Cloning,expression and cellular localization of the Doublesex gene in the water flea, Daphnia carinata, during different developmental stages

In this study, one of Doublesex genes from the common freshwater cladoceran Daphnia carinata, designated DapcaDsx1, was cloned using primers based on homologous sequences and rapid amplification of cDNA ends (RACE). qPCR was employed to quantify differences in DapcaDsx1 expression between the different sexual phases, with expression levels being higher in sexual females. The role of DapcaDsx1 in the reproductive transformation was further investigated in parthenogenetic-phase females and sexual-phase females using whole-mount in situ hybridization. This cellular localization study showed specific expression of DapcaDsx1 in the thoracic segments, second antenna and part of the ventral carapace. Higher expression levels were exhibited in sexual females compared to parthenogenetic females. This suggests that the DapcaDsx1 gene plays significant roles in switching modes of reproduction and during sexual differentiation.     

Identification and expression analysis of a doublesex1 gene in Daphnia pulex during different reproductive stages

The gene doublesex (dsx) has shown deep conservation in the sex determination in many organisms. Environmental stimuli initiate a switch in the reproductive strategy of Daphnia pulex from asexual to sexual reproduction; however, occasionally, changes in environmental conditions will not lead to this transition. So study genetic responses to environmental stimuli and the molecular basis for the switch of reproductive stages are urgently needed. Therefore, we isolated and sequenced a D. pulex doublesex1 gene (Dpdsx1) and analyzed its expression and location by quantitative polymerase chain reaction (qPCR) and whole-mount in situ hybridization in D. pulex during different stages of reproduction. The predicted amino acid sequence has 335 amino acids that contained one DM domain and one dimerization domain, which is characteristic of insect orthologs of Dsx. Real-time PCR showed that Dpdsx1 expression decreased significantly (P?<?0.05) in different reproductive stages in the following order: male, parthenogenetic female, ephippial female, resting egg, and juvenile female. Whole-mount in situ hybridization revealed that Dpdsx1 is expressed in the first antennae, first thoracic limb and compound eye in males, whereas expression levels in the corresponding sites of parthenogenetic and ephippial females were relatively weak. Dpdsx1 could not be detected in the gonads of males or ephippial and parthenogenetic females. Taken together, these different reproductive stages’ and sex specific expression patterns are regulated temporally and spatially. We speculate that Dpdsx1 may involve in switching different stages of reproduction and in sexual differentiation in D. pulex.     

A population of sexual Daphnia pulex resists invasion by asexual clones

Asexual reproduction avoids the costs associated with sex, predicting that invading asexual clones can quickly replace sexual populations. Daphnia pulex populations in the Great Lakes area are predominately asexual, but the elimination of sexual populations by invading clones is poorly understood. Asexual clones were detected at low frequency in one rare sexual population in 1995, with some increase in frequency during 2003 and 2004. However, these clones remained at low frequency during further yearly sampling (2005–2013) with no evidence that the resident sexual population was in danger of elimination. There was evidence for hybridization between rare males produced by asexual clones and sexual females with the potential to produce new asexual genotypes and spread the genetic factors for asexuality. In a short-term laboratory competition experiment, the two most common asexual clones did not increase in frequency relative to a genetically diverse sexual population due in part to a greater investment in diapausing eggs that trades-off current population growth for increased contribution to the egg bank. Our results suggest that a successful invasion can be prolonged, requiring a combination of clonal genotypes with high fitness, persistence of clones in the egg bank and negative factors affecting the sexual population such as inbreeding depression resulting from population bottlenecks.     

Meiosis genes in Daphnia pulex and the role of parthenogenesis in genome evolution

Background  

Thousands of parthenogenetic animal species have been described and cytogenetic manifestations of this reproductive mode are well known. However, little is understood about the molecular determinants of parthenogenesis. The Daphnia pulex genome must contain the molecular machinery for different reproductive modes: sexual (both male and female meiosis) and parthenogenetic (which is either cyclical or obligate). This feature makes D. pulex an ideal model to investigate the genetic basis of parthenogenesis and its consequences for gene and genome evolution. Here we describe the inventory of meiotic genes and their expression patterns during meiotic and parthenogenetic reproduction to help address whether parthenogenesis uses existing meiotic and mitotic machinery, or whether novel processes may be involved.     

Sexual reproduction of Daphnia pulex in a temporary habitat

Species of Daphnia (Crustacea: Cladocera) typically reproduce by cyclical parthenogenesis, in which a period of all-female parthenogenetic reproduction is followed by sexual reproduction. Sex in Daphnia is determined by the environment, with factors such as temperature, photoperiod and crowding stimulating the production of males and sexual females. Previous studies on Daphnia pulex from temporary pond habitats demonstrated the coexistence of male-producing and non-male-producing (NMP) females, as determined under crowding in the laboratory. A strong genetic component to this sex allocation variation suggested that sex expression in D. pulex is better described as a result of genotype-environment interaction. The present study examined the switch from parthenogenetic to sexual reproduction in two temporary-pond populations of D. pulex. Both populations showed a very early investment in sexual reproduction, independent of population density, by producing males very soon after the populations were reestablished from resting eggs in the early spring. Approximately 40% of the initial broods were male. Additional evidence for gender specialization was obtained by observing the sex of two or three successive broods for 85 individual females. Fifty-eight females produced successive broods of females, 13 females produced successive broods of males and 14 females produced successive broods which included both male and female broods. Females that produced successive female broods under natural conditions included a higher frequency of NMP females compared to a random sample of females, confirming the existence of NMP females. Sexual females were observed in both populations after the first appearence of males, suggesting that the presence of males may stimulate the production of sexual females. For D. pulex populations in a temporary environment, there appears to be an increased emphasis on sexual reproduction and a decreased influence of the environment on sex determination, compared to Daphnia populations in more permanent habitats. Received: 19 February 1996 / Accepted: 20 January 1997     

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.     

The role of hybridization in the origin and spread of asexuality in Daphnia

The molecular mechanisms leading to asexuality remain little understood despite their substantial bearing on why sexual reproduction is dominant in nature. Here, we examine the role of hybridization in the origin and spread of obligate asexuality in Daphnia pulex, arguably the best‐documented case of contagious asexuality. Obligately parthenogenetic (OP) clones of D. pulex have traditionally been separated into ‘hybrid’ (Ldh SF) and ‘nonhybrid’ (Ldh SS) forms because the lactase dehydrogenase (Ldh) locus distinguishes the cyclically parthenogenetic (CP) lake dwelling Daphnia pulicaria (Ldh FF) from its ephemeral pond dwelling sister species D. pulex (Ldh SS). The results of our population genetic analyses based on microsatellite loci suggest that both Ldh SS and SF OP individuals can originate from the crossing of CP female F₁ (D. pulex × D. pulicaria) and backcross with males from OP lineages carrying genes that suppress meiosis specifically in female offspring. In previous studies, a suite of diagnostic markers was found to be associated with OP in Ldh SS D. pulex lineages. Our association mapping supports a similar genetic mechanism for the spread of obligate parthenogenesis in Ldh SF OP individuals. Interestingly, our study shows that CP D. pulicaria carry many of the diagnostic microsatellite alleles associated with obligate parthenogenesis. We argue that the assemblage of mutations that suppress meiosis and underlie obligate parthenogenesis in D. pulex originated due to a unique historical hybridization and introgression event between D. pulex and D. pulicaria.     

Reproductive allocation in Daphnia exposed to toxic cyanobacteria

We investigated experimentally how resources were allocated to reproduction in Daphnia pulex and Daphnia longispina when varying levels of toxic Microcystis were added to higher quality food. We used multiple regression models to estimate mean offspring size and clutch size in relation to maternal size and clutch number, and analysed effects of treatments on residuals from the models. We also measured variation in per offspring investment. At a high cyanobacterial level, D.pulex was virtually unable to reproduce. At a lower level, D.pulex produced small clutches. However, the regression model residuals indicated that the presence of cyanobacteria increased the portion of available resources allocated to reproduction. The observed allocation may be a means to maximize reproduction under diminished longevity. Effects on mean offspring size were marginal in D.pulex but variation in per offspring investment sometimes decreased in cyanobacterial exposures. Daphnia longispina was affected by a higher cyanobacterial level only, where offspring sized was reduced. Deviations from the regression model indicated that effects on maternal size alone do not explain this effect. Clutch size residuals and per offspring investment were unaffected by treatments in D.longispina. The observed responses differ from theoretical models on reproductive allocation under food imitation.      

Clonal diversity and turnover in an overwintering Daphnia pulex population,and the effect of fish predation

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Diversity in the reproductive modes of European Daphnia pulicaria deviates from the geographical parthenogenesis

Background

Multiple transitions to obligate parthenogenesis have occurred in the Daphnia pulex complex in North America. These newly formed asexual lineages are differentially distributed being found predominantly at high latitudes. This conforms to the rule of geographical parthenogenesis postulating prevalence of asexuals at high latitudes and altitudes. While the reproductive mode of high-latitude populations is relatively well studied, little is known about the reproduction mode in high altitudes. This study aimed to assess the reproductive mode of Daphnia pulicaria, a species of the D. pulex complex, from high altitude lakes in Europe.

Methodology/Principal Findings

Variation at eight microsatellite loci revealed that D. pulicaria from the High Tatra Mountains (HTM) had low genotype richness and showed excess of heterozygotes and significant deviations from Hardy-Weinberg expectations, and was thus congruent with reproduction by obligate parthenogenesis. By contrast, populations from the Pyrenees (Pyr) were generally in Hardy-Weinberg equilibrium and had higher genotypic richness, suggesting that they are cyclic parthenogens. Four lakes from lowland areas (LLaP) had populations with an uncertain or mixed breeding mode. All D. pulicaria had mtDNA ND5 haplotypes of the European D. pulicaria lineage. Pyr were distinct from LLaP and HTM at the ND5 gene. By contrast, HTM shared two haplotypes with LLaP and one with Pyr. Principal Coordinate Analysis of the microsatellite data revealed clear genetic differentiation into three groups. HTM isolates were intermediate to Pyr and LLaP, congruent with a hybrid origin.

Conclusion/Significance

Inferred transitions to obligate parthenogenesis have occurred only in HTM, most likely as a result of hybridizations. In contrast to North American populations, these transitions do not appear to involve meiosis suppressor genes and have not been accompanied by polyploidy. The absence of obligate parthenogenesis in Pyr, an environment highly similar to the HTM, may be due to the lack of opportunities for hybridization.     

Mating Behavior of Daphnia: Impacts of Predation Risk,Food Quantity,and Reproductive Phase of Females

High predation risk and food depletion lead to sexual reproduction in cyclically parthenogenetic Daphnia. Mating, the core of sexual reproduction, also occurs under these conditions. Assessment of the environmental conditions and alteration of mating efforts may aid in determining the success of sexual reproduction. Here, we evaluated the impacts of predation risk, food quantity, and reproductive phase of females on the mating behavior of Daphnia obtusa males including contact frequency and duration using video analysis. Mating–related behavior involved male–female contact (mating) as well as male–male contact (fighting). Mating frequency increased while unnecessary fighting decreased in the presence of predation risk. In addition, low food concentration reduced fighting between males. Males attempted to attach to sexual females more than asexual females, and fighting occurred more frequently in the presence of sexual females. Duration of mating was relatively long; however, males separated shortly after contact in terms of fighting behavior. Thus, assessment of environmental factors and primary sexing of mates were performed before actual contact, possibly mechanically, and precise sex discrimination was conducted after contact. These results suggest that mating in Daphnia is not a random process but rather a balance between predation risk and energetic cost that results in changes in mating and fighting strategies.     

In Silico Prediction and In Vivo Validation of Daphnia pulex Micrornas

Daphnia pulex, the crustacean with the first sequenced genome, is an important organism that has been widely used in ecological and toxicological research. MicroRNAs (miRNAs) are 21–25 nucleotide small non-coding RNAs that are involved in a myriad of physiological processes. In this research, we predicted 75 D. pulex miRNAs by sequence homology and secondary structure identification from the full genome sequence. Fourteen predicted miRNAs were selected for quantitative real time polymerase chain reaction (RT-PCR) validation. Out of these, eight (mir-8, mir-9, mir-12, mir-92, mir-100, mir-133, mir-153 and mir-283) were successfully amplified and validated. Next, expression levels were quantified at three different life stages (days 4, 8 and 12 of age) using U6 spliceosomal RNA as a reference gene. The expression of mir-8, mir-9, mir-12, mir-92 and mir-100 significantly differed across time suggesting these microRNAs might play a critical role during D. pulex development. This is the first study to identify and validate miRNAs in D. pulex, which is an important first step in further studies that evaluate their roles in development and response to environmental and ecological stimuli.     

DNA transposon dynamics in populations of Daphnia pulex with and without sex

We investigate the role of recombination in transposable element (TE) proliferation in the cyclical parthenogen, Daphnia pulex. Recombination provides a mechanism by which the rate of both TE gain and loss can be accelerated, a duality that has long intrigued many biologists interested in the influence of sex on mutation accumulation. We compared TE loads among populations of D. pulex where sex occurs regularly (cyclical parthenogens or ‘sexuals’) with those in which the ability to reproduce sexually has been completely lost (obligate ‘asexuals’) for six different families of DNA transposons. Transposon display assays showed that sexuals have more TEs than asexuals, contrary to the expectations under Muller''s ratchet but consistent with the idea that sex facilitates TE spread. Sexuals also exhibit higher insertion site polymorphism among lineages, as predicted because recombination accelerates rates of loss and gain. Asexuals, however, have proportionally more singletons (loci occupied in a single isolate), which differs from previous studies where selfing and outcrossing were used as a proxy for high and low recombination. Our multi-element survey reveals that the impact of sex on TE proliferation is consistent among different Class II TE families and we discuss the genomic consequences of different reproductive strategies over long time periods.     

Does the silver moss Bryum argenteum exhibit sex-specific patterns in vegetative growth rate,asexual fitness or prezygotic reproductive investment?

Background and Aims

Expected life history trade-offs associated with sex differences in reproductive investment are often undetected in seed plants, with the difficulty arising from logistical issues of conducting controlled experiments. By controlling genotype, age and resource status of individuals, a bryophyte was assessed for sex-specific and location-specific patterns of vegetative, asexual and sexual growth/reproduction across a regional scale.

Methods

Twelve genotypes (six male, six female) of the dioecious bryophyte Bryum argenteum were subcultured to remove environmental effects, regenerated asexually to replicate each genotype 16 times, and grown over a period of 92 d. Plants were assessed for growth rates, asexual and sexual reproductive traits, and allocation to above- and below-ground regenerative biomass.

Key Results

The degree of sexual versus asexual reproductive investment appears to be under genetic control, with three distinct ecotypes found in this study. Protonemal growth rate was positively correlated with asexual reproduction and sexual reproduction, whereas asexual reproduction was negatively correlated (appeared to trade-off) with vegetative growth (shoot production). No sex-specific trade-offs were detected. Female sex-expressing shoots were longer than males, but the sexes did not differ in growth traits, asexual traits, sexual induction times, or above- and below-ground biomass. Males, however, had much higher rates of inflorescence production than females, which translated into a significantly higher (24x) prezygotic investment for males relative to females.

Conclusions

Evidence for three distinct ecotypes is presented for a bryophyte based on regeneration traits. Prior to zygote production, the sexes of this bryophyte did not differ in vegetative growth traits but significantly differed in reproductive investment, with the latter differences potentially implicated in the strongly biased female sex ratio. The disparity between males and females for prezygotic reproductive investment is the highest known for bryophytes.     

Effects of saxitoxin- and non-saxitoxin-producing strains of the cyanobacterium Cylindrospermopsis raciborskii on the fitness of temperate and tropical cladocerans

Life-table experiments were performed with two strains of Cylindrospermopsis raciborskii, a saxitoxin-producing strain (T3) and a non-saxitoxin-producing strain (NPLP-1) combined with the green algae Ankistrodesmus falcatus, aiming to detect effects on the reproduction of three cladocerans species: Daphnia pulex, Daphnia gessneri and Moina micrura. Survivorship, age at first reproduction, clutch size and population growth rate during 12–15 days were recorded. Cladocerans showed different susceptibility to saxitoxin-producing strain (T3), with D. pulex and M. micrura being negatively affected, followed by and D. gessneri, which did not show any decrease in fitness and performed even better than the controls with this strain. A reverse response was found in experiments with the non-saxitoxin-producing strain (NPLP-1): while D. pulex and M. micrura had an increase, D. gessneri showed a significant decrease in fitness, suggesting that this strain may produce some bioactive compound. The contrasting responses of the cladoceran species to saxitoxins and non-saxitoxin-producing strains of C. raciborskii suggest that the presence of this cyanobacterium may be a selective factor in determining the composition of zooplankton communities.     

Is competition important to arctic zooplankton community structure?

1. Daphnia pulex and Daphnia middendorffiana are commonly found in the Toolik Lake region of arctic Alaska. These two species are very similar morphologically, although their natural distributions differ markedly: D. pulex is restricted to shallow ponds, while D. middendorffiana is widely distributed and found in a variety of ponds and lakes. We compared the reproductive capabilities of D. pulex and D. middendorffiana grown under similar resource conditions and in the absence of the invertebrate predator Heterocope septentrionalis. In situ life table and mesocosm experiments were conducted in Toolik Lake and Dam Pond, habitats that have historically contained natural populations of D. middendorffiana, but never D. pulex. 2. Daphnia pulex exhibited a significantly higher net growth rate than D. middendorffiana in both life table and mesocosm experiments although D. pulex has never been found in either Toolik Lake or Dam Pond. Daphnia middendorffiana exhibited a negative net growth rate in Dam Pond, which had lower resource levels then Toolik Lake. Therefore, the smaller D. pulex appears to have a lower food threshold concentration than the larger D. middendorffiana. 3. Our results indicate that D. pulex is a superior resource competitor in the Toolik Lake region. These results combined with distributional patterns suggest that the restricted distribution of D. pulex in these arctic lakes and ponds cannot be explained by resource competition alone. We suggest that in the presence of H. septentrionalis, predation is an important factor structuring arctic zooplankton communities in the Toolik Lake region.