Accessory Gland Substances Research Articles

Functions of Egg-Coating Substances Secreted by Female Accessory Glands in Alderflies, Fishflies and Dobsonflies (Megaloptera).

Simple SummaryEgg masses of the insect order Megaloptera are coated with female accessory gland substances and are diverse in shape, color, and surface structure. Female alderflies (family Sialidae) and fishflies (family Corydalidae, subfamily Chauliodinae) lay eggs in a single layer on the substrate (although the eggs are occasionally multi-layered in the fishfly genus Nigronia), and the accessory gland substances are usually brown. In contrast, dobsonflies (Corydalidae, Corydalinae) lay a multi-layered hemispherical egg mass, and the accessory gland substances differ in color and chemical properties among genera and species. Egg masses of the dobsonfly genus Protohermes are coated with a sticky substance, while those of other dobsonflies are covered with hardened white powders, both of which contribute to the prevention of egg desiccation. The accessory gland substances of most species of Megaloptera also function to protect eggs from attack by oophagous predators, such as ladybird beetles and ants. The information on the parasitoids of Megaloptera egg masses are also discussed based on the present and previously reported observations.Eggs of insects are immobile and must endure harsh environmental conditions (e.g., low temperatures in winter and aridity in summer) and avoid attack by egg-eating predators, egg parasites, and microbes. Females of Megaloptera lay their eggs as a single- or multi-layered egg mass, which is coated with chemical substances secreted from the female reproductive accessory glands. In this study, we observed the egg masses laid by females of two species of Sialidae (alderflies), nine species of Chauliodinae (fishflies), and 23 species of Corydalinae (dobsonflies) belonging to the order Megaloptera and examined the functions of accessory gland substances coating the laid eggs. The female accessory gland is a single tube in alderflies and fishflies but a paired pouch in dobsonflies. The amount and color of the gland substances differ greatly among species. These substances prevent egg desiccation, inhibit egg feeding by ladybird beetles, and repel ants. Most characteristics of the egg mass structures and the effectiveness of accessory gland substances reflect the phylogeny of Megaloptera, although some differ among closely related taxa.

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

Simple SummaryIn insects, it is known that the diet during the larval stage affects traits in the adult stage. However, it is still unclear how it affects reproductive traits such as ejaculation. The ejaculate contains many proteins and therefore requires much nutrition, so the larval diet strongly influences it. Males of the West Indian sweet potato weevil Euscepes postfasciatus use accessory gland substances to inhibit remating by females. Crossing experiments were conducted using lines reared on artificial diets or sweet potato tubers during the larval stage, and the refractory period was examined. The results showed that the larval stage diet had a significant effect on the refractory period of females. We also found one protein of approximately 15 kDa in size expressed only in the treatments reared on sweet potatoes. To our knowledge, this is the first study to show that larval diet qualitatively influences male ejaculate and female refractory period.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.

Male body size does not affect the refractory period of females in the West Indian sweet potato weevil Euscepes postfasciatus (Fairmaire) (Coleoptera: Curculionidae) and the seed bug Togo hemipterus (Scott) (Heteroptera: Lygaeidae)

Multiple mating by females increases both the risk and intensity of sperm competition for males. The ability to temporarily or permanently inhibit female receptivity to further mating represents a highly adaptive strategy to reduce the risk of sperm competition. Males of the West Indian sweet potato weevil Euscepes postfasciatus (Fairmaire) (Coleoptera: Curculionidae) and the seed bug Togo hemipterus (Scott) (Heteroptera: Lygaeidae) use accessory gland substances to inhibit remating in females. It is known that there is variance in the length of the female refractory period (i.e., the effectiveness of male ejaculate substances that inhibit female remating) within a species. However, it is unclear what types of male traits affect the length of the female refractory period. The mechanism of the female refractory period is not sufficiently revealed. We focused on male body size, which is often a target of sexual selection, and investigated the relationship between male body size and both the refractory period of females and mating duration. Contrary to our thought, male body size did not significantly influence the refractory period of females or mating duration in either species. Smaller male body size was not particularly favored by post-copulatory sexual selection in either species.

Hidden female physiological resistance to male accessory gland substances in a simultaneous hermaphrodite.

ABSTRACTTo increase fertilization chances compared with rivals, males are favoured to transfer accessory gland proteins to females during mating. These substances, by influencing female physiology, cause alteration of her sperm usage and remating rate. Simultaneously hermaphroditic land snails with love-darts are a case in point. During courtship, a love-dart is pierced through the partner's body wall, thereby introducing accessory mucous gland products. This mucus physiologically increases paternity by inhibiting the digestion of donated sperm. The sperm, which are packaged in a spermatophore, are exchanged and received in an organ called the diverticulum. Because of its length, this organ was previously proposed to be a female anatomical adaptation that may limit the dart interference with the recipient's sperm usage. For reproductive success of the donor, an anatomically long spermatophore, relative to the partner's diverticulum, is beneficial as sperm can avoid digestion by exiting through the spermatophore's tail safely. However, the snail Eobania vermiculata possesses a diverticulum that is three times longer than the spermatophore it receives. Here, we report that the love-dart mucus of this species contains a contraction-inducing substance that shortens the diverticulum, an effect that is only properly revealed when the mucus is applied to another helicid species, Cornu aspersum. This finding suggests that E. vermiculata may have evolved a physiological resistance to the manipulative substance received via the love-dart by becoming insensitive to it. This provides useful insight into the evolution of female resistance to male manipulations, indicating that it can remain hidden if tested on a single species.

Satyrization without evidence of successful insemination from interspecific mating between invasive mosquitoes.

Previous research has documented low frequencies of interspecific mating in nature between the invasive vectors Aedes aegypti and Aedes albopictus. It is also known that heterospecific male accessory gland substances transferred during mating sterilize A. aegypti but not A. albopictus females, leading to satyrization, a form of reproductive interference. This paper demonstrates that satyrization of A. aegypti by A. albopictus may occur without evidence of successful insemination. Our results show that A. aegypti females, previously exposed to A. albopictus males, are rendered refractory to subsequent conspecific mating even though their spermathecae contain no heterospecific sperm. Additional experiments demonstrating transfer of labelled semen from A. albopictus males to A. aegypti females and low production of viable eggs of females housed with conspecific males, following exposure to A. albopictus males, confirm higher incidences of satyrization than expected, based on heterospecific insemination rates. We conclude that frequencies of satyrization based on detection of interspecific sperm in spermathecae may underestimate the impact of this form of reproductive interference.

Male Aedes aegypti mosquitoes use JH III transferred during copulation to influence previtellogenic ovary physiology and affect the reproductive output of female mosquitoes

The effect of male accessory gland substances on female reproductive physiology has been previously described as “activating” egg development. However, no mechanism has been described that can explain how male mosquitoes are able to influence egg development in female mosquitoes. To investigate how male mosquitoes are able to influence ovarian physiology and reproductive output we explored three main questions: (1) Do mating and male accessory gland substances affect ovarian physiology and alter markers of oocyte quality during the previtellogenic resting stage? (2) Does the male accessory gland contain JH III and is JH III transferred to the female during copulation? (3) Finally, does the nutritional history of the male affect the amount of JH III transferred to the female and alter reproductive output? By answering these questions it is clear that male mosquitoes are able to alter the female’s resource allocation priorities towards reproduction by transferring JH III during copulation; reducing the rate of previtellogenic resorption and increasing the amount of stored ovarian lipids. These changes improve an individual follicle’s likelihood of development after a blood meal. In addition, males maintained under better nutritional conditions make and transfer more JH III, prevent more follicular resorption and realize higher fecundities than other males. Together these results illustrate one mechanism behind the “activating” effect of mating described as well as the role sugar feeding plays in male mosquitoes.

Inhibition of female mating by male accessory gland substances in the ground beetle Leptocarabus procerulus

AbstractThe ground beetle Leptocarabus procerulus (Chaudoir) possesses seminal substances that have a physical function to form mating plugs and a physiological function to induce female refractory behaviour, which act together to hinder female remating. Little is known about the physiological properties of the substances inducing female refractory behaviour, especially with respect to their secretory organ, dose‐dependency, molecular characteristics and the effect of female maturity. By injecting male‐derived substances into females, substances that induce female refractory behaviour are shown to be produced in the male accessory gland but not in the testis. Injection of extracts from the accessory gland increases the female refractory period at moderate doses but not at lower or higher doses. By contrast, injection of extracts from the testis reduces the female refractory period at high doses. The lack of an effect of accessory gland substances at higher doses could be the result of an anomalous effect of unnaturally large doses of seminal products by direct injection, the toxicity of seminal substances that deter female responses, or counteraction by injected substances that promote female receptivity. The accessory gland substances lose their activity when heated, although the testis substances do not. Females without mature eggs tend to reject mating entirely, although variation in the number of mature eggs (one or more) is not associated with the female refractory period, indicating the limited effect of female reproductive maturity. These findings may help to clarify the physiological basis of the evolution of the elaborated male mating behaviour in L. procerulus.

Male accessory gland substances from Aedes albopictus affect the locomotor activity of Aedes aegypti females

Dengue is one of the world’s most important mosquito-borne diseases and is usually transmitted by one of two vector species: Aedes aegypti or Aedes albopictus . These two diurnal mosquitoes are frequently found coexisting in similar habitats, enabling interactions between adults, such as cross-mating. The objective of this study was to assess cross-mating between Ae. aegypti females and Ae. albopictus males under artificial conditions and evaluate the locomotor activity of Ae. aegypti virgin females injected with male accessory gland (MAG) homogenates to infer the physiological and behavioural responses to interspecific mating. After seven days of exposure, 3.3-16% of Ae. aegypti females mated with Ae. albopictus males. Virgin Ae. aegypti females injected with conspecific and heterospecific MAGs showed a general decrease in locomotor activity compared to controls and were refractory to mating with conspecific males. The reduction in diurnal locomotor activity induced by injections of conspecific or heterospecific MAGs is consistent with regulation of female reproductive activities by male substances, which are capable of sterilising female Ae. aegypti through satyrisation by Ae. albopictus .

Males of the seed bug Togo hemipterus (Heteroptera: Lygaeidae) use accessory gland substances to inhibit remating by females

In species in which females mate repeatedly, males can adopt several strategies to reduce the risk of sperm competition with future males. The refractory period of females significantly increased as the mating duration increased in the seed bug Togo hemipterus (Heteroptera: Lygaeidae). To elucidate the mechanisms by which mated females are inhibited from remating, we investigated the effects of male-derived substances on the inhibition of mating receptivity of virgin females by injecting the substances into their abdomens. The length of time from injection to mating in virgin females was significantly longer for females injected with accessory gland B solution than for those injected with seminal vesicle, accessory gland A, or control solutions. This is the first report showing that heteropteran males inhibit female remating by using substances from an accessory gland. We discuss and consider the adoption and evolution of this strategy by T. hemipterus males by focusing on female genitalia structures, oviposition habit, and paternity and comparing these traits with those of other heteropterids.

Identity and transfer of male reproductive gland proteins of the dengue vector mosquito, Aedes aegypti: Potential tools for control of female feeding and reproduction

Male reproductive gland proteins (mRGPs) impact the physiology and/or behavior of mated females in a broad range of organisms. We sought to identify mRGPs of the yellow fever mosquito, Aedes aegypti, the primary vector of dengue and yellow fever viruses. Earlier studies with Ae. aegypti demonstrated that “matrone” (a partially purified male reproductive accessory gland substance) or male accessory gland fluid injected into virgin female Ae. aegypti affect female sexual refractoriness, blood feeding and digestion, flight, ovarian development, and oviposition. Using bioinformatic comparisons to Drosophila melanogaster accessory gland proteins and mass spectrometry of proteins from Ae. aegypti male accessory glands and ejaculatory ducts (AG/ED) and female reproductive tracts, we identified 63 new putative Ae. aegypti mRGPs. Twenty-one of these proteins were found in the reproductive tract of mated females but not of virgin females suggesting that they are transferred from males to females during mating. Most of the putative mRGPs fall into the same protein classes as mRGPs in other organisms, although some appear to be evolving rapidly and lack identifiable homologs in Culex pipiens, Anopheles gambiae, and D. melanogaster. Our results identify candidate male-derived molecules that may have an important influence on behavior, survival, and reproduction of female mosquitoes.

Temporal patterns in reproduction may explain variationin mating frequencies in the green-veined white butterfly Pieris napi

There is genetic variation in the female mating rate in the green-veined white butterfly (Pieris napi), and females benefit from male ejaculates that contain both sperm and accessory gland substances. Although polyandry corresponds to higher lifetime fecundity than monandry, some females abstain from remating irrespective of the number of available mates. Explaining genetic variation in mating rates requires that monandrous females perform better than polyandrous ones under some conditions. We experimentally explored the reproductive performance of females either with a low or high intrinsic mating rate by allowing them to mate, feed, and lay eggs freely in a laboratory. Individual females followed different life histories: during the early days of reproduction, females with a low mating rate produced more eggs than females with a high mating rate. Hence, refraining from the benefits of multiple mating may be beneficial, if the time for reproduction is limited, or other female traits associated with polyandry are traded off against longevity. Given the day length of 10 h, a model shows that even if polyandrous females enjoy higher lifetime reproductive success, changeable and unpredictable weather will favor monandry if each period of suitable weather lasts, on average, less than 5 days. Thus, a combination of life history cost and unpredictability of fitness may explain the maintenance of monandry in the wild. Our results are also consistent with the observation that frequency of monandry increases with latitude.

Switchover to the mated state by spermathecal activation in female Anopheles gambiae mosquitoes

Both Aedes aegypti and Anopheles gambiae mosquitoes undergo physiological and behavioral changes after the females mate, but unlike Ae. aegypti, the mated state in An. gambiae is not attained through the action of male accessory gland substances. Experiments in which the spermathecae of mated females were manipulated suggest that a spermatheca filled with sperm is responsible for triggering oviposition behavior. An estimated 48% of the previously mated An. gambiae females mated again when the interval between encounters with males was less than 24 h. Unlike male Ae. aegypti that can inseminate up to seven females after their testes are removed, An. gambiae do not store sperm in the vas deferens and without testes would be unable to inseminate any females.

The Male Accessory Gland Substances of Lucilia illustris (I): Its Effect on Mating Receptivity

ABSTRACT In order to verify the physiological functions of male accessory gland (MAG) substances of the blowfly, Lucilia illustris Meigen, the growth and changes in metabolites of male accessory gland with aging, and the effect of male accessory gland extract on mating receptivity of L. illustris were examined. There was not a significant difference in length of MAG with aging, but the width of MAG was grown widely from 0 to 5‐days old apparently. Changes in glycogen, total protein and lipid of male accessory gland substances showed same patterns but total protein content was higher than the others. Therefore, male nutrition affected the production of male accessory proteins. During the mating fed unmated male transferred larger amount of protein than that of starved male. When male accessory gland homogenates injected, mating receptivity of gravid female was greatly inhibited comparing to control, suggesting that the accessory gland substances of L. illustris alters female mating receptivity after mating.

Sexual receptivity in Anopheles gambiae mosquitoes: absence of control by male accessory gland substances

Although male accessory gland substances modulate female receptivity in most mosquito genera that have been studied, they are not active in the development of sexual refractoriness in the malaria vectors, Anopheles gambiae or Anopheles albimanus. Neither the implantation of male accessory glands nor the injection of gland homogenates affected the insemination rate of unmated females. Similarly, portions of the male genital tract and spermathecae from mated females were also inactive when introduced into these females. Interruption of nervous transmission from the brain reduced the tendency of females to become inseminated and removal of the terminal abdominal ganglion completely abolished insemination. Oviposition behavior did not occur unless the spermatheca containing sperm was present, suggesting that this may be the mechanism that mediates the switchover to mated behavior.

Exogenous juvenile hormone and methoprene, but not male accessory gland substances or ovariectomy, affect the blood/nectar choice of female Culex nigripalpus mosquitoes.

When newly emerged females of the mosquito Culex nigripalpus Theobald (Diptera: Culicidae) take a sugar meal, they develop both a propensity to seek a host and resting-stage ovarian follicles. As follicle growth is indicative of Juvenile Hormone (JH) activity, we searched for possible roles of JH in the behavioural shift from nectar to blood-host odour preference by Cx. nigripalpus after emergence, using an olfactometer-choice assay. Topically applied or injected methoprene (a JH analogue) resulted in a behavioural shift and follicular growth in unfed females that increased with increasing dosages. Topical methoprene 500 ng and 4 microg resulted in a switch to bird-odour preference. JH III injection resulted in a lesser shift from honey responses to bird responses. Methoprene application caused no detectable changes in glycogen, total sugars or total lipid when assayed 24h after treatment. Additionally, as male accessory glands (MAG) have been shown to synthesize JH, we implanted intact paired MAGs from either conspecific or Aedes aegypti (L.) donors, or injected 1/3 gland pair equivalents of conspecific MAG homogenate into unfed newly emerged females. All MAG treatments failed to induce behavioural or ovarian modifications. Ovariectomy had no effect on the sugar-induced shift from nectar to host preference in the olfactometer. Thus JH, but not MAG, mimicked the effects of a sugar meal by causing both follicular growth and the shift to preference for a host.

Male butterfly investment in successive ejaculates in relation to mating system

In Lepidoptera, female mating systems range from strict monandry to strong polyandry. Males transfer an ejaculate during copulation that contains both sperm and accessory gland substances. In butterflies the male ejaculate has at least three effects: it (1) contains sperm that can fertilize the eggs of the male’s mating partner, (2) influences the refractory period of the mated female, and (3) contains nutrients that can be used by the female to increase her reproductive output. A number of recent studies have shown that males in polyandrous mating systems are endowed with adaptations to increase mating capabilities. Relative to males in more monandrous species they transfer larger first ejaculates which contain relatively more protein, and allocate proportionally more resources to reproduction. The objective of this study was to compare male reproductive investment in successive ejaculates, in terms of both mass and quality, to determine if males of polyandrous species are capable of maintaining the production of larger and more nutritious ejaculates than males of more monandrous species. We used three species of pierid butterfly, with mating systems ranging from relative monandry to polyandry. The degree of polyandry had a substantial effect on the reproductive performance of males. The cumulative protein content of ejaculates increased with the degree of polyandry. Only males of the most polyandrous species, Pieris rapae, produced three successive ejaculates of similar mass and protein content. In the relatively monandrous species, Aporia crataegi and P. brassicae, males in subsequent matings never produced another ejaculate as large as that transferred by males mating for the first time, although the protein content (mg) of ejaculates did not differ in second and third ejaculates. Moreover, the ability to remate varied with the degree of polyandry. Given the opportunity to remate, the majority of P. rapae males mated three times, whereas relatively few A. crataegi and P. brassicae males were able to perform three matings. These results suggest that male capacity to produce large, nutritious ejaculates is limited in relatively monandrous species. In this study only males belonging to the most polyandrous species had the ability to recuperate quickly from a mating event and remate. Our results suggest that males in polyandrous systems are better adapted to mating more than once.

Protein content of spermatophores in relation to monandry/polyandry in butterflies

The evolution of a mating system, and specifically mating frequency, is dependent on the costs and benefits to both sexes of mating once or several times. In butterflies, males transfer a spermatophore that contains both sperm and accessory gland products. Accessory gland substances contain nutrients which, in some species, females use to increase their reproductive output and longevity. Nutrients contained in these packaged ejaculates represent investment by males in reproduction. Consequently, the nutritional composition of spermatophores may vary depending on the mating system. There are two lines of arguments concerning the evolution of the nutrient content of ejaculates. One hypothesis argues that male nuptial gifts evolved in the context of certainty of paternity and ease of finding mates; thus spermatophores of polyandrous males (with lower certainty of paternity and greater ease of finding mates) should contain less protein than those of monandrous males, since more spermatophores are produced on average. The other hypothesis argues that polyandry evolved in the context of maximization of male transfer of nutrients to females, and hence spermatophores of polyandrous males should contain more protein than those of monandrous males. In an attempt to distinguish between these two hypotheses, we determined how protein content of ejaculates varied with the degree of polyandry in nine species of pierid and two species of satyrid butterflies. We found that both relative ejaculate mass and protein content increased with the degree of polyandry. Hence our results are consistent with the view that polyandry has evolved in the context of male transfer of nutrients to females, and provides another example of a male adaptation to multiple mating in butterflies.

Male accessory gland substances modify the host-seeking behavior of gravid Aedes aegypti mosquitoes

Male accessory gland (MAG) substances of the mosquito, Aedes aegypti (L.), when transferred during mating or injected directly into females, significantly reduced the host-seeking behavior of the females when they were gravid. The effect on female behavior was modulated by the nutritional state of the male; compared to well-fed males, starved males contained less total protein in their MAGs, transferred less protein to females during mating, and had less of an effect on the inhibition of host-seeking behavior by the gravid females to which they mated. Juvenile hormone (JH) administered to starved males increased the levels of total MAG proteins in their accessory glands, but the host-seeking behavior of gravid females mated to JH-treated males was not significantly affected, suggesting that the production of specific substances did not increase. Injection of MAG homogenates from other mosquito species into Ae. aegypti indicated that there was some degree of specificity in the inhibition of the host-seeking behavior.

Multiple mating in female mosquitoes—evidence from a field population of Anopheles freeborni (Diptera: Culicidae)

AbstractWe determined how frequently multiple mating occurs in a field population of Anopheles freeborni Aitken. We used enzyme electrophoresis to analyse field collected females and their offspring, and determined the parity rates of females mating in swarms. The genotypes of progeny in one of the 36 families examined showed conclusive evidence of two sires. 4·3% (n═93) of females mating in swarms were parous, as opposed to 33·9% of host seeking females (n═103). We conclude that multiple mating in A. freeborni occurs at low frequency in the field, and may be ascribed to incomplete transfer of sperm or male accessory gland substance, rather than to routine renewal of receptivity by females.

Anopheles culicifacies: effects of age on the male reproductive system and mating ability of virgin adult mosquitoes.

Under controlled laboratory conditions of 28-30 degrees C and 16:8 L:D photoperiod, an attempt was made to develop an age-grading technique for Anopheles culicifacies males. Mating activity was maximal when females were 5-12 days old males were 5-7 days old. The numbers of total and mature spermatocysts declined significantly with age, and the proportion of the testes occupied by the sperm reservoir increased as virgin males grew older. Mating resulted in the loss of spermatozoa and accessory gland substance from the reproductive system. Loss of mating ability of older virgin males seemed to be age-related, because the reproductive system contained ample supplies of accessory gland substance and spermatozoa. Morphological changes of the reproductive system, due to mating and age, were used to infer the age and reproductive history of unknown males in a laboratory evaluation.