Beyond Sperm and Male Accessory Gland Proteins: Exploring Insect Reproductive Metabolomes

Insect seminal fluid, the non-sperm component of the ejaculate, comprises a variegated set of molecules, including, but not limited to, lipids, proteins, carbohydrates, salts, hormones, nucleic acids, and vitamins. The identity and functional role of seminal fluid proteins (SFPs) have been widely investigated, in multiple species. However, most of the other small molecules in insect ejaculates remain uncharacterized. Metabolomics is currently adopted to deepen our understanding of complex biological processes and in the last 15years has been applied to answer different physiological questions. Technological advances in high-throughput methods for metabolite identification such as mass spectrometry and nuclear magnetic resonance (NMR) are now coupled to an expanded bioinformatics toolbox for large-scale data analysis. These improvements allow for the processing of smaller-sized samples and for the identification of hundreds to thousands of metabolites, not only in Drosophila melanogaster but also in disease vectors, animal, and agricultural pests. In this review, we provide an overview of the studies that adopted metabolomics-based approaches in insects, with a particular focus on the reproductive tract (RT) of both sexes and the ejaculate. Progress in the field of metabolomics will contribute not only to achieve a deeper understanding of the composition of insect ejaculates and how they are affected by endogenous and exogenous factors, but also to provide increasingly powerful tools to decipher the identity and molecular interactions between males and females during and after mating.

Rapid stress hardening in the Antarctic midge improves male fertility by increasing courtship success and preventing decline of accessory gland proteins following cold exposure

Rapid hardening is a process that quickly improves an animal's performance following exposure to a potentially damaging stress. In this study of the Antarctic midge, Belgica antarctica (Diptera, Chironomidae), we examine how rapid hardening in response to dehydration (RDH) or cold (RCH) improves male pre- and post-copulatory function when the insects are subsequently subjected to a damaging cold exposure. Neither RDH nor RCH improved survival in response to lethal cold stress, but male activity and mating success following sublethal cold exposure was enhanced. Egg viability decreased following direct exposure of the mating males to sublethal cold but improved following RCH and RDH. Sublethal cold exposure reduced expression of four accessory gland proteins, while expression remained high in males exposed to RCH. Though rapid hardening may be cryptic in males, this study shows that it can be revealed by pre- and post-copulatory interactions with females.

Rapid stress hardening in the Antarctic midge improves male fertility by increasing courtship success and preventing the decline of accessory gland proteins following cold exposure

Rapid hardening is a process that quickly improves animal performance following exposure to a potentially damaging stress. Features of reproduction can be improved by rapid hardening, but little is known about how rapid hardening may contribute to physiological responses in the cold environment of Antarctica. In this study of the Antarctic midge, Belgica antarctica (Diptera, Chironomidae), we examine how rapid hardening in response to dehydration (RDH) or cold (RCH) improves male pre- and post-copulatory function related to fertility when the insects are subsequently subjected to a damaging cold exposure. Neither RDH nor RCH improved survival in response to lethal cold stress, but male activity following sublethal cold exposure was enhanced. Both RCH and RDH improved mating success of males compared to those subjected directly to a sublethal bout of cold. Egg viability decreased following direct exposure to sublethal cold, but improved following RCH and RDH. Sublethal cold exposure reduced expression of four accessory gland proteins, while expression remained high in males exposed to RCH. Though rapid hardening may be cryptic in males, this study shows that it can be revealed by pre- and post-copulatory interactions with females.

FACS-based isolation and RNA extraction of Secondary Cells from the Drosophila male Accessory Gland

ABSTRACTTo appreciate the function of an organ, it is often critical to understand the role of rare cell populations. Unfortunately, this rarity often makes it difficult to obtain material for study. This is the case for the Drosophila male accessory gland, the functional homolog of mammalian prostate and seminal vesicle. In Drosophila, this gland is made up of two morphologically distinct cell types: the polygonally-shaped main cells, which compose 96% of the organ, and the larger, vacuole-containing secondary cells (SCs), which represent the remaining 4% of cells (~40 cells per lobe). Both cell types are known to produce accessory gland proteins (Acps), which are important components of the seminal fluid and are responsible for triggering multiple physiological and behavioral processes in females, collectively called the post-mating response (PMR). While a few genes are known to be specific to the SCs, the relative rarity of SCs has hindered the study of their whole transcriptome. Here, a method allowing for the isolation of SCs is presented, enabling the extraction and sequencing of RNAs from this rare cell population. The protocol consists of dissection, protease digestion and mechanical dissociation of the glands to obtain individual cells. Then, the cells are sorted by FACS, and living GFP-expressing SC singulets are isolated for RNA extraction. This procedure is able to provide SC-specific RNAs from ~40 males per condition in the course of one day. Given the speed and low number of flies required, this method enables the use of downstream RT-qPCR and/or RNA sequencing to the study gene expression in the SCs from different genetic backgrounds, ages, mating statuses or environmental conditions.SUMMARYHere, we describe the dissociation and sorting of a specific cell population from the Drosophila male accessory glands (Secondary cells), followed by RNA extraction for sequencing and RT-qPCR. The dissociation consists of dissection, proteases digestion and mechanical dispersion, followed by FACS purification of GFP-expressing cells.

Comparative genomics identifies male accessory gland proteins in five Glossina species

Accessory gland proteins (ACPs) are important reproductive proteins produced by the male accessory glands (MAGs) of most insect species. These proteins are essential for male insect fertility, and are transferred alongside semen to females during copulation. ACPs are poorly characterized in Glossina species (tsetse fly), the principal vector of the parasite that causes life-threatening Human African Trypanosomiasis and Animal trypanosomiasis in endemic regions in Africa. The tsetse fly has a peculiar reproductive cycle because of the absence of oviposition. Females mate once and store sperm in a spermathecal, and produce a single fully developed larva at a time that pupates within minutes of exiting their uterus. This slow reproductive cycle, compared to other insects, significantly restricts reproduction to only 3 to 6 larvae per female lifespan. This unique reproductive cycle is an attractive vector control strategy entry point. We exploit comparative genomics approaches to explore the diversity of ACPs in the recently available whole genome sequence data from five tsetse fly species ( Glossina morsitans, G. austeni, G. brevipalpis, G. pallidipes and G. fuscipes). We used previously described ACPs in Drosophila melanogaster and Anopheles gambiae as reference sequences. We identified 36, 27, 31, 29 and 33 diverse ACP orthologous genes in G. austeni, G. brevipalpis, G. fuscipes, G. pallidipes and G. morsitans genomes respectively, which we classified into 21 functional classes. Our findings provide genetic evidence of MAG proteins in five recently sequenced Glossina genomes. It highlights new avenues for molecular studies that evaluate potential field control strategies of these important vectors of human and animal disease.

Comparative genomics identifies male accessory gland proteins in five Glossina species

Accessory gland proteins (ACPs) are important reproductive proteins produced by the male accessory glands (MAGs) of most insect species. These proteins are essential for male insect fertility, and are transferred alongside semen to females during copulation. ACPs are poorly characterized in Glossina species (tsetse fly), the principal vector of the parasite that causes life-threatening Human African Trypanosomiasis and Animal trypanosomiasis in endemic regions in Africa. The tsetse fly has a peculiar reproductive cycle because of the absence of oviposition. Females mate once and store sperm in a spermathecal, and produce a single fully developed larva at a time that pupates within minutes of exiting their uterus. This slow reproductive cycle, compared to other insects, significantly restricts reproduction to only 3 to 6 larvae per female lifespan. This unique reproductive cycle is an attractive vector control strategy entry point. We exploit comparative genomics approaches to explore the diversity of ACPs in the recently available whole genome sequence data from five tsetse fly species (Glossina morsitans, G. austeni, G. brevipalpis, G. pallidipes and G. fuscipes). We used previously described ACPs in Drosophila melanogaster and Anopheles gambiae as reference sequences. We identified 36, 27, 31, 29 and 33 diverse ACP orthologous genes in G. austeni, G. brevipalpis, G. fuscipes, G. pallidipes and G. morsitans genomes respectively, which we classified into 21 functional classes. Our findings provide genetic evidence of MAG proteins in five recently sequenced Glossina genomes. It provides new avenues for molecular studies that evaluate potential field control strategies of these important vectors of human and animal disease.

Genetic variation in male-induced harm in Drosophila melanogaster

In Drosophila melanogaster , prolonged exposure to males reduces the longevity and fecundity of females. This harm arises from the effects of male courtship behaviours and the toxic side effects of the accessory gland proteins (Acps) in their seminal fluids. Here, we examine the relationship between male exposure and its harmful effect on the lifetime fitness of his mates, and quantify the genetic basis for this variation. We found significant additive genetic variation in the magnitude of harm that males impose on females by exposing females to males from a variety of hemiclonal backgrounds for either a brief or prolonged period of time and measuring their fecundity, a meaningful fitness index. Furthermore, we discovered a strong negative correlation between the magnitude of harm and the short-term effects of male exposure on female fitness. We discuss the evolutionary significance of these results with regards to potential life-history trade-offs in females, and its relationship to male body size.