Hormonal induction of spawning in 4 species of frogs by coinjection with a gonadotropin-releasing hormone agonist and a dopamine antagonist

<p>Abstract</p> <p>Background</p> <p>It is well known that many anurans do not reproduce easily in captivity. Some methods are based on administration of mammalian hormones such as human chorionic gonadotropin, which are not effective in many frogs. There is a need for simple, cost-effective alternative techniques to induce spawning.</p> <p>Methods</p> <p>Our new method is based on the injection of a combination of a gonadotropin-releasing hormone (GnRH) agonist and a dopamine antagonist. We have named this formulation AMPHIPLEX, which is derived from the combination of the words amphibian and amplexus. This name refers to the specific reproductive behavior of frogs when the male mounts and clasps the female to induce ovulation and to fertilize the eggs as they are laid.</p> <p>Results</p> <p>We describe the use of the method and demonstrate its applicability for captive breeding in 3 different anuran families. We tested several combinations of GnRH agonists with dopamine antagonists using Lithobates pipiens. The combination of des-Gly¹⁰, D-Ala⁶, Pro-LHRH (0.4 microrams/g body weight) and metoclopramide (10 micrograms/g BWt. MET) was most effective. It was used in-season, after short-term captivity and in frogs artificially hibernated under laboratory conditions. The AMPHIPLEX method was also effective in 3 Argentinian frogs, <it>Ceratophrys ornata</it>, <it>Ceratophrys cranwelli</it> and <it>Odontophrynus americanus</it>.</p> <p>Conclusion</p> <p>Our approach offers some advantages over other hormonally-based techniques. Both sexes are injected only once and at the same time, reducing handling stress. AMPHIPLEX is a new reproductive management tool for captive breeding in Anura.</p

Cortisol-Induced Masculinization: Does Thermal Stress Affect Gonadal Fate in Pejerrey, a Teleost Fish with Temperature-Dependent Sex Determination?

BACKGROUND: Gonadal fate in many reptiles, fish, and amphibians is modulated by the temperature experienced during a critical period early in life (temperature-dependent sex determination; TSD). Several molecular processes involved in TSD have been described but how the animals "sense" environmental temperature remains unknown. We examined whether the stress-related hormone cortisol mediates between temperature and sex differentiation of pejerrey, a gonochoristic teleost fish with marked TSD, and the possibility that it involves glucocorticoid receptor- and/or steroid biosynthesis-modulation. METHODOLOGY/PRINCIPAL FINDINGS: Larvae maintained during the period of gonadal sex differentiation at a masculinizing temperature (29 degrees C; 100% males) consistently had higher cortisol, 11-ketotestoterone (11-KT), and testosterone (T) titres than those at a feminizing temperature (17 degrees C; 100% females). Cortisol-treated animals had elevated 11-KT and T, and showed a typical molecular signature of masculinization including amh upregulation, cyp19a1a downregulation, and higher incidence of gonadal apoptosis during sex differentiation. Administration of cortisol and a non-metabolizable glucocorticoid receptor (GR) agonist (Dexamethasone) to larvae at a "sexually neutral" temperature (24 degrees C) caused significant increases in the proportion of males. CONCLUSIONS/SIGNIFICANCE: Our results suggest a role of cortisol in the masculinization of pejerrey and provide a possible link between stress and testicular differentiation in this gonochoristic TSD species. Cortisol role or roles during TSD of pejerrey seem(s) to involve both androgen biosynthesis- and GR-mediated processes. These findings and recent reports of cortisol effects on sex determination of sequential hermaphroditic fishes, TSD reptiles, and birds provide support to the notion that stress responses might be involved in various forms of environmental sex determination

Hormonal induction of spawning in 4 species of frogs by coinjection with a gonadotropin-releasing hormone agonist and a dopamine antagonist

Background: It is well known that many anurans do not reproduce easily in captivity. Some methods are based on administration of mammalian hormones such as human chorionic gonadotropin, which are not effective in many frogs. There is a need for simple, cost-effective alternative techniques to induce spawning.Methods: Our new method is based on the injection of a combination of a gonadotropin-releasing hormone (GnRH) agonist and a dopamine antagonist. We have named this formulation AMPHIPLEX, which is derived from the combination of the words amphibian and amplexus. This name refers to the specific reproductive behavior of frogs when the male mounts and clasps the female to induce ovulation and to fertilize the eggs as they are laid.Results: We describe the use of the method and demonstrate its applicability for captive breeding in 3 different anuran families. We tested several combinations of GnRH agonists with dopamine antagonists using Lithobates pipiens. The combination of des-Gly10, D-Ala6, Pro-LHRH (0.4 microrams/g body weight) and metoclopramide (10 micrograms/g BWt. MET) was most effective. It was used in-season, after short-term captivity and in frogs artificially hibernated under laboratory conditions. The AMPHIPLEX method was also effective in 3 Argentinian frogs, Ceratophrys ornata, Ceratophrys cranwelli and Odontophrynus americanus.Conclusion: Our approach offers some advantages over other hormonally-based techniques. Both sexes are injected only once and at the same time, reducing handling stress. AMPHIPLEX is a new reproductive management tool for captive breeding in Anura.Facultad de Ciencias Exacta

Processes that drive the population structuring of Jenynsia lineata (Cyprinidontiformes, Anablepidae) in the La Plata Basin

1. The distribution of genetic diversity across a species distribution range is rarely homogeneous, as the genetic structure among populations is related to the degree of isolation among them, such as isolation by distance, isolation by barrier, and isolation by environment. 2. Jenynsia lineata is a small viviparous fish that inhabits a wide range of habitats in South America. To decipher the isolation processes that drive population structuring in J. lineata, we analyzed 221 sequences of the mitochondrial cytochrome c oxidase I gene (COI), from 19 localities. Then, we examined the influence of the three most common types of isolation in order to explain the genetic variation found in this species. 3. Our results revealed a marked structuration, with three groups: (a) La Plata/ Desaguadero Rivers (sampling sites across Argentina, Uruguay, and Southern Brazil), (b) Central Argentina, and (c) Northern Argentina. A distance-based redundancy analysis, including the explanatory variables geographical distances, altitude, latitude, and basin, was able to explain up to 65% of the genetic structure. A variance partitioning analysis showed that the two most important variables underlying the structuration in J. lineata were altitude (isolation by environment) and type of basin (isolation by barrier). 4. Our results show that in this species, the processes of population diversification are complex and are not limited to a single mechanism. The processes that play a prominent role in this study could explain the high rate of diversity that characterizes freshwater fish species. And these processes in turn are the basis for possible speciation events.Instituto de Limnología "Dr. Raúl A. Ringuelet"Laboratorio de Sistemática y Biología Evolutiv

Evidence of Alternative Splicing as a Regulatory Mechanism for Kissr2 in Pejerrey Fish

Kisspeptin receptors are G-Protein-Coupled Receptors that regulate GnRH synthesis and release in vertebrates. Here, we report the gene structure of two kisspeptin receptors (kissr2 and kissr3) in pejerrey fish. Genomic analysis exposed a gene structure with 5 exons and 4 introns for kissr2 and 6 exons and 5 introns for kissr3. Two alternative variants for both genes, named kissr2_v1 and _v2, and kissr3_v1 and v2, were revealed by gene expression analyses of several tissues. For both receptors, these variants were originated by alternative splicing retaining intron 3 and intron 4 for kissr2_v2 and kissr3_v2, respectively. In the case of kissr2, the intron retention introduced two stop codons leading to a putatively truncated protein whereas for kissr3, the intron retention produced a reading shift leading to a stop codon in exon 5. Modeling and structural analysis of Kissr2 and Kissr3 spliced variants revealed that truncation of the proteins may lead to non-functional proteins, as the structural elements missing are critical for receptor function. To understand the functional significance of splicing variants, the expression pattern for kissr2 was characterized on fish subjected to different diets. Fasting induced an up-regulation of kissr2_v1 in the hypothalamus, a brain region implicated in control of reproduction and food intake, with no expression of kissr2_v2. On the other hand, fasting did not elicit differential expression in testes and habenula. These results suggest that alternative splicing may play a role in regulating Kissr2 function in pejerrey

Biogeography, habitat transitions and hybridization in a radiation of South American silverside fishes revealed by mitochondrial and genomic RAD data

Rivers and lake systems in the southern cone of South America have been widely influenced by historic glaciations events, carrying important implications on the evolution of aquatic organisms including prompting transitions between marine and freshwater habitats and by triggering hybridization among incipient species via waterway connectivity and stream capture events. Silverside fishes (Odontesthes) in the region comprise a radiation of 19 marine and freshwater species that have been hypothesized on the basis of morphological or mitochondrial DNA data to have either transitioned repeatedly into continental waters from the sea or colonized marine habitats following freshwater diversification. New ddRAD data presented here provide a robust framework to investigate biogeographic history and habitat transitions in Odontesthes. We show that Odontesthes silversides originally diversified in the Pacific but independently colonized the Atlantic three times, producing three independent marine-to-freshwater transitions. Our results also indicate recent introgression of marine mitochondrial haplotypes into two freshwater clades, with more recurring instances of hybridization among Atlantic- vs. Pacific-slope species. In Pacific freshwater drainages, hybridization with a marine species appears to be geographically isolated and may be related to glaciation events. Substantial structural differences of estuarine gradients between these two geographic areas may have influenced the frequency, intensity, and evolutionary effects of hybridization events.Laboratorio de Sistemática y Biología EvolutivaFacultad de Ciencias Naturales y Muse