Retention of Ejaculate by Drosophila melanogaster Females Requires the Male-Derived Mating Plug Protein PEBme

Within the mated reproductive tracts of females of many taxa, seminal fluid proteins (SFPs) coagulate into a structure known as the mating plug (MP). MPs have diverse roles, including preventing female remating, altering female receptivity postmating, and being necessary for mated females to successfully store sperm. The Drosophila melanogaster MP, which is maintained in the mated female for several hours postmating, is comprised of a posterior MP (PMP) that forms quickly after mating begins and an anterior MP (AMP) that forms later. The PMP is composed of seminal proteins from the ejaculatory bulb (EB) of the male reproductive tract. To examine the role of the PMP protein PEBme in D. melanogaster reproduction, we identified an EB GAL4 driver and used it to target PEBme for RNA interference (RNAi) knockdown. PEBme knockdown in males compromised PMP coagulation in their mates and resulted in a significant reduction in female fertility, adversely affecting postmating uterine conformation, sperm storage, mating refractoriness, egg laying, and progeny generation. These defects resulted from the inability of females to retain the ejaculate in their reproductive tracts after mating. The uncoagulated MP impaired uncoupling by the knockdown male, and when he ultimately uncoupled, the ejaculate was often pulled out of the female. Thus, PEBme and MP coagulation are required for optimal fertility in D. melanogaster. Given the importance of the PMP for fertility, we identified additional MP proteins by mass spectrometry and found fertility functions for two of them. Our results highlight the importance of the MP and the proteins that comprise it in reproduction and suggest that in Drosophila the PMP is required to retain the ejaculate within the female reproductive tract, ensuring the storage of sperm by mated females.     

Across-population variation in sex ratio in invasive Japanese Potamopyrgus antipodarum (Caenogastropoda: Rissooidea: Hydrobiidae)

Potamopyrgus antipodarum, a well-known invasive species, has both sexual and asexual forms in its native area, and is generally asexual in exotic areas. Potamopyrgus antipodarum was sampled from ten locations in central and western Japan, and the sex distribution was analyzed. Males were found in two of the ten localities; the percentages of males in these localities were 7.2 and 7.3 %, which is within the range of that observed in asexual populations in native and introduced areas. A flow cytometry analysis showed that populations with a low percentage of males (Kanro) and populations consisting only of females (Banyu Park, Hadano and Yugawara) and perhaps the population reported as having a high male percentage in a previous study (Chiba; 29.8 %) were triploid. Males from Chiba and Kanro produced sperm. Most of the females from Chiba stored sperm in their seminal receptacle, showing that they had copulated, but no females from Kanro stored sperm. Fertilization by males, normal development of fertilized eggs, and the fecundity of juveniles should be evaluated in further studies to demonstrate the sexual reproduction of P. antipodarum in Japan. The risk of introduction of sexual P. antipodarum individuals is further discussed in the article.     

Variation among Populations of Belonolaimus longicaudatus

Three North Carolina populations of Belonolairnus longicaudatus differed significantly from three Georgia populations in stylet measurements, the c ratio, the distance of the excretory pore from the anterior end for both sexes; the a ratio for females only; and the total body length, tail length, and spicule length for males only. The Georgia nematodes were stouter, and the females possessed sclerotized vaginal pieces. The distal portion of the spicules of North Carolina males had an indentation and hump lacking in those of the Georgia males. The haploid number of chromosomes was eight for males from all populations of B. longicaudatus and a North Carolina population of B. maritimus. Interpopulation matings of the Tarboro, N.C. and Tifton, Ga. populations indicated that the offspring produced were infertile. Morphological differences and reproductive isolation suggest that the North Carolina and the Georgia populations belong to different species.     

Molecular Characterization of a Xiphinema hunaniense Population with Morphometric Data of all Four Juvenile Stages

A population of Xiphinema hunaniense Wang and Wu, 1992 with all four juvenile stages was found in the rhizosphere of Pinus sp. in Hangzhou, Zhejiang, China. Morphometrics of 18 females and 35 juveniles of this population are given herein. Detailed morphology and morphometrics of the four juvenile stages are provided. Further comparisons based on morphometrics of the population with previous studies of the females and the first-stage juveniles of X. hunaniense with X. radicicola are given, and morphological variation in X. hunaniense populations are discussed. A revised polytomous key code of Loof and Luc (1990) for X. hunaniense identification is provided, i.e., A1- B4- C4- D4/5- E1- F2(3)- G2- H2-I3- J4- K2- L1. In addition, the sequence of the D2 and D3 expansion region of the 28S rRNA gene was analyzed and compared with sequences of closely related species downloaded from the NCBI database. Cluster analysis of sequences confirmed and supported the species identifications.     

Variations in Host Preference among and within Populations of Heterodera trifolii and Related Species

Seven populations of Heterodera trifolii from Arkansas, Kentucky, Pennsylvania, and Australia plus 3 or 4 single-cyst isolates (SCI) from each population were tested for reproduction on seven species of plants to compare the host preferences among and within populations. Common lespedeza, Kummerowia striata cv. Kobe, was a good host for all populations and isolates. Therefore, a plant was considered to be a host if the number of females produced on it was 10% or more of the number on Kobe. All seven populations reproduced on Trifolium repens and T. pratense. None reproduced on Beta vulgaris or Glycine max. One single-cyst isolate from the Australian population produced a few females on T. pratense. The Australian population maintained on carnation, Dianthus caryophyllus, produced females on carnation but not on curly dock, Rumex crispus. However, its subpopulation maintained on T. repens produced females on R. crispus but not on carnation. Four of the other six populations produced females on R. crispus, and four produced females on carnation. Differences in host range were observed among seven of the mother populations and their SCI, and among isolates within each population. Five host range patterns were found in populations and SCI of H. trifolii. Significant quantitative differences occurred among populations in the numbers of females on most hosts, between isolates and their original populations, and among isolates from the same population. SCI selected from white clover produced fewer females on a series of test hosts and had host ranges the same as or narrower than those of the original populations. However, SCI selected from Kobe lespedeza had more females on some hosts and had host ranges the same as or wider than those of the original populations. The host ranges of all populations and SCI of H. trifolii were different from those of populations and SCI of race 3 of H. glycines and H. lespedezae.     

Morphological Comparisons Between Xiphinema rivesi Daimasso and X. americanum Cobb Populations from the Eastern United States

Though in the past Xiphinema americanum has been the most commonly reported dagger nematode in the eastern United States, our studies revealed the presence in Pennsvlvania of a previously unrecognized and unreported species related to X. americanum, Morphometric data and photomicrographs establish the identity of this form as X. rivesi and show expected variations in populations of this species from various locations. Similar data and illustrations are given for X. americanum populations from Pennsylvania and other areas, showing variations and relationships. Xiphinema rivesi is widely distributed in the fruit producing area of south-central Pennsylvania and is also reported herein from raspberry in Vermont and apple in Maryland and New York. This species is frequently found in fruit growing areas of Pennsylvania associated with tomato ringspot virus-induced diseases and is also found associated with corn, bluegrass sod, and alfalfa.     

The genetics and evolution of obligate reproductive parasitism in Trichogramma pretiosum infected with parthenogenesis-inducing Wolbachia

Parthenogenesis-inducing (PI) Wolbachia belong to a class of intracellular symbionts that distort the offspring sex ratio of their hosts toward a female bias. In many PI Wolbachia-infected species sex ratio distortion has reached its ultimate expression-fixation of infection and all-female populations. This is only possible with thelytokous PI symbionts as they provide an alternative form of reproduction and remove the requirement for males and sexual reproduction. Many populations fixed for PI Wolbachia infection have lost the ability to reproduce sexually, even when cured of the infection. We examine one such population in the species Trichogramma pretiosum. Through a series of backcrossing experiments with an uninfected Trichogramma pretiosum population we were able to show that the genetic basis for the loss of female sexual function could be explained by a dominant nuclear effect. Male sexual function had not been completely lost, though some deterioration of male sexual function was also evident when males from the infected population (created through antibiotic curing of infected females) were mated to uninfected females. We discuss the dynamics of sex ratio selection in PI Wolbachia-infected populations and the evolution of non-fertilizing mutations.     

Inter- and Intraspecific Variation in Wild-type and Single Female-derived Populations of Xiphinema americanum-group Nematodes

Ten populations of Xiphinema americanum-group nematodes were reared from individual females to evaluate inter- and intraspecific variation under identical host and environmental conditions. Data indicated that morphometric variability of X. americanum was the result of genetic variation rather than phenotypic plasticity and that genetic heterogeneity was greater than previously thought. Morphometrics of single female derived (SFD) populations identified different genotypes present in the field populations. Stylet length was the least variable morphometric character of SFD populations, but collectively stylet measurements of all individuals formed an uninterrupted continuum ranging from 107-148 μm. Range and frequency of stylet measurements of field populations could be accounted for by the relative proportion of different genotypes in the population. Nine SFD populations were identified as X. americanum sensu stricto, and one SFD population was similar to X. californicum.     

Androgenesis is a maternal trait in the invasive ant Wasmannia auropunctata

Androgenesis is the production of an offspring containing exclusively the nuclear genome of the fathering male via the maternal eggs. This unusual mating system is generally considered a male trait, giving to androgenetic males a substantial fitness advantage over their sexually reproducing relatives. We here provide the first empirical study of the evolutionary outcomes of androgenesis in a haplo-diploid organism: the invasive ant Wasmannia auropunctata. Some of the populations of this species have a classical haplo-diploid sexual mating system. In other populations, females and males are produced through parthenogenesis and androgenesis, respectively, whereas workers are produced sexually. We conducted laboratory reciprocal-cross experiments with reproductive individuals from both types of populations and analysed their progenies with genetic markers, to determine the respective contribution of males and females to the production of androgenetic males. We found that androgenesis was a parthenogenetic female trait. A population genetic study conducted in natura confirmed the parthenogenetic female origin of androgenesis, with the identification of introgression events of sexual male genotypes into androgenetic/parthenogenetic lineages. We argue that by producing males via androgenesis, parthenogenetic queen lineages may increase and/or maintain their adaptive potential, while maintaining the integrity of their own genome, by occasionally acquiring new male genetic material and avoiding inbreeding depression within the sexually produced worker cast.     

Resistance of Trisomic and Diploid Hybrids of Beta vulgaris and B. procumbens to the Sugarbeet Nematode,Heterodera schachtiis Arnold

Trisomic and diploid hybrids of sugarbeet (Beta vulgaris L.) X wild beet (B. procumbens Chr. Sin.) inherited the gene for resistance to Heterodera schachtii Schm. from B. procumbens. The hybrids showed partial resistance to H. schachtii, manifested in failure of larvae to reach maturity. Although significantly greater numbers of female nematodes developed on plants inoculated with populations from the Netherlands or Italy than on plants inoculated with a population from the Salinas Valley, California. the totals for all populations on resistant plants were small. Greater numbers of males than females developed on root-slice cultures of resistant hyhrids when compared to a susceptible cultivar.     

Sperm storage mediated by cryptic female choice for nuptial gifts

Polyandrous females are expected to discriminate among males through postcopulatory cryptic mate choice. Yet, there is surprisingly little unequivocal evidence for female-mediated cryptic sperm choice. In species in which nuptial gifts facilitate mating, females may gain indirect benefits through preferential storage of sperm from gift-giving males if the gift signals male quality. We tested this hypothesis in the spider Pisaura mirabilis by quantifying the number of sperm stored in response to copulation with males with or without a nuptial gift, while experimentally controlling copulation duration. We further assessed the effect of gift presence and copulation duration on egg-hatching success in matings with uninterrupted copulations with gift-giving males. We show that females mated to gift-giving males stored more sperm and experienced 17% higher egg-hatching success, compared with those mated to no-gift males, despite matched copulation durations. Uninterrupted copulations resulted in both increased sperm storage and egg-hatching success. Our study confirms the prediction that the nuptial gift as a male signal is under positive sexual selection by females through cryptic sperm storage. In addition, the gift facilitates longer copulations and increased sperm transfer providing two different types of advantage to gift-giving in males.     

Monogamy and polygamy in two species of mirid bugs: a functional-based approach

Multiple mating in females is widespread among insects in spite of the risk of predation, disease acquisition and/or physical injury that may occur. One common consequence of female polyandry is competition among sperm from two or more males within the female to fertilize the ova. This competition is an evolutionary driving force that determines a series of adaptations in both males and females. In this work, we examine some behavioral, morphological and physiological characteristics of males and females of two Heteropteran species that are related to their monoandrous/polyandrous mating behavior. Females of Macrolophus pygmaeus (Het. Miridae), the monoandrous species, were coy about accepting a male partner, spent a short time in copula, and received only a small volume of ejaculate. Even so, with only one mating event, they received enough sperm to fertilize most of their ova (21 days after mating all females were still fertile). In contrast, females of Nesidiocoris tenuis (Het. Miridae), the polyandrous species, readily accepted any mating partner, spent a long time in copula and received a large volume of ejaculate. However, these latter females soon ran out of sperm and needed to mate periodically in order to maintain a sufficient sperm supply to fertilize their eggs. As predicted, based on current theory (Simmons, 2001b), an increased investment in spermatogenesis was detected in N. tenuis with relation to M. pygmaeus. The males of the polyandrous species had larger accessory reproductive glands, seminal vesicles, testes and sperm cells than those of the monoandrous species.     

Offspring production with cryopreserved sperm from a live-bearing fish Xiphophorus maculatus and implications for female fecundity

Xiphophorus fishes are well-established models for biomedical research of spontaneous or induced tumors, and their use in research dates back to the 1930s. Currently, 58 well-pedigreed lines exist among 24 Xiphophorus species housed as live animals at the Xiphophorus Genetic Stock Center. The technique of sperm cryopreservation has been applied to preserve these valuable genetic resources, and production of offspring has been reported with cryopreserved sperm in two species (X. helleri and X. couchianus). The goal of this research was to establish protocols for sperm cryopreservation and artificial insemination that yield live young in X. maculatus, a widely used research species. The objectives were to: 1) collect basic biological characteristics of males, and quantify the sperm production yield after crushing of dissected testis; 2) cryopreserve sperm from X. maculatus by adapting as necessary the protocols for sperm cryopreservation of X. helleri and X. couchianus; 3) use cryopreserved sperm to inseminate virgin females of X maculatus and other species (X. helleri and X. couchianus), and 4) compare experimental trials over a 3-year period to identify opportunities for improving female fecundity. In total, 117 males were used in this study with a standard length of 2.5 ± 0.3 cm (mean ± SD), body weight of 0.474 ± 0.149 g, and dissected testis weight of 7.1 ± 3.7 mg. Calculation of sperm availability showed 5.9 ± 2.8 × 10(6) sperm cells per mg of testis weight. Offspring were produced from cryopreserved sperm. Male-to-male variation (1-70%) was observed in post-thaw motility despite little variation in motility before freezing (60-90%) or genetic variation (~100 generations of sib-mating). Comparisons of biological factors of males did not have significant correlations with the production of live young, and the influence of females on production of young was identified from the comparison of artificial insemination over 3 years. Overall, this study describes offspring production from cryopreserved sperm in a third species of Xiphophorus fishes, and identifies the opportunities for improving female fecundity which is essential for establishment of germplasm repositories for Xiphophorus fishes.     

Postmating sexual selection: allopatric evolution of sperm competition mechanisms and genital morphology in calopterygid damselflies (Insecta: Odonata)

Postmating sexual selection theory predicts that in allopatry reproductive traits diverge rapidly and that the resulting differentiation in these traits may lead to restrictions to gene flow between populations and, eventually, reproductive isolation. In this paper we explore the potential for this premise in a group of damselflies of the family Calopterygidae, in which postmating sexual mechanisms are especially well understood. Particularly, we tested if in allopatric populations the sperm competition mechanisms and genitalic traits involved in these mechanisms have indeed diverged as sexual selection theory predicts. We did so in two different steps. First, we compared the sperm competition mechanisms of two allopatric populations of Calopteryx haemorrhoidalis (one Italian population studied here and one Spanish population previously studied). Our results indicate that in both populations males are able to displace spermathecal sperm, but the mechanism used for sperm removal between both populations is strikingly different. In the Spanish population males seem to empty the spermathecae by stimulating females, whereas in the Italian population males physically remove sperm from the spermathecae. Both populations also exhibit differences in genital morphometry that explain the use of different mechanisms: the male lateral processes are narrower than the spermathecal ducts in the Italian population, which is the reverse in the Spanish population. The estimated degree of phenotypic differentiation between these populations based on the genitalic traits involved in sperm removal was much greater than the differentiation based on a set of other seven morphological variables, suggesting that strong directional postmating sexual selection is indeed the main evolutionary force behind the reproductive differentiation between the studied populations. In a second step, we examined if a similar pattern in genital morphometry emerge in allopatric populations of this and other three species of the same family (Calopteryx splendens, C. virgo and Hetaerina cruentata). Our results suggest that there is geographic variation in the sperm competition mechanisms in all four studied species. Furthermore, genitalic morphology was significantly divergent between populations within species even when different populations were using the same copulatory mechanism. These results can be explained by probable local coadaptation processes that have given rise to an ability or inability to reach and displace spermathecal sperm in different populations. This set of results provides the first direct evidence of intraspecific evolution of genitalic traits shaped by postmating sexual selection.     

Morphological Differences Between Radopholus citrophilus and R. similis

SEM observations of the external morphology of populations of Radopholus citrophilus and R. similis revealed several diagnostic differences. The cloaco-spicular orifice on males of R. citrophilus had three to seven genital papillae (anterior hypoptygmata), whereas males of R. similis were either smooth or had one or two shorter genital papillae (anterior hypoptygmata). Females of R. citrophilus had four annules in the region of the vulval opening, but R. similis had five annules in the same region. The labial disc and lateral lips appeared to be of diagnostic significance, but these areas were more susceptible to artifacts due to fixation. An unknown population of Radopholus from Puerto Rico with a chromosome number of n = 4 was morphologically similar to R. similis. These morphological differences provide additional support that R. citrophilus and R. similis are distinct species.     

Diversity of Xiphinema americanum-group Species and Hierarchical Cluster Analysis of Morphometrics

Of the 39 species composing the Xiphinema americanum group, 14 were described originally from North America and two others have been reported from this region. Many species are very similar morphologically and can be distinguished only by a difficult comparison of various combinations of some morphometric characters. Study of morphometrics of 49 populations, including the type populations of the 39 species attributed to this group, by principal component analysis and hierarchical cluster analysis placed the populations into five subgroups, proposed here as the X. brevicolle subgroup (seven species), the X. americanum subgroup (17 species), the X. taylori subgroup (two species), the X. pachtaicum subgroup (eight species), and the X. lambertii subgroup (five species).     

Inhibition of female mating receptivity by male-derived extracts in two Callosobruchus species: Consequences for interspecific mating

We investigated the effects of injecting male-derived extracts on congeneric female receptivity in two species of Callosobruchus beetle, C. chinensis and C. maculatus. We also examined the influence of interspecific mating on female remating behaviour in these two species. Male-derived extracts reduced congeneric female receptivity in both species. As quick-acting components, extracts of C. chinensis male seminal vesicles reduced the receptivity of C. maculatus females, whereas extracts of C. maculatus male testes reduced the receptivity of C. chinensis females. As slow-acting components, extracts of male accessory glands of other species reduced the receptivity of both C. maculatus and chinensis females. After interspecific mating, the sperm of C. maculatus males were transferred to the reproductive organs of C. chinensis females, thereby reducing their receptivity. In contrast, no C. chinensis sperm were transferred to the reproductive organs of C. maculatus females; accordingly, the latter's receptivity was not reduced. Furthermore, the survival rate of C. chinensis females decreased markedly after interspecific mating. These results raise the possibility that under circumstances where populations of these two species share the same habitat, reproductive interference would occur only in the interactions between C. maculatus males and C. chinensis females.     

Quantitative genetic analysis suggests causal association between cuticular hydrocarbon composition and desiccation survival in Drosophila melanogaster

Survival to low relative humidity is a complex adaptation, and many repeated instances of evolution to desiccation have been observed among Drosophila populations and species. One general mechanism for desiccation resistance is Cuticular Hydrocarbon (CHC) melting point. We performed the first Quantitative Trait Locus (QTL) map of population level genetic variation in desiccation resistance in D. melanogaster. Using a panel of Recombinant Inbred Lines (RILs) derived from a single natural population, we mapped QTL in both sexes throughout the genome. We found that in both sexes, CHCs correlated strongly with desiccation resistance. At most desiccation resistance loci there was a significant association between CHCs and desiccation resistance of the sort predicted from clinal patterns of CHC variation and biochemical properties of lipids. This association was much stronger in females than males, perhaps because of greater overall abundance of CHCs in females, or due to correlations between CHCs used for waterproofing and sexual signalling in males. CHC evolution may be a common mechanism for desiccation resistance in D. melanogaster. It will be interesting to compare patterns of CHC variation and desiccation resistance in species which adapt to desiccation, and rainforest restricted species which cannot.     

Gametogenesis in Amphimictic and Parthenogenetic Populations of Aphelenchus avenae

The fetnale reproductive system of Aphelenchus avenae, studied in orcein-stained material, showed a peculiar structural pattern not yet reported in other nematodes. Chromosome morphology and behavior during gametogenesis could be studied in more detail than in other tylenchid or aphelenchid species investigated to date. In a bisexual population from Australia, gametogenesis was by normal meiosis and reproduction by amphimixis. The haploid chromosome number was n=8 in both males and females, and no sex chromosomes were detected. Three monosexual populations from Australia, California, and North Carolina underwent oogenesis by meiosis but reproduced hy parthenogenesis. The haploid chromosome number was n=8 in the Australia and the North Carolina populations, but n=9 in the California population. Spermatogenesis in temperature-induced males of the California population was by normal meiosis, and sperm had n=9 chromosomes. Most chronmsomes consisted of a central euchromatic section and two characteristic heterochromatic ends. No centromere was observed in any chronmsome. The relationship hetween the California population with n=9 and all the other populations with n=8 chromosomes is not well understood.