WW Females Research Articles

All-female crayfish populations for biocontrol and sustainable aquaculture

Snail control in aquaculture and control of snail vectors implicated in animal and human diseases are global challenges. Potential biocontrol agents could be species of crayfish, which are known to be voracious predators of snails. However, at present, the use of crayfish as biocontrol agents is limited due to their invasive nature. A promising candidate biocontrol agent is the Australian redclaw crayfish, Cherax quadricarinatus, a key species in the aquaculture industry. However, the downside of using this species for biocontrol is that C. quadricarinatus aquaculture escapees – being highly robust, fast growing, and ferocious omnivores – have become invasive, establishing feral populations around the world. Here we suggest the production of monosex C. quadricarinatus populations that open two exciting biotechnology applications—as a possible sustainable solution for snail control and as a means to improve yields and management in crayfish aquaculture, without the inherent danger of establishing invasive populations of aquaculture escapees. In the first part of this study, C. quadricarinatus was tested as a snail eradicator. Both males and females were highly efficient in predation experiments, eliminating 89% and 98% of Thiara scabra snails, respectively. The superior efficiency of the females in the predation experiments and the possible advantage for aquaculture of all-female crayfish populations provided the rationale for the second part of the study—the development of an economically feasible and non-labor-intensive biotechnology for the production of all-female C. quadricarinatus populations. To this end, we leveraged the naturally occurring intersexuality in C. quadricarinatus populations to produce female-biased populations. Females bearing only the W sex chromosome isolated from such populations using W and Z-specific genomic markers were found to be reproductive when bred with WZ intersexuals. Resulting progenies from the above crosses were 100% female populations including 50% WW females, thus paving the way to the establishment of an all-female producing brood stock. We present a quantitative scheme that demonstrates how our approach, which does not include any genomic intervention, can be implemented both for sustainable aquaculture and to fight harmful snails damaging aquaculture, agriculture, and human health.

Two Homogametic Genotypes - One Crayfish: On the Consequences of Intersexuality.

SummaryIn the Australian redclaw crayfish, Cherax quadricarinatus (WZ/ZZ system), intersexuals, although exhibiting both male and female gonopores, are functional males bearing a female genotype (WZ males). Therefore, the occurrence of the unusual homogametic WW females in nature is plausible. We developed W/Z genomic sex markers and used them to investigate the genotypic structure of experimental and native C. quadricarinatus populations in Australia. We discovered, for the first time, the natural occurrence of WW females in crustacean populations. By modeling population dynamics, we found that intersexuals contribute to the growth rate of crayfish populations in the short term. Given the vastly fragmented C. quadricarinatus habitat, which is characterized by drought-flood cycles, we speculate that intersexuals contribute to the fitness of this species since they lead to occasional increment in the population growth rate which potentially supports crayfish population restoration and establishment under extinction threats or colonization events.

Production of WW males lacking the masculine Z chromosome and mining the Macrobrachium rosenbergii genome for sex-chromosomes

The cultivation of monosex populations is common in animal husbandry. However, preselecting the desired gender remains a major biotechnological and ethical challenge. To achieve an efficient biotechnology for all-female aquaculture in the economically important prawn (Macrobrachium rosenbergii), we achieved – for the first time – WW males using androgenic gland cells transplantation which caused full sex-reversal of WW females to functional males. Crossing the WW males with WW females yielded all-female progeny lacking the Z chromosome. We now have the ability to manipulate – by non-genomic means – all possible genotype combinations (ZZ, WZ and WW) to retain either male or female phenotypes and hence to produce monosex populations of either gender. This calls for a study of the genomic basis underlying this striking sexual plasticity, questioning the content of the W and Z chromosomes. Here, we report on the sequencing of a high-quality genome exhibiting distinguishable paternal and maternal sequences. This assembly covers ~ 87.5% of the genome and yielded a remarkable N50 value of ~ 20 × 106 bp. Genomic sex markers were used to initiate the identification and validation of parts of the W and Z chromosomes for the first time in arthropods.

A Single Injection of Hypertrophied Androgenic Gland Cells Produces All-Female Aquaculture.

Monosex culture, common in animal husbandry, enables gender-specific management. Here, production of all-female prawns (Macrobrachium rosenbergii) was achieved by a novel biotechnology comprising three steps: (a) A single injection of suspended hypertrophied androgenic gland cells caused fully functional sex reversal of females into "neo-males" bearing the WZ genotype; (b) crossing neo-males with normal females (WZ) yielded genomically validated WW females; and (c) WW females crossed with normal males (ZZ) yielded all-female progeny. This is the first sustainable biotechnology for large-scale all-female crustacean aquaculture. The approach is particularly suited to species in which females are superior to males and offers seedstock protection, thereby ensuring a quality seed supply. Our technology will thus revolutionize not only the structure of the crustacean aquaculture industry but can also be applied to other sectors. Finally, the production of viable and reproducible females lacking the Z chromosome questions its role, with respect to sexuality.

Coexistence of Y, W, and Z sex chromosomes in Xenopus tropicalis

Homomorphic sex chromosomes and rapid turnover of sex-determining genes can complicate establishing the sex chromosome system operating in a given species. This difficulty exists in Xenopus tropicalis, an anuran quickly becoming a relevant model for genetic, genomic, biochemical, and ecotoxicological research. Despite the recent interest attracted by this species, little is known about its sex chromosome system. Direct evidence that females are the heterogametic sex, as in the related species Xenopus laevis, has yet to be presented. Furthermore, X. laevis' sex-determining gene, DM-W, does not exist in X. tropicalis, and the sex chromosomes in the two species are not homologous. Here we identify X. tropicalis' sex chromosome system by integrating data from (i) breeding sex-reversed individuals, (ii) gynogenesis, (iii) triploids, and (iv) crosses among several strains. Our results indicate that at least three different types of sex chromosomes exist: Y, W, and Z, observed in YZ, YW, and ZZ males and in ZW and WW females. Because some combinations of parental sex chromosomes produce unisex offspring and other distorted sex ratios, understanding the sex-determination systems in X. tropicalis is critical for developing this flexible animal model for genetics and ecotoxicology.

Consecutive Virgin Births in the New World Boid Snake, the Colombian Rainbow Boa, Epicrates maurus

Until recently, facultative automictic parthenogenesis within the squamate reptiles exhibiting ZZ:ZW genetic sex determination has resulted in single reproductive events producing male (ZZ) or female (ZW) offspring. With the recent discovery of viable parthenogenetically produced female (WW) Boa constrictors, the existence of further parthenogenetic events resulting in WW females was questioned. Here, we provide genetic evidence for consecutive virgin births by a female Colombian rainbow boa (Epicrates maurus), resulting in the production of WW females likely through terminal fusion automixis. Samples were screened at 22 microsatellite loci with 12 amplifying unambiguous products. Of these, maternal heterozygosity was observed in 4, with the offspring differentially homozygous at each locus. This study documents the first record of parthenogenesis within the genus Epicrates, a second within the serpent lineage Boidae, and the third genetically confirmed case of consecutive virgin births of viable offspring within any vertebrate lineage. Unlike the recent record in Boa constrictors, the female described here was isolated from conspecifics from birth, demonstrating that males are not required to stimulate parthenogenetic reproduction in this species and possibly other Boas.

Evidence for viable, non-clonal but fatherless Boa constrictors

Parthenogenesis in vertebrates is considered an evolutionary novelty. In snakes, all of which exhibit genetic sex determination with ZZ : ZW sex chromosomes, this rare form of asexual reproduction has failed to yield viable female WW offspring. Only through complex experimental manipulations have WW females been produced, and only in fish and amphibians. Through microsatellite DNA fingerprinting, we provide the first evidence of facultative parthenogenesis in a Boa constrictor, identifying multiple, viable, non-experimentally induced females for the first time in any vertebrate lineage. Although the elevated homozygosity of the offspring in relation to the mother suggests that the mechanism responsible may be terminal fusion automixis, no males were produced, potentially indicating maternal sex chromosome hemizygosity (WO). These findings provide the first evidence of parthenogenesis in the family Boidae (Boas), and suggest that WW females may be more common within basal reptilian lineages than previously assumed.

Introduction of Trojan sex chromosomes to boost population growth

Conservation programs that deal with small or declining populations often aim at a rapid increase of population size to above-critical levels in order to avoid the negative effects of demographic stochasticity and genetic problems like inbreeding depression, fixation of deleterious alleles, or a general loss of genetic variability and hence of evolutionary potential. In some situations, population growth is determined by the number of females available for reproduction, and manipulation of family sex ratios towards more daughters has beneficial effects. If sex determination is predominantly genetic but environmentally reversible, as is the case in many amphibia, reptiles, and fish, Trojan sex chromosomes could be introduced into populations in order to change sex ratios towards more females. We analyse the possible consequences for the introduction of XX-males (XX individuals that have been changed to phenotypic males in a XY/XX sex determination system) and ZW males, WW males, or WW females (in a ZZ/ZW sex determination system). We find that the introduction of WW individuals can be most effective for an increase of population growth, especially if the induced sex change has little or no effect on viability.

Sex determination in crayfish: are intersex Cherax quadricarinatus (Decapoda, Parastacidae) genetically females?

In the Australian red-claw crayfish Cherax quadricarinatus (von Martens) (Decapoda, Parastacidae), a gonochoristic species, seven different combinations of intersex individuals (with both male and female genital openings) have been described. However, to date, the genetic basis for this phenomenon has not been investigated. This study was designed to test a simple chromosome-based sex-determination model for C. quadricarinatus that assumes the male to be the homogametic (ZZ) sex. According to our model, intersex individuals that are functionally males are genetically females (WZ). Individual crosses were performed between intersex and female crayfish, with control crosses being performed between normal males and females. The control crosses yielded, in most cases, the expected 1:1 sex ratio in the F1 progeny. Crosses between intersex individuals and females yielded a 1:3 (male:female) sex ratio in most crosses. According to our hypothesis, one-third of the females produced in a cross of a female with an intersex animal should be WW females. The hypothesis was tested by crossing normal males with F1 females, which were progeny of intersex fathers. These crosses yielded almost 100% females, a finding that conforms to the above-suggested sex determination model for C. quadricarinatus and the female WZ genotype of intersex individuals.

Role of aromatase and androgen receptor expression in gonadal sex differentiation of ZW/ZZ-type frogs, Rana rugosa

To elucidate the mechanisms of amphibian gonadal sex differentiation, we examined the expression of aromatase and androgen receptor (AR) mRNAs for days 17–31 after fertilization. The effects of inhibitors and sex steroid hormones were also examined. In ZZ males, expression of AR decreased after day 19, while aromatase expression was low throughout the sampling period. Males treated with 17β-estradiol (E2) showed increasing aromatase expression after day 21, and formed ovaries. AR antagonist treatment also induced high-level aromatase expression and ovarian differentiation. In males co-treated with an aromatase inhibitor and E2, the undifferentiated gonads developed into testes despite high-level aromatase expression. Males treated with androgen and E2 before and during an estrogen sensitive period, respectively, also formed testes. In ZW females, AR expression persisted at a low-level, while aromatase expression increased after day 18. Short-term treatment with an aromatase inhibitor was ineffective in preventing ovarian differentiation, whereas long-term treatment resulted in testes developing from ovarian structure. Compared with the ZZ males and ZW females, WW females did not exhibit detectable expression of AR, suggesting that the active AR gene(s) itself, or a putative gene regulating AR gene expression, is located on Z chromosomes. From the time lag of aromatase expression between ZW females and ZZ males treated with E2 and the effect of AR antagonist, it was found that in males elevated AR expression suppresses aromatase expression directly or indirectly. Consequently, endogenous androgens, accumulated by blocking estrogen biosynthesis, induced testicular differentiation. The gonadogenesis of males is dependent on sex hormone, whereas that of females has evolved to hormone-independence.

Evidence for specific RNA/protein interactions in the differential segment of the W sex chromosome in the amphibian Pleurodeles waltl.

Pleurodeles exhibits a ZZ/ZW system of GSD (genotype sex determination). However, the Z and W sex chromosomes appear to be morphologically identical. A short RNA sequence is described that was specifically bound to lampbrush loops in the differential segment of the sexual bivalent IV. The distribution of these labeled loops in experimentally produced ZZ and WW females enabled us to demonstrate that such labeled loops were perfectly correlated with the W chromosome. Therefore, this RNA sequence constitutes an excellent marker for the W differential segment. Furthermore, analysis of the labeled loops under various experimental conditions suggested that their labeling is caused by specific interactions between this RNA sequence and lampbrush loop-associated proteins (RNA/protein interactions). North-western assays revealed that nuclear polypeptide(s) of 65 kDa could be responsible for such binding.

Individual Differences in the Behavior of Albino and Wild House Mice (<em>Mus musculus</em>)

Adult male and female albino (AA) and wild (WW) housemice were individually evaluated in the laboratory for their performance in exploration (EX),insect predation (PD), burrowing (BW) and food hoarding (HD) activities. The results showed that (a) in both AA and WW adult mice there are individual differences (IDs), tending to be stable at least for periods of 60 days; (b) WW tended to show higher frequencies of the activities (WW males in EX and WW females in EX, PD, and HD); (c) significant sex differences occurred both in AA (with females scoring higher in EX and BW, and males in HD) and in WW (with higher female frequencies of burrowing); (d) individual animals had distinct combinations of performance in the four behavioral activities, suggesting independence among the motivational systems responsible for the regulation of these activities.

Production of WW super females by diploid gynogenesis in Xenopus laevis

In Xenopus laevis, which does not show sex chromosomal dimorphism, the female is heterogametic (WZ) and the male is homogametic (ZZ). By activating eggs with UV-irradiated spermatozoa, and by inhibiting the formation of the second polar body gynogenetic diploids were obtained, including some WW females. These super-females are fertile and not sublethal; by gynogenetic reproduction they in turn generate only WW females, while after mating with a male they give rise to WZ females exclusively.

THE GENETICS OF ARTEMIA SALINA. II. WHITE EYE, A SEX-LINKED MUTATION

1. This paper describes the first sex-linked gene discovered among the Crustacea. The experimental results can be accounted for by the following hypotheses: (1) the mutant gene w, which determines white eyes, is recessive to its wild allele W. (2) Female brine shrimp are heterogametic. The chromosome constitution of the female will be represented as XY; the males are XX. (3) The white locus is partially sex-linked. Because it lies on the homologous segments of the sex chromosomes, both males and females may have the genotype of WW, Ww, or ww.2. In matings of Ww females to ww males, crossing over between the white locus and the sex locus may be detected. In the #9 stock, females have the genotype XW YW and the frequency of crossing over is low (1/1625 or 0.06%). This results in a mother-to-daughter inheritance of the W gene in the #9 stock.