We tested the hypothesis that mate choice is responsible for countergradient variation in the sexual coloration of Trinidadian guppies (Poecilia reticulata). The nature of the countergradient pattern is that geographical variation in the carotenoid content of the orange spots of males is counterbalanced by genetic variation in drosopterin production, resulting in a relatively uniform pigment ratio. A female hue preference could produce this pattern, because hue is the axis of colour variation most directly affected by the pigment ratio. To test this hypothesis, we crossed two populations differing in drosopterin production and produced an F₂ generation with variable drosopterin levels. When the carotenoid content of the orange spots was held constant, female guppies preferred males with intermediate drosopterin levels. This shows that females do not simply prefer males with greater orange spot pigment content; instead, the ratio of the pigments also affects male attractiveness. To our knowledge, this is the first direct evidence for a hypothesized agent of countergradient sexual selection.

Background

PCR-based surveys have shown that guppies (Poecilia reticulata) have an unusually large visual-opsin gene repertoire. This has led to speculation that opsin duplication and divergence has enhanced the evolution of elaborate male coloration because it improves spectral sensitivity and/or discrimination in females. However, this conjecture on evolutionary connections between opsin repertoire, vision, mate choice, and male coloration was generated with little data on gene expression. Here, we used RT-qPCR to survey visual-opsin gene expression in the eyes of males, females, and juveniles in order to further understand color-based sexual selection from the perspective of the visual system.

Results

Juvenile and adult (male and female) guppies express 10 visual opsins at varying levels in the eye. Two opsin genes in juveniles, SWS2B and RH2-2, accounted for >85% of all visual-opsin transcripts in the eye, excluding RH1. This relative abundance (RA) value dropped to about 65% in adults, as LWS-A180 expression increased from approximately 3% to 20% RA. The juvenile-to-female transition also showed LWS-S180 upregulation from about 1.5% to 7% RA. Finally, we found that expression in guppies' SWS2-LWS gene cluster is negatively correlated with distance from a candidate locus control region (LCR).

Conclusions

Selective pressures influencing visual-opsin gene expression appear to differ among age and sex. LWS upregulation in females is implicated in augmenting spectral discrimination of male coloration and courtship displays. In males, enhanced discrimination of carotenoid-rich food and possibly rival males are strong candidate selective pressures driving LWS upregulation. These developmental changes in expression suggest that adults possess better wavelength discrimination than juveniles. Opsin expression within the SWS2-LWS gene cluster appears to be regulated, in part, by a common LCR. Finally, by comparing our RT-qPCR data to MSP data, we were able to propose the first opsin-to-λ_max assignments for all photoreceptor types in the cone mosaic.

Background
Sexually dimorphic phenotypes are generally associated with differential gene expression between the sexes. The study of molecular evolution and genomic location of these differentially expressed, or sex-biased, genes is important for understanding inter-sexual divergence under sex-specific selection pressures. Teleost fish provide a unique opportunity to examine this divergence in the presence of variable sex-determination mechanisms of recent origin. The guppy, Poecilia reticulata, displays sexual dimorphism in size, ornaments, and behavior, traits shaped by natural and sexual selection in the wild.

Results
To gain insight into molecular mechanisms underlying the guppy’s sexual dimorphism, we assembled a reference transcriptome combining genome-independent as well as genome-guided assemblies and analyzed sex-biased gene expression between different tissues of adult male and female guppies. We found tissue-associated sex-biased expression of genes related to pigmentation, signal transduction, and spermatogenesis in males; and growth, cell-division, extra-cellular matrix organization, nutrient transport, and folliculogenesis in females. While most sex-biased genes were randomly distributed across linkage groups, we observed accumulation of ovary-biased genes on the sex linkage group, LG12. Both testis-biased and ovary-biased genes showed a significantly higher rate of non-synonymous to synonymous substitutions (dN/dS) compared to unbiased genes. However, in somatic tissues only female-biased genes, including those co-expressed in multiple tissues, showed elevated ratios of non-synonymous substitutions.

Conclusions
Our work identifies a set of annotated gene products that are candidate factors affecting sexual dimorphism in guppies. The differential genomic distribution of gonad-biased genes provides evidence for sex-specific selection pressures acting on the nascent sex chromosomes of the guppy. The elevated rates of evolution of testis-biased and female-biased genes indicate differing evolution under distinct selection pressures on the reproductive versus non-reproductive tissues.

Keywords:
Guppy; de novo transcriptome; Genome-guided transcriptome; Sex-biased genes; Sexual dimorphism; RNA-seq; Coding sequence evolution

Many aquarists, fish or rabbit breeders have at their disposal for reproduction heterogenic populations as a result of segregation, or due to crossbreeding. In many cases, they must buy new brood stock every 2-3 years due to the fact their old brood stock degenerates visible after several generations. The aim of this paper is to underline that there are some low cost and easy to understand principles available with application in animat improvement and production. Color, size and shape are important traits when we describe a product as a phenotypically uniform line, strain or breed, e.g. the albino African catfish, the red eyed swordtail, the Red Blond guppy, the large chinchilla rabbit, the New Zealand white rabbit. A standardized color, shape and size describe better an animal population and thus the said product is promoted better on the market. Two simple examples of applications will be presented in this paper, the case of purification and maintaining of thermo-resistant guppy fish, and the principle of improvement of body size and maintaining the color pattern in Transylvanian Giant rabbit. The key to success is to know what is (are) the recessive gene(s) for every relevant locus.

In Poecilia reticulata, different phenotypes appear due to dominance, codominance, poligeny, or some demonstrated interallelic interactions. Recent(both molecular and classical) investigations suggest a mechanism of expression of several different loci in a single color pattern, resulting in high numbers of possible color pattern phenotypes. The color pattern seems to be determined by complex interactions of many genes (at the same locus or not, located on the same chromosome or not), under variable environmental conditions. For example, the Maculatus color pattern is due to the presence of both Maculatus red and Maculatus black elements. On their turn, having in view the latest definitions of the gene, both Maculatus red and Maculatus black could have a composite nature, too. Sexdetermination in the guppy is studied since the 1920s. The deepest mechanism of sex determination is not clear yet, but classical studies of Winge early in the past century and recent molecular studies revealed a possible composite nature of the so called master sex determining gene, located at SDL of the Y chromosome.

In this paper we report on a special case of crossing-over in Poecilia reticulata. It was known fact that Snakeskin body trait (Ssb) can be both X-linked, or Y-linked, and also that the percent of recombination between Y and X chromosomes is 0.36, while reciprocal recombination, from X to Y, is 0.00; in other words, the gene was not known to recombine from X to Y. The aim of the current work was to present an accidental observation about the occurrence of crossing-over events from X chromosome to Y chromosome in our guppy broodstock. The results based on crossbreeding and progeny testing showed that Ssb recombination by crossing-over or gene conversion is possible both from X to Y and Y to X. These events have numerous evo lutionary implications as long the color traits and behavior are very close interrelated in many animal taxa and not only in the guppy fish.
Key words:
crossing-over, Poecilia, Snakeskin body, progeny test

Singapore is known internationally as a breeding centre for the guppy, Poecilia reticulata. About 30 domesticated colour pattern and tail shape varieties are reared, mainly for export. The culture of these varieties at two rural and eight resettled farms is described. All farms are small family concerns. The resettled farms practise monoculture of guppies while the rural ones integrated farming. Guppies are reared in large, shallow, outdoor, cement tanks and nylon cage-nets suspended in ponds. Water in aquaria and ponds has salinities from 0.5 to 9.0%,, at neutral or alkaline pH of 7.0 to 8.3. The sizes and stocking densities of aquaria and nets vary within and between farms. In breeding tanks, a male to female sex ratio of 1:3, 1:4 or 1:lO is maintained. Newborn fry are collected daily and placed in nursery aquaria. After 3 weeks, these fry are sexed: young males are transferred to grow-out cement tanks, and females to grow-out cagenets. Each farm has at least two ponds, one for water storage and the others for cagenet culture. Strains are cultured separately to maintain pure lines. Farmers improve colour patterns, size and fin shapes of the strains by continued mass selection. Fourto 6-month-old guppies are selected as brookstock and the rest which meet export quality are held in stocking aquaria and sold when demand arises.
Each farmer prepares his own formulated diet for the fish. Supplemental foods like live tubificiids, commercial dried fish flakes, or floating pellets may be given to adults, and water fleas to fry. The rural farms add pig manure to fertilise the tanks.
To remain viable in the future, farmers need to increase productivity by mechanising cleaning, draining, and filling tanks and ponds, feeding, and harvesting of the fish. This will cut down on labour and its increasing cost. Understanding the genetics of economically important characters will also help farmers produce higher quality and more exotic strains.

Many color varieties of the guppy, Poecilia reticulata, are commercially cultured in Singapore for the aquarium industry. In the group of guppy varieties called Snakeskin, males characteristically have snakeskin-like reticulations on the body and caudal fin. The snakeskin pattern on the body of male Snakeskin guppies is due to a Y-linked gene (Ssb). Female guppies, being homogametic (XX), do not carry the Ssb gene. About 90% of Yellow Snakeskin males have the typical snakeskin pattern on their bodies and tails.
The remaining males are different in that the snakeskin body pattern has been modified into four or five vertical bars on the caudal-peduncle region. F₁ and F₂ results of single-pair reciprocal matings of the Yellow Snakeskin variety show that a single gene is responsible for the vertical bar pattern. This gene, bar, is autosomal recessive. In the homozygous condition (barbar), it interacts with the Y-linked Ssb gene to give vertical barring patterns on the caudal-peduncle of Yellow Snakeskin males. This pattern is not expressed when the dominant allele, bar⁺, is present.

The Blue Snakeskin (BSS) and Red Snakeskin (RSS) varieties are popular strains commercially cultured in Singapore. The blue-black tail color of BSS guppies is determined by a dominant X-linked gene (Bit) and the silvery snakeskin pattern on the body of males is under the control of a Y-linked gene (Ssb). The Y-linked snakeskin tail pattern gene (Sst) though present in BSS males is masked by the blue-black tail color gene (Bit). The putative genotypes for males and females of the BSS variety are X B i,Y SsbSs , and X W ,X B „, respectively. The red tail color of the RSS variety is due to an X-linked dominant gene (Rdt). The snakeskin body pattern of RSS males is under the control of the Y-linked Ssb gene while the black reticulations on the tail is due to interaction between the snakeskin tail pattern gene (Sst) and the red tail gene (Rdt). The proposed genotypes for males and females of the RSS variety are X Rdl Y SsbSsl and X Rdl X Rd „ respectively. An estimate of 0.9% crossover frequency was obtained between the Y-linked Ssb and Sst genes and a 2.7% crossover rate of the Bit gene from the X-to the Y-chromosome.