Male Accessory Gland Secretions Research Articles

Plasticity in moth mating behavior and ejaculate proteomic diversity induced by male competition

In male competition, large and costly ejaculates are advantageous. Prior research on male accessory gland secretions in Plutella xylostella left open questions about how males modulate their mating behaviors and ejaculate composition allocation in response to varying levels of competition. The current study aimed to delve deeper into these unexplored facets. A totally of 928 ejaculate proteins were identified across males exposed to different competition conditions. Notably, males courting under non–, low-, and high-competition scenarios exhibited 867, 635, and 858 ejaculate proteins, respectively. Approximately 10% of these ejaculate proteins displayed variations that aligned with changes in competition intensity. Subsequent analyses focused on the proteins transferred to females, revealing that 44% of ejaculate proteins were transferred, with 37 proteins exhibiting differential expression. Functional analyses uncovered their crucial roles in sperm maturation, motility, and capacitation. Our findings reveal adaptive adjustments in ejaculate protein abundance and transmission in P. xylostella as a response to varying competition levels. Moreover, fluorescent sperm labeling indicated higher sperm transfer during low competition correlated with shorter sperm length. Furthermore, evidence suggests that males shorten their courtship duration and extend their mating duration when faced with competition. These results illustrate how competition drives ejaculate investment and behavioral plasticity, offering valuable insights for advancements in assisted reproductive technologies and pest management strategies.

Sexual inactivation induced by the mucus that covers land snail love darts: sexual selection and evolution of allohormones in hermaphrodites.

The transfer of male accessory gland secretions is a well-investigated reproductive strategy for winning in sexual selection. An example of such a strategy is the conspicuous mating behaviour of simultaneously hermaphroditic land snails, the so-called shooting of love darts, whereby a snail drives a love dart(s) into the body of its mating partner. In the land snail Euhadra quaesita, it has been shown that a specific mucus which coats the love dart is transferred into the partner's haemolymph and that it suppresses subsequent matings in the darted individual. However, how the mucus of the love dart suppresses rematings remains unclear. In the present study, we tested the hypothesis that by injection of the dart mucus, love-dart shooters manipulate the physiology of a dart recipient and make the individual sexually inactive. In an experiment in which snails were provided with opportunities to encounter a potential mating partner, we found that the latency period to achieve sexual arousal was longer in snails injected with the dart mucus than in snails of the control treatments. This finding indicates that the dart mucus delays sexual arousal in injected snails. This delay in arousal is a novel example of the effects of the mucus in simultaneously hermaphroditic land snails. The remating suppression effect of the dart mucus is likely to occur through sexual inactivation.

Unreceptivity – a phenomenon induced by male accessory gland secretions in female Helicoverpa armigera

Unreceptivity, a phenomenon generally observed in female insects following mating, was investigated by simulating a mating situation in Helicoverpa armigera females by injecting male accessory glands and duplex (MAG-duplex) extracts sans sperm. Injection of extracts into the abdomen of virgin females induced unreceptivity. The virgin females started behaving like mated females by rejecting the males by avoiding the advances made by the latter to mate. Unreceptivity was observed in about 93% of the females on the day of injection, which reduced to 82% on the second scotophase compared to controls. However, on the third day, 75% of them reverted back to receptivity that was comparable to virgin count. The Tricine–SDS-PAGE of MAG-duplex of virgin and mated male moths revealed the conspicuous absence of a low molecular weight (< 7 kDa) peptide band in the mated moths, and the possibility of being delivered to female at the time of mating. MALDI-TOF spectra of the same extracts showed the presence of several peptides < 7 kDa, further, the absence of them in mated moths.

Oviposition after sex: mated Anastrepha ludens (Diptera: Tephritidae) females increase oviposition without receiving an ejaculate

AbstractMating and receiving ejaculate can alter female insect physiology and postcopulatory behaviour. During mating, females receive both internal and external stimuli and different components in the ejaculate. In insects, these components consist mostly of sperm and male accessory gland secretions. Some of the most important changes associated with receiving male accessory gland secretions are a reduction in female sexual receptivity and an increase in oviposition. However, a clear function for these molecules has not been found in the Mexican fruit fly Anastrepha ludens (Loew) (Diptera: Tephritidae). Here, we tested how the stimulus of mating, receiving a full ejaculate, or only receiving accessory gland secretions can influence ovarian development and oviposition. Our results indicate that the stimulus of mating per se is enough to induce oviposition and increase egg laying in females even if ejaculate is not received, whereas receiving only accessory gland secretions does not increase ovarian development and is not enough to induce oviposition or increase egg production. Further research on the internal and external copulatory courtship of A. ludens will increase our understanding of the role of these secretions in stimulating oviposition independent of ejaculate effects. A biological function for male accessory gland secretions on female behaviour for A. ludens still needs to be found.

Relevance of prolonged first mating in reproductive bioactivities of Dysdercus koenigii (Fabricius, 1775) (Heteroptera: Pyrrhocoridae)

Abstract Adults of Dysdercus koenigii remain in continuous copula for up to 72h. The adaptive significance of prolonged copulation in the reproductive bioactivities of D. koenigii was investigated by a series of interrupted mating experiments. The adults were allowed to mate for a specified duration, after which mating was forcibly terminated. The study indicated that insemination in the female takes place during the first day of mating. This was reflected by the observation that 24h mated females laid fertile eggs. Prolonged mating modifies the female’s reproductive behaviour. The duration of mating had a profound effect on the onset of oviposition as well as the total number of eggs and egg batches laid by a female during her lifetime. Female fertility was also correlated with the duration of mating. Therefore, a female mated for 24h laid fewer fertile eggs in comparison to females mated for 48h or 72h. Hatching success decreased significantly in the egg batches laid by a female during the latter part of her life. A mated female exhibited rhythmic ovipositional behaviour. Our results indicated that the rhythmicity of the oviposition changed as a consequence of the different duration of mating. For instance, in females mated for 24h, there was a change in the number of eggs laid and in the rhythmicity of ovipositional peaks. Further, the rate of oocyte growth appeared to be enhanced by mating. This was evidenced by the shorter inter-oviposition period in the case of females mated for a longer period. It is suggested that throughout mating, females acquire additional sperm and non-gametic resources, such as male accessory gland secretions, which govern their reproductive behaviour and physiology.

Culex pipiens sperm motility is initiated by a trypsin-like protease from male accessory glands.

In most animals, sperm are stored in a quiescent state in the male reproductive tract and only initiate motility when released into either the female reproductive tract, or, in the case of broadcast spawners, the external environment. Male accessory gland secretions transferred into the female reproductive tract may provide factors that modulate sperm viability and storage, or aid in sperm competition, as well as activate sperm motility. In several insects, serine proteases have been implicated in activating sperm motility. Our previous studies have shown that, in Culex quinquefasciatus, either a male accessory gland extract or purified trypsin is sufficient to initiate sperm motility in vitro. The objective of this study was to identify and characterize trypsin-like enzymes produced in the Culex male accessory glands. Mass spectrometry was used to analyze accessory gland proteins and this preliminary proteomic analysis identified 4 trypsin-like proteases (trypsin, trypsin4, and two trypsin7 isoforms). When measured with the chromogenic trypsin substrate Na -benzoyl-L-arginine-ethyl-ester-hydrochloride (BAEE), trypsin-like protease activity in the accessory glands was robust, with a pH optimum of 8. The pH range for the Culex trypsin activity was substantially narrower than a mammalian homologue (porcine pancreatic trypsin). A soybean trypsin inhibitor (SBTI) -agarose affinity column was used to independently identify trypsin-like accessory gland proteins. Several proteins were enriched in the eluate, as detected by silver staining of SDS-PAGE gels. Taken together, these data demonstrate the presence of trypsin-like activity and several trypsin-like proteins in the Culex male accessory glands.

Proteome analysis of male accessory gland secretions in oriental fruit flies reveals juvenile hormone-binding protein, suggesting impact on female reproduction.

In insects, the accessory gland proteins (Acps) secreted by male accessory glands (MAGs) account for the majority of seminal fluids proteins. Mixed with sperm, they are transferred to the female at mating and so impact reproduction. In this project, we identified 2,927 proteins in the MAG secretions of the oriental fruit fly Bactrocera dorsalis, an important agricultural pest worldwide, using LC-MS analysis, and all sequences containing open reading frames were analyzed using signalP. In total, 90 Acps were identified. About one third (26) of these 90 Acps had a specific functional description, while the other two thirds (64) had no functional description including dozens of new classes of proteins. Hence, several of these novel Acps were abundant in the MAG secretions, and we confirmed their MAG-specific expression by qPCR. Finally and interestingly, one of these novel proteins was functionally predicted as juvenile hormone-binding protein, suggesting the impact of Acps with reproductive events in the female. Our results will aid in the development of an experimental method to identify Acps in insects, and in turn this information with new Acps in B. dorsalis will pave the way of further exploration their function in reproduction and potential development as new insecticide targets.

Ejaculated Mouse Sperm Enter Cumulus-Oocyte Complexes More Efficiently In Vitro than Epididymal Sperm

The mouse is an established and popular animal model for studying reproductive biology. Epididymal mouse sperm, which lack exposure to secretions of male accessory glands and do not precisely represent ejaculated sperm for the study of sperm functions, have been almost exclusively used in studies. We compared ejaculated and epididymal sperm in an in vitro fertilization setting to examine whether ejaculated sperm enter cumulus-oocyte complexes more efficiently. In order to prepare sperm for fertilization, they were incubated under capacitating conditions. At the outset of incubation, ejaculated sperm stuck to the glass surfaces of slides and the incidences of sticking decreased with time; whereas, very few epididymal sperm stuck to glass at any time point, indicating differences in surface charge. At the end of the capacitating incubation, when sperm were added to cumulus-oocyte complexes, the form of flagellar movement differed dramatically; specifically, ejaculated sperm predominantly exhibited increased bending on one side of the flagellum (a process termed pro-hook hyperactivation), while epididymal sperm equally exhibited increased bending on one or the other side of the flagellum (pro-hook or anti-hook hyperactivation). This indicates that accessory sex gland secretions might have modified Ca2+ signaling activities in sperm, because the two forms of hyperactivation are reported to be triggered by different Ca2+ signaling patterns. Lastly, over time, more ejaculated than epididymal sperm entered the cumulus oocyte complexes. We concluded that modification of sperm by male accessory gland secretions affects the behavior of ejaculated sperm, possibly providing them with an advantage over epididymal sperm for reaching the eggs in vivo.

Double insurance of paternity by a novel type of mating plug in a monandrous solitary mason beeOsmia bicornis(Hymenoptera: Megachilidae)

Males of some hymenopteran species are able to behaviourally induce unreceptivity to mating in females by a post-copulatory display (post-copulatory courtship). This method of preventing further inseminations requires a high degree of female cooperation by the female and is especially susceptible to manipulations. If mating chances are low and sexual competition between males high, males can be expected to evolve additional mechanisms to protect their fertilization opportunities. This hypothesis was checked in the red mason bee Osmia bicornis (Linnaeus, 1758; syn. Osmia rufa). Males of this species were found to use a mating plug as a reinsurance to protect their paternity, although they induce females' monandry by a post-copulatory display. Male accessory gland secretions transferred during ejaculation form a plastic, spongy plug in the females' vagina that also contains the spermatozoa. This mating plug does not hinder rivals to mate with the female but prevents intermixing of the sperm. Thus, the sperm precedence of the first male is secured. The plug is ejected by the female after approximately 1 day when the spermatheca has been completely filled with sperm. The male accessory gland supply is sufficient for the formation of two to three plugs only and is not replenished. This pattern fits well with the low mating opportunities of O. bicornis males. The evolution of a mating plug in addition to the behavioural induction of unreceptivity to mate in females is discussed. © 2015 The Linnean Society of London, Biological Journal of the Linnean Society, 2015, 115, 28–37.

Molecular characterization and functional analysis of a putative sex-peptide receptor in the tobacco cutwormSpodoptera litura(Fabricius, 1775) (Lepidoptera: Noctuidae)

The sex-peptide receptor (SPR) acts in Drosophila females to detect male sex-peptide (SP) and trigger changes in female behaviour. Further study on SPR and its ligand family from different taxa will not only provide insight into ligand–receptor coevolution but also facilitate finding new approaches for pest control. Our previous studies on the tobacco cutworm Spodoptera litura (Fabricius, 1775) showed that mating usually results in marked changes in female behaviour and determined that factors from male accessory gland (MAG) secretions play a key role in such switches in females after mating. In the present study, we report the cloning of a transcript encoding a G-protein-coupled receptor from S. litura. This 3 kb transcript contains an ORF encoding a protein of 421 amino acids that shared significant amino acid identity to the SPR identified in Drosophila. Real-time PCR showed that the expression levels of the receptor in adults correlate well with the process of sexual maturation and mating rates; mating resulted in distinct sexual dimorphic differences in gene expression. Our results suggest that this gene is involved in mediating reproductive behaviour in adult moths. To test the hypothesis, we further studied the possible function of this receptor (SplSPR) on mediating female post-mating behaviour by using RNA interference (RNAi) and treatment with MAG secretions. RNAi reduced the expression levels of SplSPR by 65–88% and significantly prevented the effect of MAG secretions. Injection of MAG secretions causes normal virgin females to behave as though they had mated (they are sexually unreceptive and start to lay eggs), whereas SPR RNAi females fail to respond to MAG factors and continue to show virgin behaviours (lay very few eggs and copulate readily). These results support the hypothesis that the SplSPR functions in mediating female post-mating behaviour and the existence of an SP-like ligand in MAG secretions in S. litura.

His Hormone, Her Oogenesis: How Male Malaria Mosquitoes Trigger Female Egg Development

A female mosquito needs two things to reproduce: your blood and a visit from a male mosquito. The blood provides the nutrient-rich meal she will use to feed her developing eggs. The male provides not only sperm, but also a complex mix of proteins and other molecules, secreted from the so-called male accessory glands (MAGs) and deposited along with the sperm during mating. The secretions form a gelatinous “mating plug,” which blocks access to the uterus. Anopheles oocytes after blood feeding. The oocyte, which is surrounded by epithelial cells (blue) and muscle cells (green), accumulates lipids (red) in the cytoplasm. This accumulation depends on the interaction between a female protein, MISO, and the ... Both the blood meal and copulation provide necessary signals for the female to begin development of eggs in her ovary. While much is known about the events triggered by feeding, relatively little is known about how mating stimulates oogenesis. A new study by Francesco Baldini, Flaminia Catteruccia, and colleagues shows that a hormone delivered by males stimulates production of a protein in females, and together the pair induces expression of multiple genes to increase egg development. Working with the malaria-carrying mosquito Anopheles gambiae, the authors had previously shown that a protein called mating-induced stimulator of oogenesis (MISO) was upregulated in the uterus, or atrium, shortly after mating. They began the current work by using RNA interference to block MISO expression in mated, blood-fed females. They found that oogenesis specifically was impaired, decreasing egg development in treated insects to the level found in virgin females. Those egg follicles that did develop did so slowly, suggesting that they were unable to accumulate lipids at the normal rate. This, the authors found, was due to decreased expression of the lipid transporter lipophorin, whose levels were again similar to those seen in virgin females, as a consequence of loss of MISO. Previous studies have suggested that MAG secretions control aspects of behavior and physiology in females after mating and that one key component of the secretions is the steroid hormone 20-hydroxy-ecdysone (20E), which has multiple effects in female mosquitoes. Control and MISO-deficient females had equivalent levels of 20E in the mating plug, but it dispersed from the plug much more slowly in the females with reduced MISO. This suggested that the two might interact, a hypothesis confirmed when the authors showed that 20E and MISO co-immunoprecipitated. Loss of circulating 20E led to reduced expression of several of its gene targets, including the yolk protein precursor vitellogeninand the ecdysone receptor (EcR), which acts as nuclear receptor for the hormone. In control mosquitoes, an increase in 20E led to an increase in production of MISO, an effect that could be blocked by loss of its receptor EcR. The picture that emerges is that male-supplied 20E binds to EcR to induce the female to produce MISO, which it then binds as well. This complex then promotes transcription of a variety of oogenic genes, leading to increased lipid accumulation in oocytes and promoting egg development. The elucidation of this pathway may have important consequences in the fight against malaria, which yearly kills over half a million people worldwide. Introduction of males deficient in 20E synthesis, for instance, could reduce reproduction in females that mate with them. Furthermore, the lipid accumulation that occurs during oogenesis reduces the ability of the female mosquitos to destroy the malaria parasites they carry; thus, blocking this pathway could reduce the deadliness of a bite from these troublesome insects. Baldini F, Gabrieli P, South A, Valim C, Mancini F, et al. (2013) The Interaction between a Sexually Transferred Steroid Hormone and a Female Protein Regulates Oogenesis in the Malaria Mosquito Anopheles gambiae. doi:10.1371/journal.pbio.1001695

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.

Stimulation of Mating‐Induced Uterine Contractions in the Bitch and Their Modification and Enhancement of Fertility by Prostatic Fluid

Little is known about the response of the bitch's reproductive tract to mating or of the role of male accessory gland secretions in the female. In this clinical study, the component stimuli causing mating-induced uterine contractions were investigated in 64 bitches. Basal uterine contractions were present during oestrus and a significant increase in the frequency of contractions was observed during natural mating. Neither teasing with a male nor stimulation of the vagina or cervix by vaginal or transcervical insemination (TCI) caused an increase in the frequency of uterine contractions. Increased contractions were however present after both vaginal and transcervical insemination when the vestibule was distended, and dorsal wall of the vaginal was manually stimulated. Interestingly, this increase in uterine contractions was partially ameliorated when prostatic fluid was used as a flushing component following transcervical insemination. Two further studies performed with 72 bitches of which 18 were each inseminated transcervically with fresh or frozen semen flushed into the uterus with either saline or prostatic fluid demonstrated that prostatic fluid significantly increased the pregnancy rate and litter size of both groups. There are important mechanisms regulating the transport and elimination of sperm from the bitch's reproductive tract. Whilst physical aspects of coitus are undoubtedly involved in initiating uterine contractions, prostatic fluid appears to have an important role in modulating uterine contractions and fertility.

Effects of sperm DNA damage on the levels of RAD51 and p53 proteins in zygotes and 2-cell embryos sired by golden hamsters without the major accessory sex glands

We previously reported that the male accessory sex gland (ASG) secretion is the main source of antioxidants to safeguard sperm genomic integrity and functional competence. Removal of all ASGs in the golden hamster can reduce male fertility by increasing embryo wastage. This study aims to investigate whether the oxidative DNA-damaged sperm from hamsters without all ASGs (TX) could successfully fertilize oocytes and to qualify the status of DNA repair by the expression of RAD51 and p53 proteins. Here we demonstrated a significantly higher DNA-base adduct formation (8-hydroxy-2′-deoxyguanosine) in sperm from TX males than those from sham-operated males. Comet assays demonstrated that all female pronuclei in both zygotes were intact, but single- and double-strand DNA damage was found in decondensed sperm in TX males only. DNA damage could also be detected in both nuclei of the TX 2-cell embryos. RAD51, a DNA repair enzyme, was found to be evenly distributed in the cytoplasm and nuclei in oocytes/zygotes, while at the 2-cell stage, a strong expression of p53 protein and a larger clear perinuclear area without RAD51 expression were found in TX embryos. In conclusion, we demonstrated for the first time DNA damage in decondensed sperm of zygotes and blastomeres of 2-cell stage embryos sired by TX males, resulting in the activation of DNA repair. Sperm DNA damage could induce the increase in p53 expression and the reduction of RAD51 expression in the TX 2-cell stage embryos.

The bitch uterine response to semen deposition and its modification by male accessory gland secretions

Little is known about the response of the bitch's reproductive tract to semen deposition. In this study, an influx of polymorphonuclear neutrophils (PMNs) into the uterus was detected after artificial insemination, but there was normal fertility. Doppler ultrasonography showed that insemination induced an increase in uterine artery blood velocity and a decrease in the resistance index of short duration, indicating vasodilation. Semen that was extended in fluid from the sperm rich fraction of the ejaculate (seminal plasma, SP), or third fraction of the ejaculate (prostatic fluid, PF), produced a similar magnitude of effect but of longer duration. It was hypothesised that vasodilation following insemination was largely induced by SP and PF which, together with PMN influx, was part of a normal uterine response. Physiological concentrations of PMNs in vitro reduced the ability of spermatozoa to attach to uterine epithelium, most likely as a result of spermatozoa becoming attached to PMNs. However, both SP and PF increased attachment of spermatozoa to the uterine epithelium by reducing sperm attachment to PMNs, and potentially by an additional mechanism that did not involve inhibition of sperm binding to PMNs. These are the first canine studies to document an apparent physiological response by the uterus to semen, associated with uterine artery vasodilation and PMN influx. Moreover, these investigations are the first to demonstrate that canine SF and PF are part of the mechanism for increasing uterine perfusion and that both fluids have a modulatory effect on PMN-induced inhibition of spermatozoal attachment to uterine epithelium, most likely mediated by reduced sperm attachment to PMNs.

Function and composition of male accessory gland secretions in Anopheles gambiae: a comparison with other insect vectors of infectious diseases

Human malaria, a major public health burden in tropical and subtropical countries, is transmitted exclusively by the bite of a female Anopheles mosquito. Malaria control strategies aimed at inducing sexual sterility in natural vector populations are an attractive alternative to the use of insecticides. However, despite their importance as disease vectors, limited information is available on the molecular mechanisms regulating fertility in Anopheles mosquitoes. In the major malaria vector, An. gambiae, the full complement of sperm and seminal fluid required for a female’s lifelong egg production is obtained from a single mating event. This single mating has important consequences for the physiology and behavior of An. gambiae females: in particular, they become refractory to further insemination, and they start laying eggs. In other insects including Drosophila, similar post-copulatory changes are induced by seminal proteins secreted by the male accessory glands and transferred to the female during mating. In this review, we analyze the current state of knowledge on the function and characterization of male seminal proteins in An. gambiae, and provide a comparative assessment of the role of these male reproductive factors in other mosquito vectors of human disease in which female post-copulatory behavior has been studied. Knowledge of the factors and mechanisms regulating fertility in An. gambiae and other vectors can help the design of novel control strategies to fight the spread of disease.

Morphology of reproductive accessory glands in eight species of blood-feeding Hemiptera (Hemiptera, Reduviidae) insect vectors of Chagas disease

This paper documents the morphology of previously undescribed adult reproductive accessory glands in eight species of blood-feeding Hemiptera, vectors of Chagas disease. These species are three Triatoma (T. dimidiata, T. klugi, T. sordida), three Rhodnius (R. brethesi, R. nasutus, R. pictipes), and one species each from Nesotriatoma (N. bruneri) and Panstrongylus (P. megistus). This survey shows that the male reproductive systems between species of four genera of Reduviidae adhere to the same general plan seen in previously described vectors of Chagas disease. This morphological similarity suggests that reproductive success of the male is contingent on the delivery of a vital set of male accessory gland secretions to the female in conjunction with material from the testes and seminal vesicle. However, variations were observed in the accessory glands of females, especially at the level of the genus. The spermathecae are morphologically distinct, and the posterior accessory glands are absent in some. The differences in spermathecae morphology likely reflect physiological adaptations associated with speciation driven by cryptic female choice in which the female determines which sperm are used for fertilization. Differences in the posterior reproductive accessory gland can be correlated with variations in ovipositioning behaviour. Since reproductive physiology is important for species success, this information also augments epidemiological studies by providing a comparison to R. prolixus, a Chagas disease vector for which the physiology is well known.

Male accessory gland proteins induce female monogamy in anopheline mosquitoes

The role of male accessory gland (MAG) secretions in inducing refractoriness to further mating in mosquitoes (Diptera: Culicidae) was established in the late 1960s. In a set of simple experiments, MAG extract was injected intra-thoraxically into the hemocoel of virgin Aedes aegypti (L.), Culex pipiens pipiens (L.) and Anopheles quadrimaculatus Say females. This subsequently caused most females to remain unmated when exposed to males. For anophelines these findings were later challenged by a study involving intra-abdominal injections of MAG extracts into Anopheles gambiae Giles s.l. and Anopheles albimanus Wiedmann females, which failed to induce refractoriness to further mating. These findings led to controversy about the respective role of sperm and accessory gland peptides in inducing female monogamy in Anopheles and are at odds with our current understanding of the mating process in Drosophila spp. (Diptera: Drosophillidae) and other dipterans. Here we confirm the function of MAG secretions in anophelines experimentally by showing that intra-thoracic injections in Anopheles stephensi Liston and in the M and S molecular forms of An. gambiae s.s. result in the expected female monogamy. Cross-injections of MAG extracts between the M and S molecular forms of An. gambiae, two cryptic taxa within An. gambiae s.s. which are thought to be undergoing incipient speciation, also elicited effective refractoriness, suggesting that the two sub-taxa have not diverged with regard to sex peptides responsible for female monogamy. Importantly, this also suggests that the rare cases of re-mating following cross-mating observed in this species may not be a form of reproductive barrier between molecular forms.

Juvenile hormone regulation of male accessory gland activity in the red flour beetle, Tribolium castaneum

Male accessory gland proteins (Acps) act as key modulators of reproductive success in insects by influencing the female reproductive physiology and behavior. We used custom microarrays and identified 112 genes that were highly expressed in male accessory glands (MAG) in the red flour beetle, Tribolium castaneum. Out of these 112 identified genes, 59 of them contained sequences coding for signal peptide and cleavage site and the remaining 53 contained transmembrane domains. The expression of 14 of these genes in the MAG but not in other tissues of male or female was confirmed by quantitative real-time PCR. In virgin males, juvenile hormone (JH) levels increased from second day post adult emergence (PAE), remained high on third day PAE and declined on fourth day PAE. The ecdysteroid titers were high soon after adult emergence but declined to minimal levels from 1 to 5 days PAE. Feeding of juvenile hormone analog, hydroprene, but not the ecdysteroid analog, RH-2485, showed an increase in size of MAGs, as well as an increase in total RNA and protein content of MAG. Hydroprene treatment also increased the expression of Acp genes in the MAG. RNAi-mediated knock-down in the expression of JHAMT gene decreased the size of MAGs and expression of Acps. JH deficiency influenced male reproductive fitness as evidenced by a less vigor in mating behavior, poor sperm transfer, low egg and the progeny production by females mated with the JH deficient males. These data suggest a critical role for JH in the regulation of male reproduction especially through MAG secretions.

Honey bee males and queens use glandular secretions to enhance sperm viability before and after storage

Internal fertilization requires live sperm to be transferred from male to female before egg fertilization. Both males and females assist the insemination process by providing sperm with glandular secretions, which have been inferred to contain subsets of proteins that maintain sperm viability. Here we show that in the honeybee ( Apis mellifera) secretions of the male accessory glands, the major contributors towards seminal fluid, enhance sperm survival. We further demonstrate that the protein fraction of the male accessory gland secretion is indeed important for achieving the maximal effect on sperm survival. After sperm storage, the queens also provide sperm with secretions from spermathecal glands and we show that these secretions have a comparable positive effect on sperm viability. SDS gels show that the proteomic profiles of accessory gland secretion and spermathecal fluid secretion hardly overlap, which suggests that males and females use different proteins to enhance sperm viability during, respectively, ejaculation and final sperm storage.