Heterologous expression of a substance which inhibits receptivity and calling in Helicoverpa armigera (Hübner).

The accessory glands of male moths secrete several proteins, which are known to affect post-mating behaviour in females such as calling, reduction in receptivity, rate of egg maturation and laying, sperm maintenance and release and formation of mating plug. Helicoverpa armigera (Hübner) is a polyphagous pest of numerous crops and it is widely distributed on the Indian subcontinent where it causes severe economic losses. In the present study, receptivity- and calling-inhibiting substance (RCIS), a peptide secreted from the accessory glands of male H. armigera, was sequenced, cloned and expressed in a prokaryote, Escherichia coli. RCIS is a peptide comprising 58 amino acids and had a theoretical molecular weight of 6.03 kDa. It showed 64% similarity with pheromonostatic peptide 1, identified in Helicoverpa zea (Kingan et al., 1995) but differed regarding deletion of four and one amino acids at positions 14-17 and 44, respectively, and insertion of one and five amino acids at position 38 and the terminal position of RCIS, respectively. H. armigera females injected with recombinant RCIS showed reduced receptivity and calling behaviour (in 70-80% of the treated individuals), and mating frequencies decreased by 80%. Recombinant RCIS may be employed to artificially induce non-receptivity in virgin females in order to prevent reproduction.

Larger males facilitate population expansion in Ophraella communa.

One of the most intriguing concepts in animal ecology is the reproductive advantages offered by larger body size, and the females prefer to mate with larger males to gain reproductive advantage. Currently, it is not clear how females recognize signs of male 'quality' and what mechanisms are involved in producing offspring with direct or indirect benefits. Our study aims to assess the preferences of females for males in Ophraella communa, determine the reproductive benefits and reveal the underlying mechanism behind this advantage. We demonstrate that male body size is an important determinant in the evolutionary process of O. communa, affecting female mate choice. Moreover, our study establishes that females prefer males with a larger body size, and this could further improve the developmental and reproductive fitness of their offspring. Finally, we focus on the seminal fluid proteins (SFPs) in O. communa, determine differentially expressed genes (i.e. OcACE, OcCBP and OcSFP) by analysing their proteomes and transcriptomes, and define the role of these SFPs-related genes through RNAi. Our study proved that the reproductive benefit of large males may be regulated by biased expression of crucial SFPs genes. The present study advances our understanding of the biological significance of preferential mating.

Alpha-2 macroglobulin as a region-specific secretory protein in male reproductive tract, and its dynamics during sperm transit toward the female spermatheca in the blue crab.

A 250-kDa protein was isolated from fluid in the middle spermatic duct (MSD) of the blue crab (Portunus pelagicus). N-terminal and partial amino acid sequences revealed that this MSD-specific protein is highly similar to the plasma-enriched protein Alpha-2 macroglobulin (α2M). The P. pelagicus ortholog (Ppα2M) is a large glycoprotein possessing mannose and N-acetylglucosamine residues. Ppa2m mRNA was detected in the spermatic duct, androgenic gland, and hematopoietic tissue, whereas the protein was primarily observed in the apical cytoplasm of MSD epithelium and in the matrix of the acrosome of MSD sperm; distally within spermatic duct, Ppα2M was lost from the sperm membrane but remained in the sperm acrosome. These results suggest that Ppα2M is expressed and glycosylated in the epithelium of spermatic ducts, secreted into MSD fluid, taken up by sperm in the MSD, and removed from the surface of sperm during its transit towards the female spermatheca. Given that Ppα2M also exhibits protease inhibitor activity, we hypothesize that acrosome localized Ppα2M may suppress premature acrosome reaction during post-testicular sperm maturation in this crab.

Seminal fluid protein genes of the brown planthopper, Nilaparvata lugens.

BACKGROUND: Seminal fluid proteins (SFPs) are produced mainly in the accessory gland of male insects and transferred to females during mating, in which they induce numerous physiological and post-mating behavioral changes. The brown plant hopper (BPH), Nilaparvata lugens, is an economically important hemipterous pest of rice. The behavior and physiology of the female of this species is significantly altered by mating. SFPs in hemipteran species are still unclear. RESULTS: We applied high-throughput mass spectrometry proteomic analyses to characterize the SFP composition in N. lugens. We identified 94 putative secreted SFPs, and the expression levels of these proteins was determined from the male accessory gland digital gene expression database. The 94 predicted SFPs showed high expression in the male accessory gland. Comparing N. lugens and other insect SFPs, the apparent expansion of N. lugens seminal fluid trypsins and carboxylesterases was observed. The number of N. lugens seminal fluid trypsins (20) was at least twice that in other insects. We detected 6 seminal fluid carboxylesterases in N. lugens seminal fluid, while seminal fluid carboxylesterases were rarely detected in other insects. Otherwise, new insect SFPs, including mesencephalic astrocyte-derived neurotrophic factor, selenoprotein, EGF (epidermal growth factor) domain-containing proteins and a neuropeptide ion transport-like peptide were identified. CONCLUSION: This work represents the first characterization of putative SFPs in a hemipeteran species. Our results provide a foundation for future studies to investigate the functions of SFPs in N. lugens and are an important addition to the available data for comparative studies of SFPs in insects.

Male-derived phospholipase A2 enhances WD46 expression and increases fertility in Ophraella communa.

Successful bisexual reproduction requires interactions between males and females. Male-derived seminal fluid proteins (SFPs) transferred to females during mating profoundly affect females from pre- to post-mating, and the subsequent shift in female physiology enhances their fertility. SFPs have important evolutionary implications for the fitness of many insects. However, little is known about how females respond to male SFPs. In this study, we identified a male-derived SFP-phospholipase A2 (PLA2) in Ophraella communa. PLA2 is a vital enzyme in eicosanoid biosynthesis; however, it has not been identified as an insect SFP. We found that OcPLA2 is specifically expressed in males, especially in the male accessory glands (MAGs); it is transferred to the female during mating and functions as an SFP to enhance fertility. The expression of a female-derived gene encoding the WD repeat-containing protein 46 (WD46) was upregulated when OcPLA2 entered the female reproductive tract, and this contributed to female egg production by increasing triacylglycerol lipase (TGL) gene expression and the triglyceride (TG) content. This is the first study to identify PLA2 as an SFP in insects. Our findings also shed light on the regulatory role of OcPLA2 in beetle reproduction; the expression of OcPLA2 is initially correlated with female WD46 expression and later with the decline in TGL gene expression and the TG content. This represents a unique mechanism of reproductive regulation by an SFP.

Effects of an entomopathogenic fungus on the reproductive potential of Drosophila males.

While mortality is often the primary focus of pathogen virulence, non-lethal consequences, particularly for male reproductive fitness, are less understood; however, they are essential for understanding how sexual selection contributes to promoting resistance. We investigated how the fungal pathogen Metarhizium brunneum affects mating ability, fertility, and seminal fluid protein (SFP) expression of male Drosophila melanogaster paired with highly receptive virgin females in non-competitive settings. Depending on sex and dose, there was a 3-6-day incubation period after infection, followed by an abrupt onset of mortality. Meanwhile, the immune response was strongly induced already 38 h after infection and continued to increase as infection progressed. Latency to mate somewhat increased during the incubation period compared to sham-treated males, but even on Day 5 post infection >90% of infected males mated within 2 h. During the incubation period, M. brunneum infection reduced male reproductive potential (the number of offspring sired without mate limitation) by 11%, with no clear increase over time. Approaching the end of the incubation period, infected males had lower ability to convert number of mating opportunities into number of offspring. After repeated mating, infected males had lower SFP expression than sham controls, more so in males that mated with few mates 24 h earlier. Overall, despite strong activation of the immune response, males' mating ability and fertility remained surprisingly little affected by the fungal infection, even shortly before the onset of mortality. This suggests that the selection for resistance acts mainly through mortality, and the scope for fertility selection to enhance resistance in non-competing settings is rather limited.

The carboxypeptidase B and carbonic anhydrase genes play a reproductive regulatory role during multiple matings in Ophraella communa.

Seminal fluid proteins (SFPs) are key factors in sexual reproduction and are transferred to females during mating with sperm. SFPs have a nutritional value because they protect and activate sperm storage and release to optimize fecundity. Multiple matings promote ovipositioning in several insect species. Therefore, insects may obtain more SFP through multiple matings to maximize reproduction, but this process has not yet been clearly confirmed. Here, the relationship between multiple matings and the SFPs in Ophraella communa (Coleoptera: Chrysomelidae), a biological control agent of the common ragweed Ambrosia artemisiifolia (Asterales: Asteraceae), was studied. Multiple matings significantly increased female fecundity and ovary egg deposition. Carboxypeptidase B (OcCpb) and carbonic anhydrase (OcCa) genes were identified as putative SFP genes in O. communa and they showed strong male-biased expression. Additionally, OcCpb and OcCa expression was upregulated in the bursa copulatrix of mating females compared to that in virgin females, but their expression gradually declined after copulation. Furthermore, OcCpb and OcCa knockdown in males led to a decrease in insect fecundity compared to that in the control. The reproductive tract of females mated with dsRNA-treated males was dissected and observed and, notably, the ovaries produced significantly fewer eggs. These data suggest that OcCpb and OcCa play regulatory roles during multiple matings in O. communa.

Effects of Larval Diet on the Male Reproductive Traits in the West Indian Sweet Potato Weevils Euscepes postfasciatus (Coleoptera: Curculionidae).

Larval diet significantly affects adult traits, although less is known about how they affect reproductive traits. Males of West Indian sweet potato weevil Euscepes postfasciatus deliver a remating inhibitor along with sperm to their mates during mating, leading to a refractory period (the period before females mate again). Crossing experiments were conducted using lines reared on artificial diets, including sweet potato powder (AD) or sweet potato tubers (SP) during the larval stage, and the refractory period was examined. We also examined whether the larval diet qualitatively or quantitatively altered male ejaculate. The results showed that the refractory period was significantly longer in the SP treatment than in the AD treatment for males and females. There was no significant difference in ejaculate volume. However, the number of sperm in the testes-seminal vesicles complex was significantly higher in the SP treatment. Additionally, SDS-PAGE revealed that the ejaculate was qualitatively different depending on the larval diet, and one protein of approximately 15 kDa in size was expressed only in the SP treatments. Revealing how larval diet affects reproductive traits in adult males will help shed light on the diverse evolution of insect mating systems and reproductive behavior.

The Drosophila seminal proteome and its role in postcopulatory sexual selection.

Postcopulatory sexual selection (PCSS), comprised of sperm competition and cryptic female choice, has emerged as a widespread evolutionary force among polyandrous animals. There is abundant evidence that PCSS can shape the evolution of sperm. However, sperm are not the whole story: they are accompanied by seminal fluid substances that play many roles, including influencing PCSS. Foremost among seminal fluid models is Drosophila melanogaster, which displays ubiquitous polyandry, and exhibits intraspecific variation in a number of seminal fluid proteins (Sfps) that appear to modulate paternity share. Here, we first consolidate current information on the identities of D. melanogaster Sfps. Comparing between D. melanogaster and human seminal proteomes, we find evidence of similarities between many protein classes and individual proteins, including some D. melanogaster Sfp genes linked to PCSS, suggesting evolutionary conservation of broad-scale functions. We then review experimental evidence for the functions of D. melanogaster Sfps in PCSS and sexual conflict. We identify gaps in our current knowledge and areas for future research, including an enhanced identification of PCSS-related Sfps, their interactions with rival sperm and with females, the role of qualitative changes in Sfps and mechanisms of ejaculate tailoring. This article is part of the theme issue 'Fifty years of sperm competition'.

Drosophila seminal sex peptide associates with rival as well as own sperm, providing SP function in polyandrous females.

When females mate with more than one male, the males' paternity share is affected by biases in sperm use. These competitive interactions occur while female and male molecules and cells work interdependently to optimize fertility, including modifying the female's physiology through interactions with male seminal fluid proteins (SFPs). Some modifications persist, indirectly benefiting later males. Indeed, rival males tailor their ejaculates accordingly. Here, we show that SFPs from one male can directly benefit a rival's sperm. We report that Sex Peptide (SP) that a female Drosophila receives from a male can bind sperm that she had stored from a previous male, and rescue the sperm utilization and fertility defects of an SP-deficient first-male. Other seminal proteins received in the first mating 'primed' the sperm (or the female) for this binding. Thus, SP from one male can directly benefit another, making SP a key molecule in inter-ejaculate interaction.

When fruit flies and other animals reproduce, a compatible male and a female mate, allowing sperm from the male to swim to and fuse with the female's egg cells. The males also produce proteins known as seminal proteins that travel with the sperm. These proteins increase the likelihood of sperm meeting an egg and induce changes in the female that increase the number, or quality, of offspring produced. Some seminal proteins help a male to compete against its rivals by decreasing their chances to fertilize eggs. However, since many of the changes seminal proteins induce in females are long-lasting, it is possible that a subsequent male may actually benefit indirectly from the effects of a prior male's seminal proteins. It remains unclear whether the seminal proteins of one male are also able to directly interact with and help the sperm of another male. Male fruit flies make a seminal protein known as sex peptide. Normally, a sex peptide binds to the sperm it accompanies into the female, increasing the female's fertility and preventing her from mating again with a different male. To test whether the sex peptide from one male can bind to and help a rival male's sperm, Misra and Wolfner mated female fruit flies with different combinations of males that did, or did not, produce the sex peptide. The experiments found that female flies that only mated with mutant males lacking the sex peptide produced fewer offspring than if they had mated with a 'normal' male. However, in females that mated with a mutant male followed by another male who provided the sex peptide, the second male's sex peptide was able to bind to the mutant male's sperm (as well as to his own). This in turn allowed the mutant male's sperm to be efficiently used to sire offspring, at levels comparable to a normal male providing the sex peptide. These findings demonstrate that the ways individual male fruit flies interact during reproduction are more complex than just simple rivalry. Since humans and other animals also produce seminal proteins comparable to those of fruit flies, this work may aid future advances in human fertility treatments and strategies to control the fertility of livestock and pests, including mosquitoes that transmit diseases.

The angiotensin converting enzyme (ACE) inhibitor, captopril disrupts the motility activation of sperm from the silkworm, Bombyx mori.

Angiotensin I-converting enzyme (also known as peptidyl dicarboxypeptidase A, ACE, and EC 3.4.15.1), which is found in a wide range of organisms, cleaves C-terminal dipeptides from relatively short oligopeptides. Mammalian ACE plays an important role in the regulation of blood pressure. However, the precise physiological functions of insect ACE homologs have not been understood. As part of our effort to elucidate new physiological roles of insect ACE, we herein report a soluble ACE protein in male reproductive secretions from the silkmoth, Bombyx mori. Seminal vesicle sperm are quiescent in vitro, but vigorous motility is activated by treatment with either a glandula (g.) prostatica homogenate or trypsin in vitro. When seminal vesicle sperm were pre-incubated with captopril, a strong and specific inhibitor of mammalian ACE, and then stimulated to initiate motility by the addition of the g. prostatica homogenate or trypsin, the overall level of acquired motility was reduced in an inhibitor-concentration-dependent manner. In the course of this project, we detected ACE-related carboxypeptidase activity that was inhibited by captopril in both the vesicular (v.) seminalis of the noncopulative male reproductive tract and in the spermatophore that forms in the female bursa copulatrix at the time of mating, just as in an earlier report on the tomato moth, Lacanobia oleracea, which belongs to a different lepidopteran species (Ekbote et al., 2003a). Two distinct genes encoding ACE-like proteins were identified by analysis of B. mori cDNA, and were named BmAcer and BmAcer2, respectively [the former was previously reported by Quan et al. (2001) and the latter was first isolated in this paper]. RT-qPCR and Western blot analyses indicated that the BmAcer2 was predominantly produced in v. seminalis and transferred to the spermatophore during copulation, while the BmAcer was not detected in the adult male reproductive organs. A recombinant protein of BmAcer2 (devoid of a signal peptide) that was expressed in Escherichia coli cells exhibited captopril-sensitive carboxypeptidase activities. Our findings show that the BmAcre2 gene encodes a secreted ACE protein included in the Bombyx seminal plasma. In particular, the silkworm ACE protein in the seminal fluid might be involved in the signaling pathway that leads to the activation and regulation of sperm motility.

Effects of atrazine exposure on male reproductive performance in Drosophila melanogaster.

Atrazine is a commonly utilized herbicide to control broadleaf weeds in the agricultural setting. It can, however, have negative effects on male reproductive performance in a variety of vertebrate species. Much less is known, however, about the effects of atrazine on invertebrates. In this study, we investigated the effects of several different concentrations of larval atrazine exposure on measures of reproductive performance in adult male Drosophila melanogaster. Atrazine exposure had significant effects on a male's mating ability and the number of eggs his partner laid when he was successful at mating. Exposed males also sired a smaller proportion of the offspring under competitive conditions when they were the first male to mate to a doubly mated female. Atrazine exposure had no measurable effect on a male's ability to prevent a mated female from mating to another male or on the proportion of offspring sired when the exposed males were the second male to mate. Exposure upregulated expression of one male reproductive gene, ovulin, but had no effect on expression of another, sex peptide. Exposed males produced and transferred more sex peptide protein to the female during mating but ovulin protein levels were not affected. In general, we observed non-monotonic responses such that the intermediate exposure levels showed the largest reduction in male reproductive performance. This study suggests that atrazine exposure affects male reproductive performance in insects and future studies should aim to understand the molecular mechanisms underlying the fitness effects of exposure.