A comparative protein profile of accessory glands of virgin and mated Leucinodes orbonalis males

Male accessory gland (MAG) secretory proteins affect female reproductive physiology and behaviour when they are delivered to the female at the time of mating along with sperm. In our study, proteomic approaches were used to identify MAG proteins of Leucinodes orbonalis, a monophagous and destructive pest of brinjal. A set of 117 and 186 MAG spots were observed in virgin and mated males, respectively, using two‐dimensional gel electrophoresis (2D‐GE). Among the identified spots, 49 are unique to the virgin, whereas 118 are unique to mated MAGs, with 68 spots found to be common. The differentially expressed MAG proteins, 14 are up‐regulated, and 16 are down‐regulated in virgin after mating. We used matrix‐assisted laser desorption ionization (MALDI)–mass spectrometry (MS) to identify the 13 unique proteins in the MAG of virgin L. orbonalis and analysed the data of the Swiss‐Prot database using the Mascot search engine. The proteins were identified as proteolysis regulators, lipid transporters, olfactory proteins, metabolism‐related proteins, DNA‐binding proteins, and hexamerins. This is the first report on the gel‐based protein analysis of L. orbonalis MAGs.     

Accessory gland proteome of male tobacco cutworm,Spodoptera litura (F.) - An approach to identify proteins influencing reproductive physiology and behaviour

Male-derived accessory glands (MAGs) in insects typically transfer a mixture of proteins and other molecules during mating that have profound effect on female physiology and reproductive behaviour. In the present study, we sought to identify MAG proteins of the serious agricultural pest Spodoptera litura as they have been extensively studied in Drosophila and other insects. A total of 566 distinct proteins across gels of virgin and mated males, as well as 91 differentially expressed proteins in mated contrast to virgin males of S. litura were detected by 2D gel electrophoresis. Here, we successfully identified 20 MAG proteins that appeared in virgin males and depleted in mated males by mass spectrometry, thus predicting the transfer of proteins from males to females during mating. We analyzed the proteins for homology to other proteins with known functions available in Swiss- Prot through Mascot search engine. The proteins were categorized into six diverse functional groups: Metabolism, protein folding, antioxidation, olfaction, DNA binding/replication and cellular activity/response. Most of the MAG proteins fall into the same functional class as in other organisms, although some appear to rapidly evolve. The probable roles of the identified proteins associated with physiology and reproduction have been discussed. Thus, in this study we establish a strong precedent for future research on the potential of MAG proteins to interfere with the pest's own mechanisms resulting in its failure to reproduce successfully.     

Reduction of mating receptivity and changes in longevity of rice leaf bug, <Emphasis Type="Italic">Trigonotylus caelestialium</Emphasis> (Heteroptera: Miridae), females after mating

To develop new and improved pest management strategies against Trigonotylus caelestialium (Kirkaldy) (Heteroptera: Miridae), a major pest of rice in Japan, it is important to understand the mating behavior of this species. In this study, we examined the effect of mating on subsequent mating receptivity and longevity in female T. caelestialium. After mating, females temporarily exhibited decreased mating receptivity. In addition, the cumulative remating frequency of females that mated with a male that had just mated with another female was higher than that of females who mated with virgin males. As a result, we hypothesized that the male ejaculate reduces female mating receptivity. Furthermore, mated females survived longer without food and water than virgin females; on the other hand, the life span of mated females with access to food and water was less than that for virgin females.     

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.     

Reduction in the attraction of males to females after mating in the rice leaf bug Trigonotylus caelestialium

The influence of mating on the extent to which males are attracted to females in Trigonotylus caelestialium (Heteroptera: Miridae) was examined. No differences in attraction of males to mated and virgin females were observed within 3–5 h of mating, but males became less attracted to females 1 to 2 days after the first mating. The difference in male attraction to mated vs virgin females disappeared at 4 days after mating. These results indicate that reduced attraction of males to mated females occurs after a certain time interval, and persists for a few days. Furthermore, males were less attracted to females that had mated with virgin vs recently mated males, i.e. males that had just mated with another female at 1 and 2 days after mating. The ejaculate expenditure of recently mated males was less than that of virgin males. Hence, the amount of male ejaculate transferred to females during mating, rather than the act of mating, might influence the attraction of males to females. The results demonstrate that mating reduces the attraction of males to females in T. caelestialium on the basis of direct observation of male behavior.     

Female mate choice and mating costs in the polyandrous butterflyPieris napi (Lepidoptera: Pieridae)

Males of the green-veined white butterfly (Pieris napi L.) transfer large ejaculates that represent on average 15% of their body mass when mating for a first time. Shortly after mating a male is able to transfer only a small ejaculate when mating a second time. Male ejaculate production plays a crucial role in the mating system ofP. napi because females use male-derived nutrients for egg production and somatic maintenance. Here we study how timing of female rematings and copulation duration are influenced by the mating history of their mates and, also, study if females exert mate choice to minimize their mating costs. Mating with a recently mated male increased female mating costs by increasing time in copula and mating frequency. Virgin females that mated with virgin males remated after an average of 6 days, whereas virgin females that mated with recently mated males remated after an average of 2 days. Moreover, copulations involving recently mated males lasted on average almost 7 h, whereas copulations involving virgin males lasted on average 2 h. Recently mated males were eager to remate, in spite of the fact that the size of the ejaculate they transfer is small and that they remain in copula for a long time. Hence it seems that males are more successful in the sexual conflict over mating decisions and that females do not minimize mating costs by choosing to mate preferentially with virgin males.     

Accessory gland secretory proteins in relation to fitness parameters of Drosophila ananassae and D. varians

Developmental morphometry, qualitative and quantitative analysis of the accessory gland secretory proteins, fecundity and productivity in relation to protein ejected during subsequent (first to fourth-time) matings have been studied in Drosophila ananassae Doleschall and Drosophila varians Bock. In both species, size and secretion of accessory glands increases from 1 to 8 days and the stored secretion ejected from males to the female genital tract during subsequent mating varies. The maximum number of eggs and flies are produced from the females mated with bachelor males and it is a minimum when virgin females are mated with fourth-time mated males. Sodium dodecylsulfate–polyacrylamide gel electrophoresis analysis of accessory gland secretory protein patterns and their glycosylation differs in both the species. Correlation coefficient analysis between gland size and quantity of secretion, percentage of secretory protein transferred per mating, and eggs and flies that emerged showed a highly significant, positive relationship. Among different matings, the number of eggs laid and flies that emerged per female between subsequent (first to fourth-time) matings of males was found to be highly significant and the difference between fecundity and productivity between the two species was highly significant.     

Spermatophore and Sperm Allocation in Males of the Monandrous Butterfly Pararge aegeria: the Female’s Perspective

In insects, spermatophore production represents a non‐trivial cost to a male. Non‐virgin males have been shown to produce small spermatophores at subsequent matings. Particularly in monandrous species, it may be an issue to receive a sufficiently large spermatophore at the first and typically only mating. Females of the monandrous Speckled wood butterfly Pararge aegeria (L.) produce fewer offspring after mating with a non‐virgin male. After mating, females spend all their active time selecting oviposition sites and typically ignore other males. Here, we show that females did not discriminate between a virgin male and a recently mated male in our laboratory experiments. We demonstrate that the number of eupyrene sperm bundles relative to spermatophore mass differed with subsequent male matings. Males transferred a significantly smaller spermatophore after the first copulation, but the spermatophore mass did not decrease further with subsequent matings. However, the number of eupyrene sperm bundles decreased linearly. Therefore, there was proportionally more eupyrene sperm in the male’s second spermatophore compared with the first and the later spermatophores. Such a pattern has been shown in polyandrous species. Hence, it suggests that differences in sperm allocation strategy between polyandrous and monandrous butterflies may be quantitative rather than qualitative. There was also a tendency for females that had mated with a recently mated male to have higher propensity to remate than did females that had mated with a virgin male. We discuss the results relative to the mating system in P. aegeria, including female remating opportunities in the field and male mate‐locating behaviour.     

Effects of delayed mating and male mating history on the reproductive potential of Leucoptera coffeella (Lepidoptera: Lyonetiidae)

Abstract 1 Despite the importance of Leucoptera coffeella (Guérin‐Mèneville) in coffee production worldwide, there is a lack of information on its reproduction. This knowledge will help in mass rearing, and support the development of behavioural control techniques for this insect. The present study determined the effects of delayed mating and previous matings of male L. coffeella on fecundity, egg viability and frequency of female remating. 2 The highest levels of fecundity and egg viability were obtained from matings of 1–3‐day‐old females. When females mated at 5 days of age, there were reductions of 40% in oviposition and of 43% in egg viability. 3 Females mated with 2‐day‐old virgin males were more fecund than those mated with older males; egg viability was also low (18%) from females mated with older males. 4 Virgin females that mated with virgin males laid a greater number of eggs than those mated with previously copulated males. Egg viability decreased with the increase in the number of previous male matings. 5 Five‐day‐old females remated in greater proportion than 2–3‐day‐old females. Females that copulated with males that had previously mated three times had higher rates of remating than those that copulated with virgin males. 6 The results obtained indicate that 1–3 days after emergence is the optimum age for mating. The implications of these findings for the control of L. coffeella by synthetic sex pheromone are discussed.     

Orientation of the Cigarette Beetle, <Emphasis Type="Italic">Lasioderma serricorne</Emphasis> (F.) (Coleoptera: Anobiidae) to Plant-Derived Volatiles

Experiments were conducted to study the orientation of adult cigarette beetles,Lasioderma serricorne (F.), to plant volatiles in a walking bioassay. Seven out of sixteen test materials that displayed attractive responses were further studied for (1) responses to whole extracts from three types of solvents and (2) the effects of sex and mating status of L. serricorne on responses to plant volatiles. Bioassays with extracts revealed that responses of L. serricorne varied among the type of solvent extract. Volatiles from different Capsicum products attracted significantly more adult beetles than did volatiles from other materials. When virgin males, virgin females and mated females were bioassayed, mated females responded significantly more than the other groups to plant volatiles. Semiochemical-based pest management methods may be developed for L. serricorne with plant volatile-based attractants that may be identified and used alone or combined with a synthetic sex pheromone to manipulate beetle behavior. Mention of product or trade name in this paper does not constitute an endorsement or recommendation for its use by Oklahoma State University     

Effects of residual chlorfluazuron on haemolymph-borne oviposition-stimulating factors in Spodoptera litura

Acylurea insecticides can have various effects on insect reproduction and may even interfere with haemolymph‐borne oviposition‐stimulating factors (OSFs). This study describes the effects of injecting haemolymph into females of the common cutworm, Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae), the effects of chlorfluazuron on the activities of OSFs, when sublethal doses (LD₁₀: 1.00 ng larva^?1 or LD₃₀: 3.75 ng larva^?1) were applied topically to newly ecdysed fifth instars under laboratory conditions. The haemolymph of moths of varying age, sex, and mating status resulted in the following increases in oviposition rates within the first 24 h, listed in decreasing order: the haemolymph of mated females injected into mated females (56%), mated females into newly emerged virgin females (53%), virgin males into virgin females (49%), and virgin females into mated females (29%), compared with uninjected mated or virgin females. Two factors are involved in the activation of ovipositon: OSF‐I is found in virgin females, whereas OSF‐II may be formed or received by males during mating. By contrast, the haemolymph taken from chlorfluazuron‐treated adults (treated as fifth instars with sublethal doses) injected into females resulted in the following percentage decreases in oviposition rates within the first 24 h, listed in decreasing order: the haemolymph of LD₁₀‐ and LD₃₀‐treated virgin males injected into untreated virgin females (77 and 84%, respectively), LD₁₀ and LD₃₀‐treated virgin females into untreated mated females (70 and 80%, respectively), LD₁₀ and LD₃₀‐treated mated females into untreated mated females (61 and 69%, respectively), LD₁₀ and LD₃₀‐treated mated females into untreated virgin females (59 or 68%, respectively), compared with untreated ones. Hence, residual chlorfluazuron decreases the activities of OSFs by significantly decreasing the oviposition rates. Moreover, virgin females’ or males’ OSFs are more sensitive to chlorfluazuron than those of mated cutworms. Both OSFs are most likely proteins.     

The impact of mating and sugar feeding on blood-feeding physiology and behavior in the arbovirus vector mosquito Aedes aegypti

BackgroundAedes aegypti mosquitoes are globally distributed vectors of viruses that impact the health of hundreds of millions of people annually. Mating and blood feeding represent fundamental aspects of mosquito life history that carry important implications for vectorial capacity and for control strategies. Females transmit pathogens to vertebrate hosts and obtain essential nutrients for eggs during blood feeding. Further, because host-seeking Ae. aegypti females mate with males swarming near hosts, biological crosstalk between these behaviors could be important. Although mating influences nutritional intake in other insects, prior studies examining mating effects on mosquito blood feeding have yielded conflicting results.Methodology/Principal findingsTo resolve these discrepancies, we examined blood-feeding physiology and behavior in virgin and mated females and in virgins injected with male accessory gland extracts (MAG), which induce post-mating changes in female behavior. We controlled adult nutritional status prior to blood feeding by using water- and sugar-fed controls. Our data show that neither mating nor injection with MAG affect Ae. aegypti blood intake, digestion, or feeding avidity for an initial blood meal. However, sugar feeding, a common supplement in laboratory settings but relatively rare in nature, significantly affected all aspects of feeding and may have contributed to conflicting results among previous studies. Further, mating, MAG injection, and sugar intake induced declines in subsequent feedings after an initial blood meal, correlating with egg production and laying. Taking our evaluation to the field, virgin and mated mosquitoes collected in Colombia were equally likely to contain blood at the time of collection.Conclusions/SignificanceMating, MAG, and sugar feeding impact a mosquito’s estimated ability to transmit pathogens through both direct and indirect effects on multiple aspects of mosquito biology. Our results highlight the need to consider natural mosquito ecology, including diet, when assessing their physiology and behavior in the laboratory.     

Influence of previous mating experience on future mating success in maleLucilia cuprina (Diptera: Calliphoridae)

Receptive and virginL. cuprina females were placed with a virgin male and an experienced male that had mated between one and five times previously. The experienced males secured significantly more matings than virgin males. Males with previous matings also gained experience in competing with males. Males directed mating attempts at each other, seemingly in the context of intrasexual competition. Experienced males directed more mating attempts at virgin males than vice versa. As their number of previous matings increased, experienced males made the first mating attempt at females more often and directed more mating attempts at females compared with virgin males. Females did not actively discriminate against experienced males, even though the proportion of matings secured on the first attempt by experienced males declined with increasing mating experience. Alternative behavioral explanations are discussed.     

Effect of mating status on the fecundity of a cricket,Teleogryllus emma

Abstract Fecundity in some insects is affected by mating status. The effect of mating status on the fecundity and total egg production of Teleogryllus emma (Ohmachi et Matsumura) (Orthoptera: Gryllidae) was examined in this study. The results showed that the pre‐oviposition period was shorter for amphigonic females than that for virgin females. However, no significant difference in pre‐oviposition was found between amphigonic females and those that had mated with a male with either the phallodeum or testes extirpated. There is no difference in adult longevity between the above four groups. The fecundity and total egg production were much higher in amphigonic females than in those controlled under the three non‐amphigonic treatments. The females of T. emma that mated with the testes‐extirpated males produced more eggs (up to two‐fold more) than both the virgin females and those that mated with the phallodeum‐extirpated males, but there was no difference between them. The fecundity‐enhancing substances transferred from male to female can stimulate the female to produce more eggs, but this stimulation has to occur in collaboration with sperm.     

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.     

Volatiles from host plant brinjal attract the brinjal Fruit and Shoot Borer -Leucinodes orbonalis Guenee

Brinjal Fruit and Shoot Borer- Leucinodes orbonalis Guenee is a major insect pest on brinjal- Solanum melongena worldwide. An effective strategy used in developing pest controlling agents is the synergism between insect pheromones and host plant volatiles, which can increase the attraction of insect pest. The present study was aimed at investigating the chemical constituents and attractant effects of the volatiles extracted from different parts of the host plant brinjal on the behavior of adult L. orbonalis. Bioassay using Y-shaped olfactometer revealed that the one-day old virgin female, gravid female and male insects respond positively to the host plant volatiles extracted from fruits, leaves and shoots but not to that of flowers. It was shown that the gravid females were significantly attracted to all three volatiles (p < 0.05). Bioassay using X-shaped olfactometer identified that all three types of insects highly preferred the volatiles from fruits (p < 0.05). Gas chromatography-mass spectrometry analysis of volatiles indicated that brinjal plant produces volatile secondary metabolites, which include 2,2′-(Ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl) dibenzoate (12.11%), 3,7-dimethylocta-1,6-dien-3-ol (22.38%), Benzyl alcohol (22.9%) and Benzyl alcohol (27.06%) as major constituents from fruits, shoots, leaves and flowers respectively. Responses of insects to the volatiles from host plant in the absence of visual cues direct us to focus on the importance of host plant volatiles to locate the plant. Results of this study emphasize the major role that host plant volatiles play in the attraction of insect pests towards the plant.     

Laboratory and field studies on the reproductive ecology of Parocneria orienta (Lepidoptera: Lymantriidae) in China

To lay a foundation for the monitoring, prevention and control of Parocneria orienta, a pest moth in China, we investigated the reproductive ecology of P. orienta, including the environmental factors affecting reproduction and the effects of pheromones in field traps. The results showed that the mating duration is shorter when the temperature is higher, with the optimal temperature range for P. orienta reproduction being 22–27°C. The sex ratio had an effect on the male mating capacity, which significantly increased when the sex ratio was ♀:♂ ≥ 3:1. The average number of matings was 2.44 for males, while female P. orienta mated only once in a lifetime. Males were more attracted to virgin females than to mated females, and the temperature and light period had an effect on the release of sex pheromones by females. Field trap tests using virgin females supported the results obtained in the laboratory, showing that the release of sex pheromones by P. orienta follows a distinct circadian rhythm.     

Differential mating success of virgin female katydidsRequena verticalis (Orthoptera: Tettigoniidae)

Male katydids (Orthoptera: Tettigoniidae) invest in offspring through nutrients provided in a large spermatophore. Previous research with Requena verticalishad shown that almost all of the investment of males mating with recently mated (4 days previously) females is in eggs fertilized by the female's previous mate. Thus males are predicted to discriminate against such females as mates. In experiments placing males with both a virgin and a female mated 4–5 days previously, virgin females obtained almost all matings. Although male discrimination of mates was noted in the experiments, there was no evidence that such discrimination was against nonvirgins in both this experiment and one in which a single virgin or mated female was placed with a male. Instead, the results suggest that the differential mating was a result of interfemale competition. The mating advantage held by virgin females over nonvirgins appeared to be lost once the latter had oviposited. Finally, there was no evidence from both single- and paired-female experiments that males preferred larger females as mates.     

Multiple mating reduces male survivorship but not ejaculate size in the polygamous insect Stenomacra marginella (Heteroptera: Largidae)

In polygamous species, successful males should be able to inseminate multiple females, to defeat sperm from previous males, to avoid sperm displacement by other males, and to induce females to use his sperm during fertilization. Since resources are limited, adaptations to perform any of these functions may conflict with each other (and with other life-history traits) and trade-offs are expected to evolve. We studied if males of the polygamous true bug Stenomacra marginella face a trade-off between multiple mating and survivorship, by comparing the survivorship of virgin and multiply mated males. We also looked for physiological costs of ejaculate production by examining ejaculate production in consecutive matings in multiple mated males. Multiply mated males were able to produce ejaculates of similar size in up to six consecutive copulations but they had decreased survivorship in comparison with virgin males. There was no difference in survivorship between males mated three and six consecutive times, suggesting that the negative relation between survivorship and number of copulations is not linear. The decrease in survivorship seems to be a cost of mating and ejaculate production. This cost could favor the evolution of prudence in the allocation of resources to ejaculate production (e.g., cryptic male choice).     

Mating Status Effects on Sexual Response of Males and Females in the Parasitoid Wasp <Emphasis Type="Italic">Urolepis rufipes</Emphasis>

Although mate preferences are most commonly examined in females, they are often found in both sexes. In the parasitoid wasp Urolepis rufipes, both female and male mating status affected certain aspects of sexual interactions. Female mating status mattered only in the later stages of mating. Males did not discriminate between virgin and mated females in terms of which they contacted or mounted first. However, once mounted, most virgin females were receptive to copulation, whereas very few mated females were. Whether a male’s mating status affected his own sexual response depended on the female’s ability to respond and the stage of mating. Examining male behavior toward dead females allowed elimination of the role of female behavior in how males responded. Virgin and mated males are both attracted to dead females as evidenced by their fanning their wings at such females. However, mated males were quicker than virgin males to contact and to mount in an experiment with dead females, whereas there was no such differential response in an experiment with live females. This difference is consistent with greater female sexual responsiveness to virgin males. Male mating status also affected female receptivity to copulate. Once mounted, live virgin females were less likely to become receptive to copulation by mated males than to virgin males, but only in a choice experiment, not in a no-choice experiment.