone response in Drosophila

Pheromones are chemical signals that induce innate responses in individuals of the same species that may vary with physiological and developmental state. In Drosophila melanogaster, the most intensively studied pheromone is 11-cis-vaccenyl acetate (cVA), which is synthezised in the male ejaculatory bulb and is transferred to the female during copulation. Among other effects, cVA inhibits male courtship of mated females. We found that male courtship inhibition depends on the amount of cVA and this effect is reduced in male flies derived from eggs covered with low to zero levels of cVA. This effect is not observed if the eggs are washed, or if the eggs are laid several days after copulation. This suggests that courtship suppression involves a form of pre-imaginal conditioning, which we show occurs during the early larval stage. The conditioning effect could not be rescued by synthetic cVA, indicating that it largely depends on conditioning by cVA and other maternally-transmitted factor(s). These experiments suggest that one of the primary behavioral effects of cVA is more plastic and less stereotypical than had hitherto been realised.

Evolutionary dynamics of male reproductive genes in the Drosophila virilis subgroup

Postcopulatory sexual selection (PCSS) is a potent evolutionary force that can drive rapid changes of reproductive genes within species, and thus has the potential to generate reproductive incompatibilities between species. Male seminal fluid proteins (SFPs) are major players in postmating interactions, and likely the main targets of PCSS in males. The virilis subgroup of Drosophila exhibits strong interspecific gametic incompatibilities, and can serve as a model to study the genetic basis of PCSS and gametic isolation. However, reproductive genes in this group have not been characterized. Here we use short-read RNA sequencing of male reproductive organs to examine the evolutionary dynamics of reproductive genes in members of the virilis subgroup: D. americana, D. lummei, D. novamexicana, and D. virilis. For each of the three male reproductive organs (accessory glands, ejaculatory bulb, and testes), we identify genes that show strong expression bias in a given tissue relative to the remaining tissues. We find that the majority of male reproductive transcripts are testes-biased, accounting for ~15% of all annotated genes. Ejaculatory bulb-biased transcripts largely code for lipid metabolic enzymes, and contain orthologs of the D. melanogaster ejaculatory bulb protein, Peb-me, which is involved in mating-plug formation. In addition, we identify 71 candidate SFPs, and show that this set of genes has the highest rate of nonsynonymous codon substitution relative to testes- and ejaculatory bulb-biased genes. Furthermore, these SFPs are underrepresented on the X chromosome and are enriched for proteolytic enzymes, which is consistent with SFPs in other insect species. Surprisingly, we find 35 D. melanogaster SFPs with conserved accessory gland expression in the virilis group, suggesting these genes may have conserved reproductive roles in Drosophila. Finally, we show that several of the SFPs that have the highest rate of nonsynonymous codon substitutions reside on the centromeric half of chromosome 2, which contributes to paternal gametic incompatibility between species. Our results suggest that SFPs are under strong selection in the virilis group, and likely play a major role in PCSS and/or gametic isolation.

Loss of Drosophila pheromone reverses its role in sexual communication in Drosophila suzukii

The Drosophila pheromone cis -11-octadecenyl acetate ( c VA) is used as pheromone throughout the melanogaster group and fulfils a primary role in sexual and social behaviours. Here, we found that Drosophila suzukii , an invasive pest that oviposits in undamaged ripe fruit, does not produce c VA. In fact, its production site, the ejaculatory bulb, is atrophied. Despite loss of c VA production, its receptor, Or67d, and cognate sensillum, T1, which are essential in c VA-mediated behaviours, were fully functional. However, T1 expression was dramatically reduced in D. suzukii , and the corresponding antennal lobe glomerulus, DA1, minute. Behavioural responses to c VA depend on the input balance of Or67d neurons (driving c VA-mediated behaviours) and Or65a neurons (inhibiting c VA-mediated behaviours). Accordingly, the shifted input balance in D. suzukii has reversed c VA's role in sexual behaviour: perfuming D. suzukii males with Drosophila melanogaster equivalents of c VA strongly reduced mating rates. c VA has thus evolved from a generic sex pheromone to a heterospecific signal that disrupts mating in D. suzukii , a saltational shift, mediated through offsetting the input balance that is highly conserved in congeneric species. This study underlines that dramatic changes in a species' sensory preference can result from rather ‘simple’ numerical shifts in underlying neural circuits.

Sex-specific triacylglycerides are widely conserved in Drosophila and mediate mating behavior

Pheromones play an important role in the behavior, ecology, and evolution of many organisms. The structure of many insect pheromones typically consists of a hydrocarbon backbone, occasionally modified with various functional oxygen groups. Here we show that sex-specific triacylclyerides (TAGs) are broadly conserved across the subgenus Drosophila in 11 species and represent a novel class of pheromones that has been largely overlooked. In desert-adapted drosophilids, 13 different TAGs are secreted exclusively by males from the ejaculatory bulb, transferred to females during mating, and function synergistically to inhibit courtship from other males. Sex-specific TAGs are comprised of at least one short branched tiglic acid and a long linear fatty acyl component, an unusual structural motif that has not been reported before in other natural products. The diversification of chemical cues used by desert-adapted Drosophila as pheromones may be related to their specialized diet of fermenting cacti.