Trophic morphology and diet of the endangered fish Tlaloc hildebrandi (Cyprinodontiformes: Profundulidae) / Morfología trófica y dieta de Tlaloc hildebrandi (Cyprinodontiformes: Profundulidae), especie amenazada de extinción

Abstract Introduction: Tlaloc hildebrandi is a freshwater killifish, endemic to Southern Mexico and under threat of extinction; the knowledge of the trophic morphology and diet is needed by conservation managers. Objective: To analyse and describe the anatomy of the visceral skeleton, visceral musculature, digestive tract and its adjoining glands of T. hildebrandi; as well as its diet. Methods: We performed the trophic anatomy on 20 adult specimens of both sexes, through manual dissection; as well as gut content analysis in 60 individuals to describe the diet. Results: As notable characters of the visceral skeleton of T. hildebrandi we found the posterior notch of the premaxillary, the presence of the "coronoid cartilage", the tricuspid shape of the gill rakers of the first branchial arch, and the presence of the coronomeckelian bone; some outstanding characters of the visceral musculature are the origin of the retractor dorsalis muscle from the first four vertebral centra, and the division of the pharyngoclavicularis externus muscle into two sections. The notable characters of the digestive tube are the absence of stomach and pyloric caeca, and the presence of the "intestinal valve". Insects (IVI = 66.6 %) and ostracods (13 % IVI) were the dominant prey items of the Tlaloc hildebrandi diet; larvae and adults of the family Chironomidae were the most dominant insects in the diet (53 % IVI). Conclusions: The organization of the digestive system of T. hildebrandi corresponds to the general morphologic pattern of the Cyprinodontiformes; however, we register as new information for these fish, the presence of the "coronoid cartilage" and the "intestinal valve". The structures of the trophic morphology and the components of the diet, confirms us that T. hildebrandi is a carnivorous-insectivorous fish.

Resumen Introducción: Tlaloc hildebrandi es un killi de agua dulce, endémico del sur de México y bajo amenaza de extinción; el conocimiento de la morfología trófica y la dieta son necesarios para los administradores de la conservación. Objetivo: Analizar y describir la anatomía del esqueleto visceral, la musculatura visceral, el tracto digestivo y las glándulas adyacentes de T. hildebrandi; así como los componentes de su dieta. Métodos: Mediante la técnica del descarnado manual, realizamos la descripción de la anatomía trófica en 20 especímenes adultos de ambos sexos, y el análisis del contenido estomacal en 60 individuos para describir la dieta. Resultados: Como caracteres sobresalientes del esqueleto visceral de T. hildebrandi está la escotadura posterior del premaxilar, la presencia del "cartílago coronoides", la forma tricúspide de las branquiespinas del primer arco branquial y la presencia del hueso coronomeckeliano; como caracteres de la musculatura visceral sobresalen el origen del músculo retractor dorsalis de los cuatro primeros centros vertebrales, y la división del músculo pharyngoclavicularis externus en dos secciones. Los caracteres notables del tubo digestivo son la ausencia de estómago y de ciegos pilóricos y la presencia de la "válvula intestinal". Los insectos (IVI = 66.6 %) y ostrácodos (13 % IVI) fueron los componentes dominantes de la dieta de T. hildebrandi; particularmente las larvas y adultos de la familia Chironomidae fueron los insectos más abundantes en la dieta (53 % IVI). Conclusiones: La organización del sistema digestivo de T. hildebrandi corresponde al patrón morfológico general de los Cyprinodontiformes, sin embargo, se registra como nueva información para estos peces, la presencia del cartílago coronoides y la válvula intestinal. Las estructuras de la morfología trófica y los componentes de la dieta nos confirman que T. hildebrandi es un pez carnívoro-insectívoro.

Descripción morfológica e histológica del tejido hepático de Gambusia holbrooki (Pisce: Poeciliidae) que habita en la Provincia de Arica - Chile / Morphological and histological description of the liver tissue of Gambusia holbrooki (Pisce: Poeciliidae) inhabiting the Province of Arica - Chile

Gambusia holbrooki es el pez de agua dulce con mayor distribución en Chile y el mundo, pero los estudios que abordan la morfología e histología hepática del pez son escasos. El hígado es utilizado para evidenciar efectos subletales de contaminantes ambientales y es preciso contar con una descripción histomorfológica del hígado para futuros estudios comparativos. El presente estudio tiene como objetivo describir patrones histomorfológicos del hígado de Gambusia holbrooki, para ello se colectaron 97 individuos adultos de los sistemas límnicos de los valles de Lluta y Azapa (Extremo norte de Chile), para observar patrones morfológicos comunes en ambas poblaciones, se utilizó técnicas histológicas de rutina e histoquímica. Las evidencias demostraron que el hígado contiene tejido pancreático y su arquitectura tisular es trabecular con mayor presencia de capilares sinusoides.

Gambusia holbrooki is the freshwater fish with the greatest distribution in Chile and the world. However, studies dealing with morphology and liver histology of fish are scarce. The liver commonly shows the sublethal effects of environmental pollutants and there should be a histomorphological description of the liver for further comparative studies. The present study aims to describe histomorphological patterns of the liver of Gambusia holbrooki. A total of 97 adult individual specimens were collected from the ecosystems in the valleys of Lluta and Azapa (Region of Arica and Parinacota), to observe patterns morphologically common in both populations. Routine histological and histochemical techniques were used for analysis. The evidence showed that the liver contains pancreatic tissue, and that tissue architecture is trabecular with greater presence of capillary sinusoids.

Rapid masculinization and effects on the liver of female western mosquitofish (Gambusia affinis) by norethindrone.

Natural and synthetic progestins in receiving streams can disrupt the normal endocrine systems of fish. Norethindrone (NET) is a widely used synthetic progestin that often appears in wastewater effluents. For this research, adult female western mosquitofish (Gambusia affinis) were exposed to NET at three concentrations. The effects of NET on the following biological factors were evaluated: the histology of the ovaries and livers, the anal fin morphology, and transcription of genes related to steroidogenesis signaling pathways in the livers. After 42 d exposure to NET at 33.0 ng L-1 and 347.5 ng L-1, rapid masculinization, an increase in the number of atretic and postovulatory follicles in the ovary, enhanced vascularization, degenerated hepatocytes and irregular nuclei in the livers were observed. Exposure to NET did not affect the expression of the androgenic and estrogenic receptor genes and Cyp19a except for a significant up-regulation of Erα. However, the expression of Vtg A, Vtg B, and Vtg C were markedly inhibited in the females exposed to three concentrations of NET. Compared to the control female, exposure to NET at 33.0 ng L-1 and 347.5 ng L-1 caused a 4.4- and 5.8-fold increase in the expression of Hsd17ß3 in the livers, respectively. The results demonstrate that NET can cause rapid masculinization of female G. affinis, hepatopathological alterations and inhibited expressions of Vtg A, Vtg B, and Vtg C. The results imply that G. affinis populations might be threatened in NET-contaminated environment.

Response of PXR signaling pathway to simvastatin exposure in mosquitofish (Gambusia affinis) and its histological changes.

As a widely used lipid lowering agent, simvastatin recently has been frequently detected in aquatic environment and the potential adverse effects from simvastatin exposure to non-target organisms such as fish is worthy of more attention. The aim of this study was to reveal the responses of detoxification system in fish to simvastatin exposure. In this investigation a ubiquitous small freshwater fish, mosquito fish (Gambusia affinis), was employed as test organism, and the transcriptional expression of nucleus transcriptional factor pregnane X receptor (PXR) and its downstream genes, including P-glycoprotein (P-gp), cytochrome 3A (CYP3A), multidrug resistance protein 2 (MRP2), UDP-glucuronosyl transferase (UGT) in mosquito fish were investigated by qRT-PCR methods under the exposure of concentrations of simvastatin (0.5 µg L-1, 5 µg L-1, 50 µgL-1, 500 µg L-1) for 24 h, 72 h and 168 h. The related enzyme activity (Erythromycin-N-Demethylase, ERND), the protein expression of PXR and the histological changes of liver tissues in fish were also determined via west blotting and transmission electron microscope approaches in the same conditions. Results showed that the mRNA expression of PXR, CYP3A and P-gp showed significantly changes under simvastatin exposure, exhibiting an obvious time/dose-effect relationship with the prolong of exposure time. ERND activity also showed time-effect at 24 h, and western blotting showed PXR protein displaying a dose-effect relationship to some extent. Hepatocyte cellular of mosquito fish exposed to simvastatin (5 µg L-1, 168 h) exhibited obvious histological changes in form of swelling, incomplete fragmentary structure etc. Overall, simvastatin altered the expression of PXR signaling pathway and subsequently bring about changes in high-levels of mosquito fish.

Origins of female genital diversity: Predation risk and lock-and-key explain rapid divergence during an adaptive radiation.

The study of male genital diversity has long overshadowed evolutionary inquiry of female genitalia, despite its nontrivial diversity. Here, we identify four nonmutually exclusive mechanisms that could lead to genital divergence in females, and potentially generate patterns of correlated male-female genital evolution: (1) ecological variation alters the context of sexual selection ("ecology hypothesis"), (2) sexually antagonistic selection ("sexual-conflict hypothesis"), (3) female preferences for male genitalia mediated by female genital traits ("female-choice hypothesis"), and (4) selection against inter-population mating ("lock-and-key hypothesis"). We performed an empirical investigation of all four hypotheses using the model system of Bahamas mosquitofish inhabiting blue holes that vary in predation risk. We found unequivocal support for the ecology hypothesis, with females exhibiting a smaller genital opening in blue holes containing piscivorous fish. This is consistent with stronger postmating female choice/conflict when predators are present, but greater premating female choice in their absence. Our results additionally supported the lock-and-key hypothesis, uncovering a pattern of reproductive character displacement for genital shape. We found no support for the sexual conflict or female choice hypotheses. Our results demonstrate a strong role for ecology in generating female genital diversity, and suggest that lock-and-key may provide a viable cause of female genital diversification.

Conservation status of an endangered annual fish Hypsolebias antenori (Rivulidae) from Northeastern Brazil / Status de conservação de Hypsolebias antenori (Rivulidae) um peixe anual ameaçado do Nordeste brasileiro

This work presents information regarding the biology and state of conservation of an endangered annual fish Hypsolebias antenori. Fish were captured from small seasonal pools located in Northeastern Brazil. The total body length, body mass, sex ratio, first sexual maturity were investigated. The sampled population showed sexual dimorphism. There was a predominance of females (60%) over males (40%) with a sex ratio of 1: 1.4 and males were bigger and heavier than the females. Amplitude of total length of males ranged from 2.6 to 7.1cm (4.1±1.15) and that of females from 2.2 to 5.4cm (3.6±0.9). Amplitude of body mass of males varied from 0.25 to 7g (1.3g±1.4) and that of females from 0.12 to 2.1g (0.7g±0.5). The total weight-length equation of males was Wt=0.0108Lt3.172 with r = 0.9826 and of females was Wt=0.0122Lt3.0114 with r = 0.9608. Females attained first sexual maturity at 3.2 cm (±0.25) total length and males at 3.3 cm (±0.08) total length. All temporary pools surveyed in Northeastern Brazil were in a high degree of degradation, suffering due to anthropogenic action. Reduced rainfall resulting from global climate change prevents the filling up of these pools, thus preventing the completion of the reproductive cycle of annual fishes. One factor hindering development of conservation strategies is limited literature on biology and conservation status of annual fishes. There is a need for conservation measures to protect annual fish populations, especially creation of protected areas in the Brazilian semiarid region.

Este trabalho apresenta informações sobre a biologia e estado de conservação de Hypsolebias antenori, um peixe anual ameaçado. Os peixes foram capturados em pequenas poças temporárias localizadas no Nordeste do Brasil. O comprimento total, peso total, proporção sexual e a primeira maturação sexual foram investigados. A espécie em estudo apresentou dimorfismo sexual bem evidente. Houve um predomínio de fêmeas (60%) sobre os machos (40%), com uma razão sexual de 1M: 1,4F. Os machos foram significativamente maiores e mais pesados do que as fêmeas. A amplitude do comprimento total dos machos variou de 2,6 a 7,1 centímetros (4,1 ± 1,15) e as fêmeas de 2,2 a 5,4 centímetros (3,6 ± 0,9). A amplitude de peso dos machos variou de 0,25 a 7 g (1,4 ± 1,3 g), enquanto que as fêmeas de 0,12 a 2,1 g (0,5 ± 0,7 g). A equação da relação peso total/comprimento dos machos foi Wt = 0.0108Lt3.172 com r = 0,9826 e para fêmeas foi Wt = 0.0122Lt3.0114 com r = 0,9608. As fêmeas alcançaram a primeira maturação sexual com 3,2 cm ( ± 0,25) de comprimento total e os machos com 3,3 centímetros ( ± 0,08 ) de comprimento total. Todas as poças temporárias amostradas neste trabalho estavam em um alto grau de degradação, principalmente devido à ação antrópica. Observamos que diminuição das chuvas e a desertificação decorrente das mudanças climáticas globais que também são alarmantes e poderão impedir a conclusão do ciclo reprodutivo dos peixes anuais. As informações sobre a biologia e o estado de conservação dos peixes anuais ainda são limitadas, portanto há uma grande necessidade estudos e de medidas de conservação para proteger as populações de peixes anuais, entre elas especialmente a criação de áreas proteção nos ambientes aquáticos efêmeros do semiárido brasileiro.

Diferencias osteológicas entre los subgéneros Profundulus y Tlaloc (Teleostei: Profundulidae) / Osteological differences between the subgenus Profundulus and Tlaloc (Teleostei: Profundulidae)

El género Profundulus es uno de los grupos de peces más antiguos, distribuidos al norte de América Central en México, Guatemala y Honduras. No obstante, son pocos los trabajos que se han realizado para conocer el número de especies que lo conforman y sus relaciones filogenéticas. Históricamente se reconoció que este taxón estaba conformado por dos grupos naturales, actualmente considerados como los subgéneros Profundulus y Tlaloc. Recientes investigaciones genéticas han revelado un alto grado de diferenciación entre ambos taxones y algunas de sus especies, lo cual probablemente este asociado a cambios morfológicos. De esta manera, se realizó la comparación osteológica entre seis especies de los subgéneros Profundulus y Tlaloc (Cyprinodontidae: Profundulidae). Se revisaron 17 huesos ubicados en el cráneo y la cintura pélvica, se encontraron diferencias cualitativas en el mesetmoides, vómer, lacrimal, premaxilar, articular, cuadrado, interopérculo y basipterigio. Los patrones de variación observados permiten diagnosticar ambos subgéneros, corroborando lo encontrado en estudios morfológicos y moleculares. Las evidencias permiten suponer que ambos taxones podrían ser reubicados como géneros, como previamente se había establecido.

The genus Profundulus is one of the oldest fish groups, distributed north of Central America in Mexico, Guatemala and Honduras. However, there are few studies that determine the number of species and their phylogenetic relationships. Historically it has been recognized that this taxon comprises two natural groups, defined as the subgenus Profundulus and Tlaloc. Recent genetic researches have revealed a high degree of differentiation between the two taxa and some species, probably associated with the morphological changes. In this way, we made the osteological comparison between six species of the subgenus Profundulus and Tlaloc (Cyprinodontidae: Profundulidae). We reviewed 17 bones located in the skull and pelvic girdle; qualitative differences were found in eight bones: the mesethmoid, vomer, lachrymal, premaxilla, articular, quadrate, interoperculum, and basipterygium. The variation patterns permit to diagnose both subgenus, corroborating the previous morphological and molecular studies. The evidence suggests that both taxa could be relocated as genus, such as previously was established.

Ultrastructural features of spermatogenesis in Melanorivulus punctatus (Cyprinodontiformes: Rivulidae).

Fish belonging to the family Rivulidae possess one of the most complex reproductive systems. Rivulus, a genus of freshwater fish in the Rivulidae family, was recently reclassified into five genera, including Melanorivulus. Its type species, M. punctatus, is widely distributed and probably represents a species complex. The ultrastructure of sperm has been broadly used in systematics, and we hereby describe the ultrastructural features of spermatogenesis in M. punctatus. Ten M. punctatus males were collected from the reservoir of Parque Estadual da Quineira, municipality of Chapada dos Guimarães, Mato Grosso, Brazil, and prepared for analysis by light microscopy and transmission electron microscopy. M. punctatus undergoes cystic spermatogenesis. Its cysts consist of groups of germ cells that are in synchronous development and are surrounded by cytoplasmic projections of Sertoli cells. With the breakdown of the cysts, the spermatozoa are released and their maturation is completed in the duct, where part of the cytoplasmic material is discarded through the vesicles. The mature spermatozoon is characterized by a spherical head with homogeneously condensed chromatin, a symmetric midpiece consisting of a pair of perpendicular centrioles, a ring of mitochondria, several vesicles, and one flagellum medial to the nucleus. Early stages of spermatogenesis show no peculiarities; however, in spermiogenesis, we observed that the spermatids remain interconnected by cytoplasmic bridges and have pockets of residual cytoplasm. The sperm is of the aquasperm type and is similar to that observed in the members of the family Rivulidae. The spermatozoa have a single flagellum that consists of a classic axoneme (9+2), as found in most groups of fish, despite the lateral extensions.

Reproductive strategies of two Neotropical killifish, Austrolebias vandenbergi and Neofundulus ornatipinnis (Cyprinodontiformes: Rivulidae) in the Bolivian Gran Chaco / Estrategias reproductivas de dos especies de rivulidos Neotropicales Austrolebias vandenbergi y Neofundulus ornatipinnis (Cyprinodontiformes: Rivulidae) en el Gran Chaco Boliviano

The dry Chaco, a semiarid thorn forest, is experiencing some of the highest deforestation rates globally, coupled with the fact that small-bodied fish are at the highest risk of extinction, the killifish inhabiting this region may be some of the most threatened taxa. Yet, aspects of ecology and life history for Neotropical killifishes in the Bolivian Gran Chaco region are completely lacking, and basic life-history data is of critical importance for the design and implementation of conservation measures. Collections were conducted during the early (January 2011) and late (March-April 2011) rainy season using an area-based sampler and dip net surveys. Fish standard length and body depth were measured as well as the number of oocytes per size class, mean oocyte diameter per size class, and total fecundity for the females of each species. A total of 490 specimens of rivulids were captured; Austrolebias vandenbergi: 85 females, 105 males and 39 juveniles, and N. ornatipinnis: 62 females, 113 males, 86 juveniles. Sexual size dimorphism, absolute fecundity, oocyte developmental stages, oocyte diameter, and population sex ratios were determined for each species. Both species exhibited sexual size dimorphism. Male A. vandenbergi exhibited longer standard length (mean±SD; males: 27.07±3.89mm, females: 23.6±2.02mm) and body depth (males: 8.9±1.7mm, females: 7.2±1.1mm) as compared to females. Male N. ornatipinnis had a similar pattern for both standard length (males: 26.0±7.1mm, females: 19.1±5.83mm) and body depth (males: 5.6±1.9mm, females: 4.7±1.0mm). Austrolebias vandenbergi had fewer and smaller oocytes per female (47±31.6) than N. ornatipinnis (206±131.2). There was a positive relationship between fecundity and female body size in both species. The presence of multiple developmental stages of oocytes (immature, maturing, and mature) suggest that both species of rivulids exhibit fractional spawning, a reproductive strategy that enhances reproductive success in these extreme habitats. Neofundulus ornatipinnis exhibited a higher mean oocyte diameter for all three developmental stages (immature, maturing, and mature) as compared to A. vandenbergi. Austrolebias vandenbergi exhibited an equal adult sex ratio (males:females, 1:1), but there was a slight female biased ratio for N. ornatipinnis (males:females, 1:1.8). These results provide fundamental and valuable information for ensuring rivulid conservation in tropical regions, and also improve the knowledge on the biology and ecology of these poorly known species. To our knowledge, this study represents the first contribution on the reproductive biology of two Neotropical annual rivulid fishes (Austrolebias vandenbergi and Neofundulus ornatipinnis) inhabiting semi-permanent and ephemeral ponds in the Gran Chaco of Southeastern Bolivia.

Información sobre la ecología e historia de vida de varias especies de rivulidos no ha sido estudiada, por lo tanto se analiza la biología reproductiva de dos especies neotropicales (Austrolebias vandenbergi y Neofundulus ornatipinnis) que habitan pozos semipermanentes y temporales en el Gran Chaco Boliviano. Las recolectas se realizaron al inicio y al final de la estación lluviosa. Dimorfismo sexual, fecundidad absoluta, diámetro de los ovocitos y proporción sexual fue determinada para cada especie de rivulido. Machos en las dos especies presentaron mayor tamaño de longitud estándar y altura del cuerpo cuando fue comparado con las hembras. Austrolebias vandenbergi tuvo menor fecundidad por hembra y huevos más pequeños que N. ornatipinnis, pero hembras en ambas especies presentaron una relación estrecha y positiva entre fecundidad y tamaño cuerpo. La presencia de varios estadios gonadales sugiere un desove fraccionado, lo cual es una estrategia reproductiva que promueve la viabilidad y sobrevivencia de estos peces en hábitats de extremas condiciones. La proporción sexual no varió entre machos y hembras de A. vandenbergi (1:1), pero en N. ornatipinnis la proporción sexual fue dominada por las hembras (1:1.8). Este estudio representa uno de los primeros que investiga aspectos de la biología reproductiva de A. vandenbergi y N. ornatipinnis, lo cual es importante para la conservación de rivulidos en regiones tropicales.

Evolution of male coloration during a post-Pleistocene radiation of Bahamas mosquitofish (Gambusia hubbsi).

Sexual signal evolution can be complex because multiple factors influence the production, transmission, and reception of sexual signals, as well as receivers' responses to them. To grasp the relative importance of these factors in generating signal diversity, we must simultaneously investigate multiple selective agents and signaling traits within a natural system. We use the model system of the radiation of Bahamas mosquitofish (Gambusia hubbsi) inhabiting blue holes to test the effects of resource availability, male body size and other life-history traits, key aspects of the transmission environment, sex ratio, and predation risk on variation in multiple male color traits. Consistent with previous work examining other traits in this system, several color traits have repeatedly diverged between predation regimes, exhibiting greater elaboration in the absence of predators. However, other factors proved influential as well, with variation in resource levels, body size, relative testes size, and background water color being especially important for several color traits. For one prominent signaling trait, orange dorsal fins, we further confirmed a genetic basis underlying population differences using a laboratory common-garden experiment. We illustrate a promising approach for gaining a detailed understanding of the many contributing factors in the evolution of multivariate sexual signals.

Pigmentation in Xiphophorus: an emerging system in ecological and evolutionary genetics.

The genus Xiphophorus has great potential to contribute to the study of vertebrate pigmentation and elucidating the relative influence of ecology, physiology, and behavior on evolution at the molecular level. More importantly, the association between pigmentation and a functional oncogene offers the potential to understand the evolution and maintenance of cancer-causing genetic elements. Using criteria laid out recently in the literature, I demonstrate the power of the Xiphophorus system for studying pigment evolution through integrative organismal biology. Using the most recent phylogeny, the phylogenetic distribution of several important pigmentation loci are reevaluated. I then review support for existing hypotheses of the functional importance of pigmentation. Finally, new observations and hypotheses regarding some of the characteristics of pigment patterns in natural populations and open questions and future directions in the study of the evolution of these traits are discussed.

A fish eye out of water: epithelial surface projections on aerial and aquatic corneas of the 'four-eyed fish' Anableps anableps.

BACKGROUND: Vertebrate corneas feature a variety of microprojections, to which a tear film adheres. These microprojections are formed by folds in epithelial cell membranes, which increase surface area, stabilise the tear film and enhance movement of nutritional and waste products across cell membranes. Differences in corneal microprojections among vertebrates have been correlated with habitat and differ markedly between terrestrial and aquatic species. METHODS: This study investigated epithelial microprojections of both the aerial (dorsal) and aquatic (ventral) corneal surfaces of the 'four-eyed fish' Anableps anableps using scanning electron microscopy. RESULTS: The central region of the dorsal cornea, which projects above the water, had a density of 16,387 ± 3,995 cells per mm(2) , while the central region of the ventral cornea (underwater) had a density of 22,428 ± 6,387 cells per mm(2), a difference that suggests an environmental adaptation along the two visual axes. Both corneal surfaces were found to possess microridges rather than microvilli or microplicae characteristic of terrestrial/aerial vertebrates. Microridges were 142 ± 9 nm wide and did not differ (p = 0.757) between dorsal and ventral corneas. Microridges were consistently separated by a distance of 369 ± 9 nm across both corneas. CONCLUSION: Dorsal-ventral differences in corneal epithelial cell density in Anableps anableps suggest a difference in osmotic pressure of the two corneas. The modest differences in the microprojections indicate that the need to secure the tear film underlying each optical axis is of prime importance, due to the likelihood that a persistent layer of water normally covers both dorsal and ventral corneal surfaces and that maintaining a transparent optical pathway for vision is critical for a species prone to predation from both above and below the water's surface.

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.

Ovarian structure and oogenesis of the oviparous goodeids Crenichthys baileyi (Gilbert, 1893) and Empetrichthys latos Miller, 1948 (teleostei, Cyprinodontiformes).

The cyprinodontiform family Goodeidae comprises two biogeographically disjunct subfamilies: the viviparous Goodeinae endemic to the Mexican Plateau, and the oviparous Empetrichthyinae, known only from relict taxa in Nevada and California. Ovarian characteristics of two oviparous species of goodeid, Crenichthys baileyi and Empetrichthys latos, studied using museum collections, are compared with those of viviparous species of goodeids. Both subfamilies have a single, cystovarian ovary. The ovary in the viviparous Goodeinae has an internal septum that divides the ovarian lumen into two compartments, and it may possess oogonia. There is no ovarian septum in the oviparous C. baileyi and E. latos. Oogenesis is similar in both subfamilies with regard to the proliferation of oogonia, initiation of meiosis, primary growth and development of an oocyte during secondary growth in which fluid yolk progressively fuses into a single globule. Notably, eggs of C. baileyi and E. latos are approximately double the size of those of the viviparous Goodeinae in which embryos develop inside the ovarian lumen and are nourished, in part, by nutrients transferred from the maternal tissues, a mode of embryo development called matrotrophy. Egg envelopes of the two subfamilies differ in that those of C. baileyi and E. latos have a relatively thick zona pellucida, attachment fibrils or filaments that develop between the follicle cells during oogenesis, and a micropyle observed only in E. latos. In contrast, viviparous goodeid eggs have a relatively thin zona pellucida, but lack adhesive fibrils, and a micropyle was not observed. These reproductive characters are compared with those of species of the eastern North American Fundulus, a representative oviparous cyprinodontiform. One newlyrecognized shared, derived character, a single, median ovoid ovary with no obvious external evidence of fusion, supports monophyly of the Goodeidae. Differences among the goodeid subfamilies and Fundulus are interpreted relative to the oviparous versus viviparous modes of reproduction.

Mucus cells in the digestive tract in prenatal platyfish larvae (Poeciliidae, Teleostei).

The appearance and histochemical properties of mucus cells in various parts of digestive tract in prenatal larvae of platyfish (Xiphophorus maculatus), a stomachless teleost, are described and compared. Such cells occurred regularly throughout the entire epithelial cell layer in mouth, pharynx, oesophagus and intestine in 6 mm long larvae i.e. approximately 1 week before delivery, but were very scarce in 4 mm long larvae. The mucin in these cells was strongly stained by alcian blue at pH 2.5 and weakly coloured by this dye at pH 1.0. It displayed a clean blue colour after high iron diamine followed by alcian blue (pH 2.5), except in some very few cells which displayed a purple-brown colour after this treatment. The mucus cells displayed various colours between purple-blue and magenta when the alcian blue (pH 2.5) was followed by periodic acid-Schiff (PAS) solution; the magenta-coloured cells were mainly confined to oesophagus. Together these findings suggest that the mucin in the digestive tract of prenatal platyfish consists mainly of non-sulphated, carboxylated glycoconjugates. The observations from the present lectin histochemistry suggest that the amount of galactosamine and glucosamine in this mucin varies much from cell to cell. Also, the various colours after PAS suggest a significant cellular heterogeneity in the mucus cell population. This may reflect different stages in development and maturation or a true and permanent cellular heterogeneity, suggesting that this mucus is able to play a number of important roles even a week before the larvae start to feed and digest.

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.

A new species of poeciliid fish, Poeciliopsis santaelena, from Peninsula Santa Elena, Area de Conservación Guanacaste, Costa Rica

An undescribed freshwater fish was recently discovered during a biological inventory of Area de Conservación Guanacaste, northwestern Costa Rica. This new species of Poeciliopsis is closely related to a group of widespread species of small spotted fishes designated as the Poeciliopsis gracilis complex in this study. The complex extends on both versants from southern Mexico to Nicaragua, and with this new finding, extends into Costa Rica. The new species is distinguished from its Central American relatives by its lack of lateral and ventral markings, higher pectoral and caudal-fin counts and more posterior placement of the dorsal fin. Poeciliopsis santaelena is endemic to the Río Potrero Grande, a river whose flow is reduced to just a few pools during the long dry season and flows fully only during very rainy weather. It is just another in a growing list of threatened or endangered species found in this unique peninsular environment. Rev. Biol. Trop. 56 (2): 829-838. Epub 2008 June 30.

Recientemente se descubrió una especie no descrita de pez dulceacuícola durante un inventario biológico en Area de Conservación Guanacaste (ACG) en el noroeste de Costa Rica. Esta especie nueva del género Poeciliopsis tiene un parentesco cercano con un grupo de especies con puntos en los costados ampliamente distribuidas que se designan en este informe como complejo de Poeciliopsis gracilis. El complejo se extiende en ambas vertientes desde el sur de México hasta Nicaragua. La especie nueva se distingue de sus parientes centroamericanos por su falta de manchas negras laterales y ventrales, mayor número de radios pectorales y caudales, y posición más posterior de la aleta dorsal. Poeciliopsis santaelena, especie endémica de un sólo riachuelo intermitente, es una especie más in la lista de especies en peligro que se encuentra en este peculiar ambiente peninsular.

Exposure to pesticides impairs the expression of fish ornaments reducing the availability of attractive males.

Ornament magnitude often reflects a local balance between sexual selection and other sources of natural selection opposing their elaboration. Human activity may disrupt this balance if it modifies the costs of producing, maintaining or displaying the ornaments. When costs are increased, a shortage of acceptable partners may ensue, with consequences commensurate with how stringent (and effective) the process of mate choice is. Here, we show that the expression of ornaments in the viviparous amarillo fish (Girardinichthys multiradiatus) is influenced by embryonic exposure to low concentrations of an organophosphorus insecticide. Male ornamental fin size, dimorphic yellow coloration and display rates were all compromised in exposed fish, but unaffected in their paternal half-sibling controls and in their sisters (morphology and colour). Exposure resulted in smaller fish of both sexes, thus the differential effect by sex was restricted to attributes such as fin size only above the naturally selected magnitude shown by females. Father phenotype predicted offspring morphology of controls, but not of exposed males, which were discriminated against by both control and exposed females. Since stringent female mate choice can result in females refusing to mate with suboptimal mates, this sub-lethal developmental effect can reduce the effective population size of amarillo fish populations.

Skeletal morphology and maturation of male Gambusia holbrooki exposed to sewage treatment plant effluent.

Sewage effluent has been identified as a major source of endocrine disrupting chemicals (EDCs) in the aquatic environment. The modified hemal spines (modified skeletal structures important in reproduction) of mosquitofish, Gambusia spp. have been shown to be under androgenic control and to be affected by exposure to estrogen. The current study aimed to investigate the effects of two sewage treatment plants (STPs) in Sydney, Australia on the morphology of hemal spines in populations of Gambusia holbrooki and on the ability of the fish to reach sexual maturity. The effluent from the two STPs had different effects on the hemal spines of males. At St. Marys differences in hemal spine morphology between fish upstream and downstream of the STP were not attributable to effluent from the STP. At Quakers Hill, results suggest that the effluent is generally estrogenic to G. holbrooki. There was a decrease in the proportion of males that were morphologically mature downstream of both STPs indicating potential population level effects that were associated with the presence of the STPs.

Assessment of the reproductive-endocrine disrupting potential of chlorine dioxide oxidation products of plant sterols.

This study examined the hypothesis that chlorine dioxide bleaching used in pulp and paper production causes the formation of reproductive-endocrine disrupting compounds from plant sterols. This was tested by conducting a laboratory simulation of the chlorine dioxide oxidation of two plant sterols, beta-sitosterol and stigmasterol. Oxidation products of the plant sterol beta-sitosterol were purified and identified and found to be cholestan-24-ethyl-3-one, 4-cholestene-24-ethyl-3-one, and 4-cholestene-24-ethyl-3,6-dione. The first two compounds were found in a number of pulp and paper effluents and biosolids. The sterols and their oxidation products were tested in vitro using bioassays for androgenicity and estrogenicity. A 28 d in vivo bioassay was employed to examine masculinization in female mosquitofish. In vitro bioassays revealed little estrogenic activity in the parent sterols or in mixtures of their oxidation products. Androgenic activity as measured by the androgen receptor binding bioassay was in the order of 19-96 microg/g testosterone equivalents but with no increase or decrease with chlorine dioxide oxidation. The mosquitofish bioassay did not show significant masculinization for any of the preparations tested. A number of androstane steroids were identified in the sterols tested, however, those compounds could only account for a small fraction of the androgenic activity in the sterols. It was clear that the parent sterols were not themselves acting as androgens in the bioassays used. This study indicated that chlorine dioxide oxidation of sterols produced predominantly oxidized sterols that were not likely to act through androgenic or estrogenic mechanisms.