Effect of Male Accessory Gland Products on Egg Laying in Gastropod Molluscs

In internally fertilizing animals, seminal fluid is usually added to the spermatozoa, together forming the semen or ejaculate. Besides nourishing and activating sperm, the components in the seminal fluid can also influence female physiology to augment fertilization success of the sperm donor. While many studies have reported such effects in species with separate sexes, few studies have addressed this in simultaneously hermaphroditic animals. This video protocol presents a method to study effects of seminal fluid in gastropods, using a simultaneously hermaphroditic freshwater snail, the great pond snail Lymnaea stagnalis, as model organism. While the procedure is shown using complete prostate gland extracts, individual components (i.e., proteins, peptides, and other compounds) of the seminal fluid can be tested in the same way. Effects of the receipt of ejaculate components on egg laying can be quantified in terms of frequency of egg laying and more subtle estimates of female reproductive performance such as egg numbers within each egg masses. Results show that seminal fluid proteins affect female reproductive output in this simultaneous hermaphrodite, highlighting their importance for sexual selection.     

A Syringe-Like Love Dart Injects Male Accessory Gland Products in a Tropical Hermaphrodite

Sexual conflict shapes the evolution of many behaviours and processes involved in reproduction. Nearly all evidence supporting this comes from species where the sexes are separated. However, a substantial proportion of animals and most plants are hermaphroditic, and theoretical work predicts that sexual conflict plays an important role even when the sexes are joined within one individual. This seems to have resulted in bizarre mating systems, sophisticated sperm packaging and complex reproductive morphologies. By far the best-known example of such a strategy in hermaphrodites is the shooting of so-called love-darts in land snails. All known love darts carry a gland product on their outside and enter this into the partner’s hemolymph by stabbing. Here, we show that species of the snail genus Everettia possess a syringe-like dart that serves as a real injection needle. Their dart is round in cross-section, contains numerous channels, and has perforations along its side. Histology and electron microscopy show that these holes connect to the channels inside the dart and run all the way up to the elaborate mucus glands that are attached to the dart sac. This is the first report on a love dart that is used as a syringe to directly inject the gland product into the partner’s hemolymph. Although the exact use and function of this dart remains to be demonstrated, this clearly adds to the complexity of the evolution of reproductive strategies in hermaphrodites in general. Moreover, the perforations on the outside of the love dart resemble features of other injection devices, thus uncovering common design and repeated evolution of such features in animals.     

Egg Trading in the Chalk Bass,Serranus tortugarum,a Simultaneous Hermaphrodite

The simultaneously hermaphroditic fish, Hypoplectrus nigricans, engages in a form of reciprocal spawning called “egg trading”, where one partner gives up eggs for fertilization in exchange for the opportunity to fertilize those of the partner. Since egg trading appears to be important in the evolutionary maintenance of hermaphroditism in H. nigricans, an attempt was made to determine if another species, Serranus tortugarum, behaved in a similar fashion. S. tortugarum do trade eggs, and the phenomenon seems to be widespread in simultaneously hermaphroditic serranids. However, the cues for spawning are much subtler than in H. nigricans and other simultaneously hermaphroditic serranids. This trait appears to be an evolutionary response to the occurrence of heavy sperm competition in the form of streaking.     

Receipt of Seminal Fluid Proteins Causes Reduction of Male Investment in a Simultaneous Hermaphrodite

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昆虫雄性生殖腺分泌物的功能

昆虫两性成功交配后,雄性生殖腺分泌物使雌性昆虫的生理和行为都发生了巨大的改变。昆虫雄性生殖腺分泌物含有多种具有生物活性的分子,这些生物活性分子通过成功交配转移到雌虫生殖道后,对雌虫的生殖活动产生影响,使交配雌虫一段时间内不再交配,使已转移的精子易于在雌虫生殖道内储藏,使卵与精子完成受精过程,还可刺激雌虫产卵和卵的发育,调控排卵和产卵等生殖过程。在精子的转移过程中,雄性生殖腺分泌物中的抗菌媒介质能使雌虫的生殖导管提供友好的环境。此外,一些昆虫的雄性生殖腺分泌物还含有一些有毒的化学物质,保护已产下的卵不被天敌取食和病原体侵染。     

Accessory Gland as a Site for Prothoracicotropic Hormone Controlled Ecdysone Synthesis in Adult Male Insects

Insect steroid hormones (ecdysteroids) are important for female reproduction in many insect species and are required for the initiation and coordination of vital developmental processes. Ecdysteroids are also important for adult male physiology and behavior, but their exact function and site of synthesis remains unclear, although previous studies suggest that the reproductive system may be their source. We have examined expression profiles of the ecdysteroidogenic Halloween genes, during development and in adults of the flour beetle Tribolium castaneum. Genes required for the biosynthesis of ecdysone (E), the precursor of the molting hormone 20-hydroxyecdysone (20E), are expressed in the tubular accessory glands (TAGs) of adult males. In contrast, expression of the gene encoding the enzyme mediating 20E synthesis was detected in the ovaries of females. Further, Spookiest (Spot), an enzyme presumably required for endowing tissues with competence to produce ecdysteroids, is male specific and predominantly expressed in the TAGs. We also show that prothoracicotropic hormone (PTTH), a regulator of E synthesis during larval development, regulates ecdysteroid levels in the adult stage in Drosophila melanogaster and the gene for its receptor Torso seems to be expressed specifically in the accessory glands of males. The composite results suggest strongly that the accessory glands of adult male insects are the main source of E, but not 20E. The finding of a possible male-specific source of E raises the possibility that E and 20E have sex-specific roles analogous to the vertebrate sex steroids, where males produce primarily testosterone, the precursor of estradiol. Furthermore this study provides the first evidence that PTTH regulates ecdysteroid synthesis in the adult stage and could explain the original finding that some adult insects are a rich source of PTTH.     

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.     

Seeking a Sex‐Specific Coolidge Effect in a Simultaneous Hermaphrodite

In polygamous mating systems, a capability to discriminate against familiar mates may be beneficial to both sexes. Polyandrous females, for instance, may enhance the odds of finding sires with optimal genetic compatibility or high genetic quality by mating with multiple different males; polygynous males, in addition, may more efficiently invest their limited ejaculates across multiple (rather than single) females. The Coolidge effect facilitates this kind of mate discrimination, as sexual motivation declines across consecutive copulations with a familiar partner but resurrects with a novel mate. In simultaneous hermaphrodites, we expect the Coolidge effect to show sex role‐dependent patterns and vary with previous sex‐specific mating activity. Using the promiscuous hermaphroditic sea slug Chelidonura sandrana, we investigated (1) whether sexual motivation indeed declines when repeatedly exposed to familiar partners, (2) whether the Coolidge effect occurs in a sex‐specific manner and (3) whether ejaculation is strategic with respect to partner familiarity. We found neither mating latency, nor penis intromission duration, mating propensity or the frequency of sex role alternations to vary significantly with treatments. Furthermore, slugs did not donate larger ejaculates to novel than to familiar partners. Partner novelty thus elicited no detectable response in sexual motivation or mating effort in C. sandrana. We suggest that the sea slugs' promiscuous mating system in often large mating aggregations makes mate discrimination based on novelty obsolete in comparison with more relevant criteria such as partner body size or mating history.     

Seminal Fluid-Mediated Manipulation of Post-mating Behavior in a Simultaneous Hermaphrodite

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Variation in Sperm Displacement and Its Association with Accessory Gland Protein Loci in Drosophila Melanogaster

Genes that influence mating and/or fertilization success may be targets for strong natural selection. If females remate frequently relative to the duration of sperm storage and rate of sperm use, sperm displacement may be an important component of male reproductive success. Although it has long been known that mutant laboratory stocks of Drosophila differ in sperm displacement, the magnitude of the naturally occurring genetic variation in this character has not been systematically quantified. Here we report the results of a screen for variation in sperm displacement among 152 lines of Drosophilia melanogaster that were made homozygous for second and/or third chromosomes recovered from natural populations. Sperm displacement was assayed by scoring the progeny of cn;bw females that had been mated sequentially to cn;bw and tested males in either order. Highly significant differences were seen in both the ability to displace sperm that is resident in the female's reproductive tract and in the ability to resist displacement by subsequent sperm. Most lines exhibited nearly complete displacement, having nearly all progeny sired by the second male, but several lines had as few as half the progeny fathered by the second male. Lines that were identified in the screen for naturally occurring variation in sperm displacement were also characterized for single-strand conformation polymorphisms (SSCP) at seven accessory gland protein (Acp) genes, Glucose dehydrogenase (Gld), and Esterase-6 (Est-6). Acp genes encode proteins that are in some cases known to be transmitted to the female in the seminal fluid and are likely candidates for genes that might mediate the phenomenon of sperm displacement. Significant associations were found between particular Acp alleles at four different loci (Acp26Aa/Ab, Acp29B, Acp36DE and Acp53E) and the ability of males to resist displacement by subsequent sperm. There was no correlation between the ability to displace resident sperm and the ability to resist being displaced by subsequent sperm. This lack of correlation, and the association of Acp alleles with resisting subsequent sperm only, suggests that different mechanisms mediate the two components of sperm displacement.     

Ecdysteroid Stimulated Protein Synthesis in the Male Accessory Reproductive Glands of Spodoptera litura

Summary

20-hydroxyecdysone stimulates protein synthesis in the male accessory reproductive glands of pharate adults of Spodoptera litura both in vivo and in vitro. Juvenile hormone-I has no synergistic effect when given in conjunction with ecdysone in vivo but it inhibits ecdysone-stimulated protein synthesis in vitro.     

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.     

The Female Post-Mating Response Requires Genes Expressed in the Secondary Cells of the Male Accessory Gland in Drosophila melanogaster

Seminal proteins from the Drosophila male accessory gland induce post-mating responses (PMR) in females. The PMR comprise behavioral and physiological changes that include increased egg laying, decreased receptivity to courting males, and changes in the storage and use of sperm. Many of these changes are induced by a “sex peptide” (SP) and are maintained by SP’s binding to, and slow release from, sperm. The accessory gland contains two secretory cell types with distinct morphological and developmental characteristics. Products of these “main” and “secondary” cells work interdependently to induce and maintain the PMR. To identify individual genes needed for the morphology and function of secondary cells, we studied iab-6^cocu males, whose secondary cells have abnormal morphology and fail to provide products to maintain the PMR. By RNA-seq, we identified 77 genes that are downregulated by a factor of >5× in iab-6^cocu males. By functional assays and microscopy, we tested 20 candidate genes and found that at least 9 are required for normal storage and release of SP in mated females. Knockdown of each of these 9 genes consequently leads to a reduction in egg laying and an increase in receptivity over time, confirming a role for the secondary cells in maintaining the long-term PMR. Interestingly, only 1 of the 9 genes, CG3349, encodes a previously reported seminal fluid protein (Sfp), suggesting that secondary cells may perform essential functions beyond the production and modification of known Sfps. At least 3 of the 9 genes also regulate the size and/or abundance of secondary cell vacuoles, suggesting that the vacuoles’ contents may be important for the machinery used to maintain the PMR.     

Neuroendocrine Regulation of Egg Laying in Aplysia californica

Two clusters of neurons, the bag cells, associated with thecentral nervous system of Aplysia californica play an essentialrole in the induction of egg laying by the animal. Studies concernedwith the morphology, electrophysiology, biochemistry, and functionof these cells are reviewed and discussed. The unusually favorablecharacteristics of this preparation suit it for developmentas a model neuroendocrine effector system.     

A Mutation in the Age-1 Gene in Caenorhabditis Elegans Lengthens Life and Reduces Hermaphrodite Fertility

age-1(hx546) is a recessive mutant allele in Caenorhabditis elegans that results in an increase in mean life span averaging 40% and in maximal life span averaging 60% at 20°; at 25° age-1(hx546) averages a 65% increase in mean life span (25.3 days vs. 15.0 days) and a 110% increase in maximum life span (46.2 days vs. 22.0 days for wild-type hermaphrodites). Mutant males also show extended life spans. age-1(hx546) is associated with a 75% decrease in hermaphrodite self-fertility as compared to the age-1(+) allele at 20°. Using two novel strategies for following the segregation of age-1, we present evidence that longer life results from a mutation in a single gene that increases the probability of survival at all chronological ages. The long-life and reduced-fertility phenotypes cosegregate and are tightly linked to fer-15, a locus on linkage group II. age-1(hx546) does not affect the timing of larval molts, the length of embryogenesis, food uptake, movement, or behavior in any way tested. Although age-1(hx546) lowers hermaphrodite self-fertility, it does not markedly affect the length of the reproductive period with all the increase in life expectancy due to an increase in the length of postreproductive life. In so far as we are aware, this mutant in age-1 is the only instance of a well-characterized genetic locus in which the mutant form results in lengthened life. It is likely that the action of age-1 in lengthening life results not from eliminating a programmed aging function but rather from reduced hermaphrodite self-fertility or from some other unknown metabolic or physiologic alteration.