Genus Xiphophorus Research Articles

Tactical Release of a Sexually-Selected Pheromone in a Swordtail Fish

BackgroundChemical communication plays a critical role in sexual selection and speciation in fishes; however, it is generally assumed that most fish pheromones are passively released since most fishes lack specialized scent glands or scent-marking behavior. Swordtails (genus Xiphophorus) are widely used in studies of female mate choice, and female response to male chemical cues is important to sexual selection, reproductive isolation, and hybridization. However, it is unclear whether females are attending to passively produced cues, or to pheromones produced in the context of communication.Methodology/Principal FindingsWe used fluorescein dye injections to visualize pulsed urine release in male sheepshead swordtails, Xiphophorus birchmanni. Simultaneous-choice assays of mating preference showed that females attend to species- and sex-specific chemical cues emitted in male urine. Males urinated more frequently in the presence and proximity of an audience (conspecific females). In the wild, males preferentially courted upstream of females, facilitating transmission of pheromone cues.Conclusions/SignificanceMales in a teleost fish have evolved sophisticated temporal and spatial control of pheromone release, comparable to that found in terrestrial animals. Pheromones are released specifically in a communicative context, and the timing and positioning of release favors efficient signal transmission.

Determination of Onset of Sexual Maturation and Mating Behavior by Melanocortin Receptor 4 Polymorphisms

Polymorphisms in reproductive strategies are among the most extreme and complex in nature. A prominent example is male body size and the correlated reproductive strategies in some species of platyfish and swordtails of the genus Xiphophorus. This polymorphism is controlled by a single Mendelian locus (P) that determines the onset of sexual maturity of males. Because males cease growth after reaching puberty, this results in a marked size polymorphism. The different male size classes show pronounced behavioral differences (e.g., courtship versus sneak mating), and females prefer large over small males. We show that sequence polymorphisms of the melanocortin receptor 4 gene (mc4r) comprise both functional and non-signal-transducing versions and that variation in copy number of mc4r genes on the Y chromosome underlies the P locus polymorphism. Nonfunctional Y-linked mc4r copies in larger males act as dominant-negative mutations and delay the onset of puberty. Copy number variation, as a regulating mechanism, endows this system with extreme genetic flexibility that generates extreme variation in phenotype. Because Mc4r is critically involved in regulation of body weight and appetite, a novel link between the physiological system controlling energy balance and the regulation of reproduction becomes apparent.

Xmrks the spot: life history tradeoffs, sexual selection and the evolutionary ecology of oncogenesis

In a classic paper, George Williams (1957) argued that alleles promoting reproductive success early in life may be favoured by selection, even if they reduce the lifespan of individuals that bear the allele. A variety of evidence supports the theory that such 'antagonistic pleiotropy' is a major factor contributing to the evolution of senescence (Ljubuncic & Reznick 2009), but examples of specific alleles known to fulfil Williams' criteria remain rare, in both humans and other animals (e.g. Alexander et al. 2007; Kulminski et al. 2010). An intriguing example in this issue of Molecular Ecology (Fernandez & Bowser 2010) demonstrates that both natural and sexual selection may favour melanoma-promoting oncogene alleles in the fish genus Xiphophorus.

Genetic and environmental melanoma models in fish

Experimental animal models are extremely valuable for the study of human diseases, especially those with underlying genetic components. The exploitation of various animal models, from fruitflies to mice, has led to major advances in our understanding of the etiologies of many diseases, including cancer. Cutaneous malignant melanoma is a form of cancer for which both environmental insult (i.e., UV) and hereditary predisposition are major causative factors. Fish melanoma models have been used in studies of both spontaneous and induced melanoma formation. Genetic hybrids between platyfish and swordtails, different species of the genus Xiphophorus, have been studied since the 1920s to identify genetic determinants of pigmentation and melanoma formation. Recently, transgenesis has been used to develop zebrafish and medaka models for melanoma research. This review will provide a historical perspective on the use of fish models in melanoma research, and an updated summary of current and prospective studies using these unique experimental systems.

On two Jenynsia taxa (Cyprinodontiformes: Anablepidae) from Argentina, originally placed in the poeciliid genus Xiphophorus

On two Jenynsia taxa (Cyprinodontiformes: Anablepidae) from Argentina, originally placed in the poeciliid genus Xiphophorus

DNA Damage, Repair and Photoadaptation in a Xiphophorus Fish Hybrid

Exposure to sunlight is responsible for most cutaneous malignant melanomas in the human population. It is very likely that DNA damage is an initial event in melanomagenesis, however, the role played by this damage is an open question. To this end, we used a hemipigmented F(1) hybrid of the fish genus Xiphophorus and HPLC tandem mass spectrometry to examine the effects of melanin on the induction and repair of the predominant UV-induced photoproducts formed in skin cell DNA. We found that heavily pigmented skin cells had about half the damage of nonpigmented cells and the relative induction of the major photoproducts was independent of the degree of pigmentation. The efficiency of photoenzymatic repair was the same in nonpigmented and pigmented areas of the fish. We found no evidence of residual damage at 10 days after the last exposure. Most striking was that repeated exposure to multiple doses of UVB caused a very significant photoadaptive response. Rather than an accumulation of damage after five doses of UVB we saw a significant reduction in the amount of damage induced after the final dose compared with the initial dose. The relevance of these observations is discussed in the context of melanoma susceptibility and UVB thresholds.

Identification of novel genes involved in the development of the sword and gonopodium in swordtail fish

Male swordtail fish of the genus Xiphophorus develop a sword, a colourful extension of the caudal fin, that evolved by sexual selection through female choice. Swords and gonopodia, an intromittent organ developing from the male anal fin, can be prematurely induced by exogenous testosterone, offering the opportunity to examine the identity and expression profiles of genes required during various stages of fin metamorphosis. Here, we employed suppression subtractive hybridisation to identify genes specifically up-regulated during two early stages of sword and gonopodium development. We identified 128 different sequences with significant similarity to known genes and characterized the rack1, dusp1, klf2, and tmsbeta-like genes as specifically up-regulated in developing as well as regenerating fin rays of the sword and gonopodium. We show that some of these genes follow distinct expression profiles in swords and gonopodia, suggesting differences in the genetic networks underlying the development of anal and caudal fin modifications.

On two Jenynsia taxa (Cyprinodontiformes: Anablepidae) from Argentina, originally placed in the poeciliid genus Xiphophorus

The type material of Xiphophorus heckelii Weyenbergh, 1874 and X. obscurus Weyenbergh, 1877 has apparently been re-examined for the first time. Xiphophorus heckelii is confirmed as a junior synonym of Jenynsia multidentata (Jenyns, 1842). Xiphophorus obscurus is recognized as a senior synonym of Jenynsia pygogramma Boulenger, 1902, which is placed in synonymy. A lectotype and paralectotypes are designated for Jenynsia obscura (Weyenbergh, 1877).

Quantitative Differential Proteome Analysis in an Animal Model for Human Melanoma

In fish of the genus Xiphophorus, different grades of pigment cell lesions from nevi to melanoma can be gained by simple crossbreeding. With this model, one can easily access tissues of different malignancies from animals with highly identical genetic background. To find protein expression differences between healthy, benign and malignant tissues, we performed 2D PAGE and DIGE and found among regulated proteins antioxidant proteins that were overexpressed with increasing malignancy.

Sperm cryopreservation of a live-bearing fish, Xiphophorus couchianus: Male-to-male variation in post-thaw motility and production of F 1 hybrid offspring

Fishes of the genus Xiphophorus are well-studied biomedical research models, and some species, such as X. couchianus, are currently listed as endangered in the wild. Sperm cryopreservation in these live-bearing fishes has begun recently. Thus far, live young have been produced with cryopreserved sperm only in one species ( Xiphophorus helleri). In this study, the goal was to develop a practical protocol for sperm cryopreservation of Xiphophorus couchianus, and to produce live young with cryopreserved sperm. Sperm were collected by crushing of testis in Hanks' balanced salt solution at an osmolality of 500 mOsmol/kg (HBSS500), and were cryopreserved with 14% glycerol (v/v) as cryoprotectant at a cooling rate of 20 °C/min from 5 to − 80 °C in 250-µL French straws. For artificial insemination, samples were thawed at 40 °C for 5 s in a water bath, washed once using fresh HBSS500 by centrifuging at 1000 g for 5 min at 4 °C, concentrated into ~ 5 µL, and injected into virgin females of Xiphophorus maculatus. The inseminated females were monitored for 90 days for subsequent discharge of live young. Results from 2006 and 2007 showed considerable male-to-male variation in post-thaw motility (from 1 to 70%). Offspring were produced by cryopreserved sperm in two tanks (of three) at 36 and 66 days after insemination in 2007. Paternity was confirmed via phenotypes (body color) and genotypes (microsatellite genetic marker) of the hybrid offspring. Overall, a practical protocol for sperm cryopreservation and artificial insemination is provided to preserve X. couchianus, which is an important biomedical research model, and also currently listed as an endangered species in the International Union for Conservation of Nature (IUCN) red list.

Fgfr1 signalling in the development of a sexually selected trait in vertebrates, the sword of swordtail fish

BackgroundOne of Darwin's chosen examples for his idea of sexual selection through female choice was the "sword", a colourful extension of the caudal fin of male swordtails of the genus Xiphophorus. Platyfish, also members of the genus Xiphophorus, are thought to have arisen from within the swordtails, but have secondarily lost the ability to develop a sword. The sustained increase of testosterone during sexual maturation initiates sword development in male swordtails. Addition of testosterone also induces sword-like fin extensions in some platyfish species, suggesting that the genetic interactions required for sword development may be dormant, rather than lost, within platyfish. Despite considerable interest in the evolution of the sword from a behavioural or evolutionary point of view, little is known about the developmental changes that resulted in the gain and secondary loss of the sword. Up-regulation of msxC had been shown to characterize the development of both swords and the gonopodium, a modified anal fin that serves as an intromittent organ, and prompted investigations of the regulatory mechanisms that control msxC and sword growth.ResultsBy comparing both development and regeneration of caudal fins in swordtails and platyfish, we show that fgfr1 is strongly up-regulated in developing and regenerating sword and gonopodial rays. Characterization of the fin overgrowth mutant brushtail in a platyfish background confirmed that fin regeneration rates are correlated with the expression levels of fgfr1 and msxC. Moreover, brushtail re-awakens the dormant mechanisms of sword development in platyfish and activates fgfr1/msxC-signalling. Although both genes are co-expressed in scleroblasts, expression of msxC in the distal blastema may be independent of fgfr1. Known regulators of Fgf-signalling in teleost fins, fgf20a and fgf24, are transiently expressed only during regeneration and thus not likely to be required in developing swords.ConclusionOur data suggest that Fgf-signalling is involved upstream of msxC in the development of the sword and gonopodium in male swordtails. Activation of a gene regulatory network that includes fgfr1 and msxC is positively correlated with fin ray growth rates and can be re-activated in platyfish to form small sword-like fin extensions. These findings point towards a disruption between the fgfr1/msxC network and its regulation by testosterone as a likely developmental cause for sword-loss in platyfish.

Etiology of MNU-induced melanomas in Xiphophorus hybrids

Genetic hybrids of the genus Xiphophorus have historically been useful models for study of the genetic aspects of tumor formation. In the most studied Xiphophorus tumor model, two-gene loci, XMRK and DIFF, are implicated as critical both to UV-induced and spontaneous melanoma formation in BC 1 hybrids of crosses between X. maculatus and X. helleri, with X. helleri as the recurrent backcross parent. In addition to UV, the direct-acting carcinogen N-methyl- N-nitrosourea (MNU) has been used to induce tumors in Xiphophorus BC 1 hybrids from several cross types. In the present study, we address the hypothesis that excess melanomas in MNU-treated BC 1 hybrids may have been generated by direct mutation of CDKN2AB, a candidate gene for DIFF. MNU treatment of F 1 and BC 1 hybrid fish significantly increased tumor incidence at 6 months; however, no association was found between MNU-induced tumor formation and zygosity of the candidate tumor tumor-suppressor CDKN2AB in BC 1 hybrids, consistent with previously reported results. Sequence analysis of the X. maculatus CDKN2AB locus of heterozygous individuals (both BC 1 and F 1 hybrids) did not reveal any mutations caused by MNU, suggesting that the mechanism of MNU-induced melanoma formation in this Xiphophorus model does not involve direct mutation of CDKN2AB but may result from mutation of other critical genes.

An organizer controls the development of the “sword,” a sexually selected trait in swordtail fish

Male swordtail fish of the genus Xiphophorus (Poeciliidae) possess a "sword" that is composed of several colored elongated ventral fin rays of the caudal fin. The sword is a secondary sexual trait that evolved through sexual selection by female preference. To uncover the developmental mechanisms underlying the metamorphosis from a juvenile caudal fin to the sword, we have devised a transplantation protocol to assay the fate of single transplanted fin rays and their interactions with flanking rays. These experiments provide evidence for the existence of a previously unrecognized inductive signal that originates in those rays that develop into the two longest sword rays. This "sword organizer" causes adjacent fin rays to grow and become integrated into the sword and induces the development of an additional, typically pigmented sword in grafts to the dorsal part of the caudal fin. We show that the potential to develop a sword is restricted to certain parts of the caudal fin. Our findings suggest that the evolution of swords in swordtails required the acquisition of two developmental mechanisms: the establishment of signaling competence in prospective sword rays in the embryo or early larva, and its activation through androgen signaling in adult male fish.

Production of F1 interspecies hybrid offspring with cryopreserved sperm from a live-bearing fish, the swordtail Xiphophorus helleri.

Despite study of sperm cryopreservation in more than 200 fish species, production of broods from cryopreserved sperm in live-bearing fish has not been demonstrated. This has not been due to a lack of effort, but instead is a result of the unique morphology, biology, and biochemistry of reproduction in viviparous fishes. For example, sperm of Xiphophorus helleri have a cylindrical nucleus, can swim for days after being activated, have glycolytic capabilities, and can reside in the female reproduction tract for months before fertilization. These traits are not found in fishes with external fertilization. The long-standing research use of the genus Xiphophorus has led to development of over 60 pedigreed lines among the 26 species maintained around the world. These species and lines serve as contemporary models in medical research, although they must be maintained as live populations. Previous attempts at establishing sperm cryopreservation protocols for Xiphophorus have not produced live young. To address this we have been studying the parameters surrounding cryobiology of Xiphophorus sperm and applying this information to an improved understanding of internal fertilization and reproduction. Here we report the first successful fertilization and offspring production by cryopreserved sperm in any live-bearing fish. This claim is supported by our use of artificial insemination between two species that yield distinct hybrid offspring to verify paternity via cryopreserved sperm. We provide a practical approach for preservation of valuable genetic resources from live-bearing fish species, a group that is rapidly being lost due to destruction of native habitats.

Conservation of Synteny Between Guppy andXiphophorusGenomes

The guppy and fish in the genus Xiphophorus have both been important model systems for the study of natural and sexual selection for over 50 years. Whereas the guppy is unique in the degree to which the environmental variables shaping phenotypic variation are known, Xiphophorus has the advantage that genomic resources have been developed due to the utility of this taxon for the study of melanoma. If linkage maps for the guppy and Xiphophorus are similar, genomic resources developed in Xiphophorus will be useful in the guppy. The authors used an F2 mapping cross of divergent populations of the guppy to construct partial female and male genetic linkage maps incorporating microsatellite markers derived from Xiphophorus mapping efforts. Flanking regions for a sample of microsatellites occurring in maps for both taxa were sequenced in the guppy and compared to published sequences from Xiphophorus. This confirmed that these loci were homologous and estimated the divergence in neutral nuclear DNA to be 0.21 substitutions per site. The female map comprises 16 linked markers on six linkage groups, and the male map comprises 24 markers on nine linkage groups. Linkage relationships among loci homologous in the guppy and Xiphophorus primarily show conservation of genetic architecture between species, but several major changes were detected.

The Genus Xiphophorus in Mexico and Central America

The genus Xiphophorus is found from northeastern Mexico (Coahuila) for about 2200 Km as far as Honduras. There are 26 species, of which 21 occupy headwaters on the eastern slope of the Sierra Madre Oriental and continuing Cordillera to the southeast. Virtually all the species in the headwaters occupy limited ranges, often in rivers traversing karst country that are separated from lowland streams by underground passages. Only the three forms in the coastal plain are more widely distributed. Nineteen taxa occur within 400 Km of the Mexican Trans Volcanic Belt, suggesting that the genus may have evolved in this region. In many localities two species are sympatric, but natural hybrids are only known from three or four sites. Four monophyletic groups have been identified: the northern platyfish and the northern swordtail groups, north of the Mexican Trans Volcanic Axis, and to the south the helleri and the clemenciae swordtail groups. The status of the three southern platyfish is still not resolved and the phylogenetic relationship of the different groups to each other is still not fully understood.

Photocarcinogenesis inXiphophorusHybrid Models

The poeciliid fish genus Xiphophorus provides important models for investigating the etiology and genetics of sunlight-induced melanoma. Interspecific hybrids generated among platyfish and swordtails have been used as genetic tumor models, particularly for cutaneous malignant melanoma (CMM), for more than 6 decades. Oncogene and tumor suppressor gene involvement in a variety of spontaneous and carcinogen-induced tumors has been and continues to be extensively studied. Select hybrids develop melanoma spontaneously or after acute or chronic exposure to ultraviolet radiation. Many scientists believe that the etiology of CMM and particularly its initiation is different from other types of sunlight-induced skin cancers, and may involve free radical chemistry rather than the direct absorption of UVB by DNA. Xiphophorus offers a unique platform to scrutinize this question and determine the types of DNA damage that are involved, the solar wavelength ranges that are important, and the role of DNA repair genes in early tumorigenesis. The diverse photochemical and photobiological responses observed in the different Xiphophorus species and interspecies hybrids suggest that heritable traits governing DNA damage induction and repair may be involved in the susceptibility of Xiphophorus hybrids to melanomagenesis.

Melanin pattern morphs do not differ in metabolic rate: implications for the evolutionary maintenance of a melanophore polymorphism in the green swordtail, Xiphophorus helleri

Variation in melanin patterns among individuals, populations, and species is common in fishes of the genus Xiphophorus. In the variable platyfish, Xiphophorus variatus, variation in metabolic rate is associated with melanin coloration and the color morphs appear to be physiological specialists adapted to particular environmental conditions. This study investigates whether a melanin polymorphism in the green swordtail, Xiphophorus helleri, is likewise associated with variation in metabolic rate. We measured metabolic rate as oxygen consumption rate of both adult male and juvenile X. helleri in static respirometers. The oxygen consumption rate does not differ significantly between the spotted and nonspotted morphs in either group, suggesting that-unlike in X. variatus-selection on metabolic rate is not involved in maintaining the polymorphism in X. helleri. We suggest that explanations need to be sought for the evolution of melanophore diversity in Xiphophorus that are pertinent to each melanin pattern polymorphism or groups of similar polymorphisms.

Subzero water permeability parameters and optimal freezing rates for sperm cells of the southern platyfish, Xiphophorus maculatus

This study reports the subzero water transport characteristics (and empirically determined optimal rates for freezing) of sperm cells of live-bearing fishes of the genus Xiphophorus, specifically those of the southern platyfish Xiphophorus maculatus. These fishes are valuable models for biomedical research and are commercially raised as ornamental fish for use in aquariums. Water transport during freezing of X. maculatus sperm cell suspensions was obtained using a shape-independent differential scanning calorimeter technique in the presence of extracellular ice at a cooling rate of 20 °C/min in three different media: (1) Hanks’ balanced salt solution (HBSS) without cryoprotective agents (CPAs); (2) HBSS with 14% (v/v) glycerol, and (3) HBSS with 10% (v/v) dimethyl sulfoxide (DMSO). The sperm cell was modeled as a cylinder with a length of 52.35 μm and a diameter of 0.66 μm with an osmotically inactive cell volume ( V b) of 0.6 V 0, where V 0 is the isotonic or initial cell volume. This translates to a surface area, SA to initial water volume, WV ratio of 15.15 μm −1. By fitting a model of water transport to the experimentally determined volumetric shrinkage data, the best fit membrane permeability parameters (reference membrane permeability to water at 0 °C, L pg or L pg [cpa] and the activation energy, E Lp or E Lp [cpa]) were found to range from: L pg or L pg [cpa] = 0.0053–0.0093 μm/min atm; E Lp or E Lp [cpa] = 9.79–29.00 kcal/mol. By incorporating these membrane permeability parameters in a recently developed generic optimal cooling rate equation (optimal cooling rate, B opt = 1009.5 exp ( - 0.0546 E Lp ) ( L pg ) ( SA WV ) where the units of B opt are °C/min, E Lp or E Lp [cpa] are kcal/mol, L pg or L pg [cpa] are μm/min atm and SA/WV are μm −1), we determined the optimal rates of freezing X. maculatus sperm cells to be 28 °C/min (in HBSS), 47 °C/min (in HBSS + 14% glycerol) and 36 ° C/min (in HBSS + 10% DMSO). Preliminary empirical experiments suggest that the optimal rate of freezing X. maculatus sperm in the presence of 14% glycerol to be ∼25 °C/min. Possible reasons for the observed discrepancy between the theoretically predicted and experimentally determined optimal rates of freezing X. maculatus sperm cells are discussed.

Strong Reproductive Skew Among Males in the Multiply Mated Swordtail Xiphophorus multilineatus (Teleostei)

Male swordtails in the genus Xiphophorus display a conspicuous ventral elongation of the caudal fin, the sword, which arose through sexual selection due to female preference. Females mate regularly and are able to store sperm for at least 6 months. If multiple mating is frequent, this would raise the intriguing question about the role of female choice and male-male competition in shaping the mating system of these fishes. Size-dependent alternate mating strategies occur in Xiphophorus; one such strategy is courtship with a sigmoid display by large dominant males, while the other is gonopodial thrusting, in which small subordinate males sneak copulations. Using microsatellite markers, we observed a frequency of multiple paternity in wild-caught Xiphophorus multilineatus in 28% of families analyzed, but the actual frequency of multiple mating suggested by the correction factor PrDM was 33%. The number of fathers contributing genetically to the brood ranged from one to three. Compared to other species in the family Poeciliidae, both frequency and degree of multiple paternity were low. Paternity was found to be highly skewed, with one male on average contributing more than 70% to the offspring. Hence in this Xiphophorus mating system, typically one male dominates and sneaker males do not appear to be particularly effective. Postcopulatory mechanisms, however, such as sperm competition, are also indicated by our data, using sex-linked phenotypes among the offspring.