A Sexually Conditioned Switch of Chemosensory Behavior in C. elegans

In sexually reproducing animals, mating is essential for transmitting genetic information to the next generation and therefore animals have evolved mechanisms for optimizing the chance of successful mate location. In the soil nematode C. elegans, males approach hermaphrodites via the ascaroside pheromones, recognize hermaphrodites when their tails contact the hermaphrodites'' body, and eventually mate with them. These processes are mediated by sensory signals specialized for sexual communication, but other mechanisms may also be used to optimize mate location. Here we describe associative learning whereby males use sodium chloride as a cue for hermaphrodite location. Both males and hermaphrodites normally avoid sodium chloride after associative conditioning with salt and starvation. However, we found that males become attracted to sodium chloride after conditioning with salt and starvation if hermaphrodites are present during conditioning. For this conditioning, which we call sexual conditioning, hermaphrodites are detected by males through pheromonal signaling and additional cue(s). Sex transformation experiments suggest that neuronal sex of males is essential for sexual conditioning. Altogether, these results suggest that C. elegans males integrate environmental, internal and social signals to determine the optimal strategy for mate location.     

Size-dependent sex allocation and sexual selection in Aplysia kurodai, a hermaphrodite with nonreciprocal mating

Abstract. For simultaneous hermaphrodites, a male-to-female shift in sex allocation with growth, and weak sexual selection on the male function, is predicted by many theories, although empirical data for both predictions are insufficient for internally fertilizing hermaphrodites with nonreciprocal mating. To address these issues, I studied mating and egg-laying behavior of the sea hare, Aplysia kurodai (Gastropoda: Opisthobranchia) in the laboratory. Both frequency and duration of egg laying increased with body weight, indicating that fecundity increased with weight. On the other hand, frequency and duration of mating as males did not increase with body weight, suggesting that sperm usage was independent of weight. Therefore, sex allocation shifted from male to female functions with growth. The lack of a relationship between body weight and mating activities as males also suggests that there was no "female" choice for large partners. However, the frequency of mating as females increased with body weight, suggesting "male" choice for large partners. This "male" choice is further supported by the presence of size-assortative mating and a longer duration of mating when the female partner was large. In addition, the variance in mating frequency as females was larger than that as males. As a whole, the mating behavior in A. kurodai can be summarized as choosy as males and unchoosy as females, the opposite of the patterns known in most gonochoric and hermaphroditic animals.     

Sex allocation in a simultaneously hermaphroditic marine shrimp

Two fundamental questions dealing with simultaneous hermaphrodites are how resources are optimally allocated to the male and female function and what conditions determine shifts in optimal sex allocation with age or size. In this study, I explored multiple factors that theoretically affect fitness gain curves (that depict the relationship between sex-specific investment and fitness gains) to predict and test the overall and size-dependent sex allocation in a simultaneously hermaphroditic brooding shrimp with an early male phase. In Lysmata wurdemanni, sperm competition is absent as hermaphrodites reproducing in the female role invariably mated only once with a single other shrimp. Shrimps acting as females preferred small over large shrimps as male mating partners, male mating ability was greater for small compared to large hermaphrodites, and adolescent males were predominant in the population during the breeding season. In addition, brooding constraints were not severe and varied linearly with body size whereas the ability to acquire resources increased markedly with body size. Using sex allocation theory as a framework, the findings above permitted to infer the shape of the male and female fitness gain curves for the hermaphrodites. The absence of sperm competition and the almost unconstrained brooding capacity imply that both curves saturate, however the male curve levels off much more quickly than the female curve with increasing level of investment. In turn, the predominance of adolescent males in the population implies that the absolute gain of the female curve is greater than that of the male curve. Last, the size-dependent female preference and male mating ability of hermaphrodites determines that the absolute gain of the male curve is greater for small than for large hermaphrodites. Taking into consideration the inferred shape of the fitness gain curves, two predictions with respect to the optimal sex allocation were formulated. First, overall sex allocation should be female biased; it permits hermaphrodites to profit from the female function that provides a greater fitness return than the male function. Second, sex allocation should be size-dependent with smaller hermaphrodites allocating more than proportionally resources to male reproduction than larger ones. This size-dependent sex allocation permits hermaphrodites to profit from male mating opportunities that are the greatest at small body sizes. Size-dependent sex allocation is also expected because the male fitness gain curve decelerates more quickly than the female gain curve and experiments indicated that resources are greater for large than small hermaphrodites. These two predictions were tested when determining the sex allocation of hermaphrodites by dissecting their gonad and quantifying ovaries versus testes mass. Supporting the predictions above, hermaphrodites allocated, on average, 118 times more to the female than to the male gonad and the proportion of resources devoted to male function was higher in small than in large hermaphrodites. A trade-off between male and female allocation is assumed by theory but no negative correlation between male and female reproductive investment was observed. In L. wurdemanni, the relationship between sex-specific investment and fitness changes during ontogeny in a way that is consistent with an adjustment of sex allocation to improve size-specific reproductive success.     

Is there sperm storage in the clam shrimp Eulimnadia texana?

Abstract. Androdioecy is a rare form of mating system in which species comprise males and hermaphrodites. One recently described case of androdioecy is the freshwater crustacean Eulimnadia texana. A mathematical model of the mating system of this shrimp suggests that males and hermaphrodites should only coexist under limited circumstances. One possible factor not considered in this model would extend the conditions for coexistence: the possibility of sperm storage in the hermaphrodites. Here we use genetically marked matings between males and hermaphrodites to determine if hermaphrodites can store male sperms. Eggs were collected from hermaphrodites both in the presence of a male and after the male was removed. A total of 30 of these matings had successful hatches, but only 14 of these 30 could be used to test for sperm storage. In these 14 cases, an average of 35% of the eggs were outcrossed when males were present, but only 0.4% were outcrossed after males were removed. Thus, sperm storage by hermaphrodites was an insignificant factor in the production of offspring. These data suggest that sperm storage cannot help explain the coexistence of males and hermaphrodites in natural populations of this crustacean.     

Density-dependent regulation of the sex ratio in an annual plant

Sex ratios are subject to strong frequency-dependent selection regulated by the mating system and the relative male versus female investment. In androdioecious plant populations, where males co-occur with hermaphrodites, the sex ratio depends on the rate of self-fertilization by hermaphrodites and on the relative pollen production of males versus hermaphrodites. Here, we report evolutionary changes in the sex ratio from experimental mating arrays of the androdioecious plant Mercurialis annua. We found that the progeny sex ratio depended strongly on density, with fewer males in the progeny of plants grown under low density. This occurred in part because of a plastic adjustment in pollen production by hermaphrodites, which produced more pollen when grown at low density than at high density. Our results provide support for the prediction that environmental conditions govern sex ratios through their effects on the relative fertility of unisexual versus hermaphrodite individuals.     

Sperm transfer is affected by mating history in the simultaneously hermaphroditic snail Lymnaea stagnalis

Abstract. Males are predicted to strategically allocate sperm across mating partners in order to maximize their chances of paternity. This requires that males have the ability to detect aspects of their partner's mating history or the number of potential mates. We investigated whether simultaneous hermaphrodites mating in the male role strategically adjust sperm transfer depending on rearing conditions. The pond snail Lymnaea stagnalis (Basommatophora) is known to donate sperm repeatedly to different partners during a breeding season and store received sperm for >3 months. The rearing conditions of the donor as well as the recipient affect the amount of sperm transferred. Sperm donors raised in isolation transfer more sperm than those raised in groups. Furthermore, isolated sperm donors transfer less sperm to partners that were raised in groups than to those raised in isolation, i.e., virgins. These findings suggest that snails raised in isolation shift their sex allocation toward the male function and indicate that they can somehow assess the mating status of their partner.     

Barriers to outcrossing success in the primarily self-fertilizing clam shrimp, Eulimnadia texana (Crustacea, Branchiopoda)

Abstract. The expected proportion of males in androdioecious populations (those comprised of males and hermaphrodites) largely depends on the fertilization opportunities of males. If male mating opportunities are low due to restricted access to hermaphroditic eggs, then populations will be hermaphrodite-biased. Hermaphrodites have two mechanisms available to limit male mating success: (1) pre-mating barriers to outcrossing, in which hermaphrodites choose not to pair with males and (2) post-mating barriers to outcrossing, in which hermaphrodite sperm has greater access to eggs than male sperm. In this study, we measured male mating success in the androdioecious clam shrimp Eulimnadia texana when pre-mating barriers to outcrossing were removed. These branchiopod crustaceans are small (5–8 mm), filter feeders that live in ephemeral pools in the deserts of the southwestern United States. Using genetic markers, we measured male mating success in laboratory experiments in two populations of these shrimp. We correlated mating success with clasping time, clasping during egg transfer, and male thrusting during egg transfer. Males fertilized an average of 24–40% of the hermaphrodites' eggs. Outcrossing success was positively correlated with clasping duration, and was nearly an order of magnitude higher for males thrusting during egg transfer relative to thrusting at other times during pairing. Because these estimates of mating success were similar to previously reported estimates (in which both pre- and post-mating barriers to outcrossing were potentially important), we deduced that pre-mating barriers to outcrossing do not greatly decrease male outcrossing success in E. texana ; the low fertilization (25–50% of available eggs) by males is thus due to post-mating barrier(s) to outcrossing.     

Adaptation to experimental alterations of the operational sex ratio in populations of Drosophila melanogaster

Theory predicts that males adapt to sperm competition by increasing their investment in testis mass to transfer larger ejaculates. Experimental and comparative data support this prediction. Nevertheless, the relative importance of sperm competition in testis size evolution remains elusive, because experiments vary only sperm competition whereas comparative approaches confound it with other variables, in particular male mating rate. We addressed the relative importance of sperm competition and male mating rate by taking an experimental evolution approach. We subjected populations of Drosophila melanogaster to sex ratios of 1:1, 4:1, and 10:1 (female:male). Female bias decreased sperm competition but increased male mating rate and sperm depletion. After 28 generations of evolution, males from the 10:1 treatment had larger testes than males from other treatments. Thus, testis size evolved in response to mating rate and sperm depletion, not sperm competition. Furthermore, our experiment demonstrated that drift associated with sex ratio distortion limits adaptation; testis size only evolved in populations in which the effect of sex ratio bias on the effective population size had been compensated by increasing the numerical size. We discuss these results with respect to reproductive evolution, genetic drift in natural and experimental populations, and consequences of natural sex ratio distortion.     

Sex Attraction and Mating in Bursaphelenchus okinawaensis and B. xylophilus

The fungal feeding, hermaphroditic Bursaphelenchus okinawaensis is a laboratory model to understand the biology of Bursaphelenchus. The extent to which B. okinawaensis can be used to model Bursaphelenchus xylophilus mating was investigated. A chemotaxis assay was conducted to examine whether B. xylophilus and B. okinawaensis produce and respond to volatile sex attractants. Unmated B. xylophilus females were found to attract B. xylophilus males. Similarly, old (sperm depleted) but not young (sperm repleted) B. okinawaensis hermaphrodites attract B. okinawaensis males. Thus, in both species, sperm status corresponds to its ability to attract males. B. xylophilus males also produce a volatile pheromone that attracts both mated and unmated females. A second assay, in which the behavior of males on petri plates in the presence of different females or hermaphrodites of Bursaphelenchus was observed, revealed that B. xylophilus unmated females attract B. okinawaensis males, and B. okinawaensis old hermaphrodites attract B. xylophilus males. These observations suggested that the pheromones of Bursaphelenchus work to some extent across species. Mating behavior through spicule insertion occurs across species, suggesting that postcopulatory mechanisms prevent production of interspecific progeny. The hermaphroditic B. okinawaensis will be a useful model to conduct genetic studies for the understanding of the molecular mechanisms underlying mating behavior in Bursaphelenchus nematodes.     

An alpha-tubulin mutant demonstrates distinguishable functions among the spindle assembly checkpoint genes in Saccharomyces cerevisiae

The free-living nematode worm Caenorhabditis elegans reproduces primarily as a self-fertilizing hermaphrodite, yet males are maintained in wild-type populations at low frequency. To determine the role of males in C. elegans, we develop a mathematical model for the genetic system of hermaphrodites that can either self-fertilize or be fertilized by males and we perform laboratory observations and experiments on both C. elegans and a related dioecious species C. remanei. We show that the mating efficiency of C. elegans is poor compared to a dioecious species and that C. elegans males are more attracted to C. remanei females than they are to their conspecific hermaphrodites. We postulate that a genetic mutation occurred during the evolution of C. elegans hermaphrodites, resulting in the loss of an attracting sex pheromone present in the ancestor of both C. elegans and C. remanei. Our findings suggest that males are maintained in C. elegans because of the particular genetic system inherited from its dioecious ancestor and because of nonadaptive spontaneous nondisjunction of sex chromosomes, which occurs during meiosis in the hermaphrodite. A theoretical argument shows that the low frequency of male mating observed in C. elegans can support male-specific genes against mutational degeneration. This results in the continuing presence of functional males in a 99.9% hermaphroditic species in which outcrossing is disadvantageous to hermaphrodites.     

Implications for the Maintenance of Androdioecy in the Freshwater Shrimp, Eulimnadia texana Packard: Encounters between Males and Hermaphrodites are not Random

Androdioecy is a mixed‐mating system in which there are males and hermaphrodites but no pure females. Few species exhibit such a mating system. Eulimnadia texana is a branchiopod crustacean that has recently been identified as an androdioecious species. This system is ideal for testing questions related to the evolution of sexual reproduction. We are testing a model that predicts androdioecy to be a stable mixed‐mating system under certain conditions. Specifically, we investigated whether encounters between males and hermaphrodites are random or if either sex seeks out the other for mating. Focal male or hermaphrodite clam shrimp were presented with stimulus shrimp of the other sex or kept alone. Swimming speed and time spent within different areas of a test chamber were recorded. Males did not alter mean swimming speed or spend more time than expected by chance near partitioned hermaphrodites. Hermaphrodites, however, decreased mean swimming speed in the presence of males and also spent more time than expected by chance near partitioned males, suggesting that hermaphrodites respond to male chemical and/or visual stimuli. Modified swimming behaviour probably facilitates inter‐sexual contact, thereby increasing opportunities for out‐crossing above that expected by random encounters.     

Diversity in mating behavior of hermaphroditic and male-female Caenorhabditis nematodes

In this study, we addressed why Caenorhabditis elegans males are inefficient at fertilizing their hermaphrodites. During copulation, hermaphrodites generally move away from males before they become impregnated. C. elegans hermaphrodites reproduce by internal self-fertilization, so that copulation with males is not required for species propagation. The hermaphroditic mode of reproduction could potentially relax selection for genes that optimize male mating behavior. We examined males from hermaphroditic and gonochoristic (male-female copulation) Caenorhabditis species to determine if they use different sensory and motor mechanisms to control their mating behavior. Instead, we found through laser ablation analysis and behavioral observations that hermaphroditic C. briggsae and gonochoristic C. remanei and Caenorhabditis species 4, PB2801 males produce a factor that immobilizes females during copulation. This factor also stimulates the vulval slit to widen, so that the male copulatory spicules can easily insert. C. elegans and C. briggsae hermaphrodites are not affected by this factor. We suggest that sensory and motor execution of mating behavior have not significantly changed among males of different Caenorhabditis species; however, during the evolution of internal self-fertilization, hermaphrodites have lost the ability to respond to the male soporific-inducing factor.     

Male–Male Competition in a Mixed‐Mating Fish

Mating systems that comprise a mixture of pure males and self‐fertilising hermaphrodites remain an evolutionary enigma. In particular, our understanding of the sexual selection pressures associated with such mating systems is nascent. Males can only reproduce by fertilising hermaphrodites’ eggs, but hermaphrodites can also fertilise their own eggs and gain a genetic advantage by doing so. Consequently, there should be intense competition among males to access hermaphrodites. Here, we test the importance of male size, colour and heterozygosity in predicting the outcome of male–male competition using the mangrove rivulus, which has a male‐hermaphrodite mixed‐mating system. We pitted males against one another in dyadic laboratory trials to develop a dominance score for each male. We then correlated these scores with male length, several components of male colour, and heterozygosity. Male size was the only significant correlate of dominance: larger males dominated smaller males, implying selection for large male size. However, male mangrove rivulus are similar in size to hermaphrodites, indicating that directional selection for large body size in males is no greater than it is in hermaphrodites. Across all trials, colour was unrelated to dominance, but contests between similarly sized males were usually won by more colourful individuals. As mangrove rivulus are dichromatic, we suspect that male colour may prove to be more important in mate choice than we found it to be in intrasexual competition. Heterozygosity did not explain dominance directly, but correlated strongly with male size, implying an indirect role in intrasexual competition.     

Male age, mating status and nuptial gift quality in a bushcricket

Female mate choice occurs in many animals, and in some species females prefer older males. Because older males have demonstrated their survival ability, they may be of higher genetic quality, providing genetic benefits to the offspring of their mates. However, in species where females receive direct benefits of matings, younger males may be more likely to provide more fertile or more nutritious ejaculates, so females may discriminate against older males. Males of the bushcricket Ephippiger ephippiger (Orthoptera: Tettigoniidae) produce large spermatophores at mating (>30% of body weight, circa 10% protein content). Female E. ephippiger discriminate against the song of older males. We examined the effects of male age and mating history on male reproductive investment (spermatophore size, sperm number, nitrogen content). Males produced spermatophores with significantly fewer sperm and of lower nitrogen content on their fourth mating, despite free access to food and a 1-week interval between matings, indicating that there is a cost of mating to males. There was no indication that older virgin males produced lower-quality spermatophores. Rather, older males produced bigger spermatophores of higher nutritional value and containing more sperm. Male age and mating history seem likely to be strongly correlated in the field. We conclude that female E. ephippiger probably prefer the songs of younger males, because in the field, this preference correlates with male mating history and therefore resources provided at mating. Thus, female preference for younger males could reflect discrimination against low-quality nuptial gifts.     

Plasticity in sex allocation in the plant Mercurialis annua is greater for hermaphrodites sampled from dimorphic than from monomorphic populations

Plants are notoriously variable in gender, ranging in sex allocation from purely male through hermaphrodite to purely female. This variation can have both a genetic and an adaptive plastic component. In gynodioecious species, where females co‐occur with hermaphrodites, hermaphrodites tend to shift their allocation towards greater maleness when growing under low‐resource conditions, either as a result of hermaphrodites shifting away from an expensive female function, or because of enhanced siring advantages in the presence of females. Similarly, in the androdioecious plant Mercurialis annua, where hermaphrodites co‐exist with males, hermaphrodites also tend to enhance their relative male allocation under low‐resource conditions. Here, we ask whether this response differs between hermaphrodites that have been evolving in the presence of males, in a situation analogous to that supposed for gynodioecious populations, vs. those that have been evolving in their absence. We grew hermaphrodites of M. annua from populations in which males were either present or absent under different levels of nutrient availability and compared their reaction norms. We found that, overall, hermaphrodites from populations with males tended to be more female than those from populations lacking males. Importantly, hermaphrodites' investment in pollen and seed production was more plastic when they came from populations with males than without them, reducing their pollen production at low resource availability and increasing their seed production at high resource availability. These results are consistent with the hypothesis that plasticity in sex allocation is enhanced in hermaphrodites that have likely been exposed to variation in mating opportunities due to fluctuations in the frequency of co‐occurring males.     

The possibility of sex and nymph discrimination by the male cockroach,Nauphoeta cinerea

The possibility of sex and nymph discrimination by males was investigated in the cockroach,Nauphoeta cinerea (Olivier). A sexually mature male takes a courting position toward a sexually mature female when he comes into contact with her and recognizes her through antennal contact. In contrast, males often behave aggressively toward each other: they bite at each others; wings and/or legs, chase each other and antennate mutually. The male, however, does not show conspicuous behavior (mating behavior or aggressive behavior) toward nearby nymphs. The male produces audible sounds when he courts a sexually mature but non-receptive female who does not respond to his courtship behavior. We found that the male also stridulates after he repeatedly courts immature teneral females, males and (last-instar) nymphs. After the bodies of teneral insects are sclerotized, the male shows courship and stridulation behavior toward sexually mature but non-receptive females but not toward mature males and nymphs. At this stage the male begins to behave aggressively toward other post-teneral mature males. We think that the variability of the sexually mature male's behavior toward other conspecifics (courtship behavior toward female, aggressive behavior toward male and no conspicuous response toward nymph) results from the male's recognition of adult and nymph.     

Maintenance of androdioecy in the freshwater shrimp Eulimnadia texana: sexual encounter rates and outcrossing success

The clam shrimp Eulimnadia texana has a rare mating system known as androdioecy, in which males and hermaphrodites cooccur butthere are no pure females. In this species, reproduction takesplace by outcrossing between males and hermaphrodites, or byselfing within a hermaphrodite; this system provides a uniqueopportunity to examine the adaptive significance of out-crossingand selfing in animals. Our study examined mating behavior in hermaphrodites and males from two populations to understandthe propensity of these shrimp to mate and to estimate a parameterof a model developed by Otto et al. (American Naturalist 141:329-337),which predicts the conditions for stability of the mixed matingsystem in E. texana. Here we present evidence that mating frequencyis environmentally sensitive, with greater numbers of encountersand matings per male when males are rare and in younger males.However, the effects of shrimp density, relative male frequency,and shrimp age interact in a complex way to determine malemating success. Overall, mating frequency was determined bya combination of encounter rates between the sexes and theproportion of encounters resulting in mating. The mating rateswere then used to estimate one of four parameters of the Ottoet al. model, and these estimates were combined with previousestimates of the other three parameters to examine the fitof the predicted to the observed sex ratios in the two populations.     

Costs of receipt and donation of ejaculates in a simultaneous hermaphrodite

Background  

Sexual conflicts between mating partners can strongly impact the evolutionary trajectories of species. This impact is determined by the balance between the costs and benefits of mating. However, due to sex-specific costs it is unclear how costs compare between males and females. Simultaneous hermaphrodites offer a unique opportunity to determine such costs, since both genders are expressed concurrently. By limiting copulation of focal individuals in pairs of pond snails (Lymnaea stagnalis) to either the male role or the female role, we were able to compare the fecundity of single sex individuals with paired hermaphrodites and non-copulants. Additionally, we examined the investment in sperm and seminal fluid of donors towards feminized snails and hermaphrodites.     

spe-12 encodes a sperm cell surface protein that promotes spermiogenesis in Caenorhabditis elegans

During spermiogenesis, Caenorhabditis elegans spermatids activate and mature into crawling spermatozoa without synthesizing new proteins. Mutations in the spe-12 gene block spermatid activation, rendering normally self-fertile hermaphrodites sterile. Mutant males, however, are fertile. Surprisingly, when mutant hermaphrodites mate with a male, their self-spermatids activate and form functional spermatozoa, presumably due to contact with male seminal fluid. Here we show that, in addition to its essential role in normal activation of hermaphrodite-derived spermatids, SPE-12 also plays a supplementary but nonessential role in mating-induced activation. We have identified the spe-12 gene, which encodes a novel protein containing a single transmembrane domain. spe-12 mRNA is expressed in the sperm-producing germ line and the protein localizes to the spermatid cell surface. We propose that SPE-12 functions downstream of both hermaphrodite- and male-derived activation signals in a spermatid signaling pathway that initiates spermiogenesis.