Validation of qPCR reference genes in the endangered annual killifish Austrolebias charrua considering different tissues, gender and environmental conditions.

The annual killifish Austrolebias charrua is an endangered species, endemic to the southern region of South America, which inhabits temporary ponds that emerges in the rainy season. The main anthropogenic threat driving the extinction of A. charrua stems from extensive agriculture, primarily due to the widrespread use of glyphosate-based herbicides near their habitats. Annual killifishes have been used as models for ecotoxicological studies but, up to now, there are no studies about reference genes in any Austrolebias species. This represents an obstacle to the use of qPCR-based technologies, the standard method for gene expression quantification. The present study aimed to select and validate potential reference genes for qPCR normalization in the annual killifish Austrolebias charrua considering different tissues, gender and environmental conditions. The candidate reference genes 18 s, actb, gapdh, ef1a, shox, eif3g, and the control gene atp1a1 were evaluated in male and female individuals in three different tissues (brain, liver, and gills) under two experimental conditions (control and acute exposition to Roundup Transorb®). The collected tissues were submitted to RNA extraction, followed by cDNA synthesis, cloning, sequencing, and qPCR. Overall, 18 s was the most stable reference gene, and 18 s and ef1a were the most stable combination. Otherwise, considering all variables, gapdh and shox were the least stable candidate genes. Foremost, suitable reference genes were validated in A. charrua, facilitating accurate mRNA quantification in this species, which might be useful for developing molecular tools of ecotoxicological assessment based on gene expression analysis for environmental monitoring of annual killifish.

Assessment of oxidative stress biomarkers in the threatened annual killifish Austrolebias charrua exposed to Roundup.

This study aimed to analyze the toxic effects of Roundup Transorb® on the endangered Neotropical annual killifish Austrolebias charrua through the assessment of molecular and biochemical biomarkers. The fish were collected in temporary ponds and exposed to environmentally realistic concentrations of the herbicide (5 mg.L-1 for 96 h). The production of ROS, lipid peroxidation, DNA damage, and membrane fluidity were evaluated in the blood cells by flow cytometry. The mRNA expression of the antioxidant-related genes sod2, cat, gstα, atp1a1, gclc, and ucp1 across the brain, liver, and gills was quantified. The acute exposure of annual killifish to Roundup significantly increased ROS production, lipid peroxidation, and DNA damage in their erythrocytes. Likewise, Roundup Transorb® decreased membrane fluidity in the blood cells of the exposed fish. Gene expression analysis revealed that Roundup exposure alters the relative expression of genes associated with oxidative stress and antioxidant defense. Our results give rise to new insights into adaptive mechanisms of A. charrua in response to Roundup. Since Brazilian annual killifishes strongly risk extinction, this study paves the way for developing novel biotechnologies applied to environmental monitoring and aquatic toxicology assessment.

Two new annual fishes (Cyprinodontiformes: Rivulidae) unexpectedly discovered in the highlands of southern Brazil.

Two new species of Austrolebias are described based on specimens collected from temporary pools located in natural grassland landscape within the Araucaria Forest domain at exceptionally high altitudes (~1000 meters a.s.l.). Austrolebias botocudo sp. n. and Austrolebias nubium sp. n. occur, respectively, in drainages of upper rio Apuaê-Inhandava (upper rio Uruguay basin) and upper rio Taquari-Antas (upper rio Jacuí, Laguna dos Patos basin), in the Meridional Plateau of southern Brazil. Despite an intensive survey conducted in the area, only two populations of each species were recorded. Both new species occurs at altitudes that are among the higher recorded for species of the genus, and both are assigned to the subgenus Acrolebias. The new species described herein are easily distinguished for its congeners by the colour pattern of males, by presence of melanophores irregularly distributed in different parts of the body, contact organs cover the body and anal fins, position of fins related with vertebrae, by preopercular and mandibular series of neuromasts united, by a series of morphometric features and by larger maximum standard length. Austrolebias botocudo and A. nubium are distinguished from each other by colour pattern of males, length of contact organs in the flank and number of contact organs in scales of lateral line, dorsal profile of head, number of neuromasts in the preopercular + mandibular series, body depth in females, and by basihyal cartilage length. Additionally, we discuss the conservation status of the new species, and provided an identification key for the species of the subgenus Acrolebias.

Molecular phylogeny of the Austrolebias/ adloffi/ group (Cyprinodontiformes, Rivulidae), with description of two new endangered and highly endemic species of annual killifishes from the Laguna dos Patos system, southern Brazil.

The Austrolebias adloffi species group encompasses a diverse lineage of annual killifishes that occurs along the Laguna dos Patos/Lagoa Mirim system, in both Brazilian and Uruguayan territories. We herein employ an integrative taxonomy approach to describe two new species of the group, inferring their phylogenetic relationships and evaluating their conservation status. Austrolebias cheffei sp. nov. and Austrolebias lourenciano sp. nov. are herein described from the western portion of the Laguna dos Patos system. Austrolebias cheffei is distinguished from the remaining species of the A. adloffi species group by presenting a yellowish green or yellowish blue dorsal fin, with wide black to dark brown bars extending from the base to the middle portion of the dorsal and anal fins in the males. Austrolebias lourenciano is distinguished from the remaining species of the A. adloffi species group by presenting a yellowish green dorsal fin, with light yellow or light bluish bars forming small triangles, interspersed with small dark brown rows of blotches in the dorsal fin base, and greenish blue anal fin, sometimes with lighter elongated yellowish iridescent blotches, limited to the basal region. According to mitochondrial cytb sequences, both species are reciprocally monophyletic relative to other species of the A. adloffi species group, and present positive barcoding gap values. Interestingly, both new species form a grade that is closely related to Austrolebias aff. minuano 1, an undescribed species that occurs at the opposite margin of the Laguna dos Patos. Among the other evaluated species, A. bagual, A. aff. minuano 1, A. nigrofasciatus, A. pelotapes, A. pongondo, A. arachan, and A. viarius also revealed to be reciprocally monophyletic, whereas A. minuano and A. adloffi revealed to be paraphyletic in regard to A. charrua and A. aff. minuano 2, respectively, and A. nachtigalli is subdivided in two clades, one of which including A. reicherti, which points to the need of a taxonomic review of the group. In addition, we discussed the conservation status of the new species, corrected the type locality of A. pongondo, and provided a dichotomous identification key of the A. adloffi species group.

Organic and conventional agriculture: Conventional rice farming causes biochemical changes in Astyanax lacustris.

World food production is directly related to human population growth. Chemicals are constantly applied to pest control in crops to increase productivity. Therefore, sustainable alternatives are needed to reduce environmental impacts. The biochemical responses in liver and muscle of Astyanax lacustris collected in different rice planting systems were analysed. Ten fish were collected in organic rice cultivation systems and conventional as well as water and sediment for pesticide analysis. In water from conventional system, bentazon (56.1 µg L-1), fipronil (0.226 µg L-1) and propoxur (0.141 µg L-1) were found, while azoxystrobin and quinclorac were below the limit of quantification (LOQ). There were no pesticides in the sediment from the conventional system. In the water of the organic system, only propoxur (below the LOQ) was registered. Metalaxil (0.025 µg kg-1) were verified in the sediment, while diphenoconazole, dinoxifene and tebuconazole were below the LOQ. The presence of these pesticides in crops with an organic production system may be related to proximity to crops to conventional production systems. Besides this, the amount is very low as compared with conventional system. Muscle glycogen, protein and amino acid levels were higher in fish collected in organic ponds. Lactate and ammonia levels were higher in conventional cultures. Lipids and proteins had greater oxidative damage in both tissues in the conventional system. Although pesticides were detected in organic sediments, the parameters of metabolic and oxidative damage were probably related by exposure to higher concentrations of pesticide in the water or by the lower oxygen content of conventional systems. However, fish mortality was not observed during collections. Despite being an anthropic environment, areas of organic cultivation seem to present better conditions for the survival of A. lacustris.

Austrolebias ephemerus (Cyprinodontiformes: Rivulidae), a new annual fish from the upper Rio Paraguai basin, Brazilian Chaco.

A new species of Austrolebias belonging to the A. bellottii species group is herein described from the Brazilian Chaco, Mato Grosso do Sul state, constituting the northernmost record of the genus in Brazil, as well as the first record of this genus on the left bank of the Rio Paraguai. The new species is distinguished from all other species of the A. bellottii group by the following combination of characters: pectoral fin posterior margin reaching vertical between base of 4th and 7th anal fin rays in females, a high number of gill rakers in the first branchial arch, a lower head width in both sexes, and a small number of neuromasts in the preopercular series. Additionally, we provide information on ecology and the conservation status of the new species.

Two new species of Melanorivulus (Cyprinodontiformes: Cynolebiidae) from Rio Verde drainage, Upper Rio Paraná basin, Brazil.

Two new species of the genus Melanorivulus are herein described from the middle Rio Verde drainage, upper Rio Paraná basin, Mato Grosso do Sul, Brazil. Both new species are members of the Melanorivulus pictus clade, diagnosed by having ventral process of angulo-articular vestigial and flanks intense greenish blue or greenish golden to purplish blue above anal fin base in males. Melanorivulus nigropunctatus, new species, from wetlands of a small drainage tributary of right side of the Rio Verde, differs from all other congeners by possessing black dots over the head and body in both sexes and pectoral fin orange with a dark grey margin in males. Melanorivulus ofaie, new species, is found in a similar environment, but at the opposite margin of the Rio Verde. It is distinguished by males presenting flank greenish blue to light blue, with seven to nine oblique chevron-like red bars, ventral portion of head whitish with dark brown spots, dorsal fin yellow with two to three transverse broad red oblique stripes and distal region red, anal fin light orangish yellow, basal area light blue with short red bars and distal portion with a dark red margin, and caudal fin yellow or orangish yellow with three to four vertical red bars in the dorsal and middle portions, sometimes with a orange distal margin. Both new species are considered endangered due to the loss and degradation of their habitat.

First record of blackfin pearlfish Austrolebias nigripinnis (Regan, 1912 (Cyprinodontiformes, Cynolebiidae) from Brazil.

Austrolebias Costa is a genus of annual fish inhabiting temporary wetlands in the Chaco-Pampasic region of southern Brazil, Uruguay, Paraguay, Bolivia and Argentina (Costa, 2006, Nielsen & Pillet, 2015, Alonso et al., 2016). Currently, about 45 species of Austrolebias are known (Costa, 2006, Volcan et al., 2014, Nielsen & Pillet, 2015), and of these, 24 occur in Rio Grande do Sul, the southernmost state of Brazil, distributed across the Rio Uruguay basin and the Patos-Mirim lagoon system and adjacent coastal areas (Volcan et al., 2015).

A new annual fish of the genus Austrolebias (Cyprinodontiformes: Rivulidae) from Rio Camaquã basin, Laguna dos Patos system, Brazilian Pampa.

A new species of Austrolebias is described from the middle course of the Rio Camaquã, Laguna dos Patos system, Rio Grande do Sul, Brazil. The new species belongs to the Austrolebias alexandri species group, which is distinguished from the remaining congeners by the presence of a dark gray pectoral fin with bright blue iridescence in males. The new species is distinguished from all remaining species of the A. alexandri species group by the following combination of characters: presence of well-defined bright blue bars on the flank in males, shorter caudal fin length in males and females, contact organs in the first three rays of the pectoral fin in males, one to two post-optic neuromasts (rarely three), basihyal width of 50% of the length, and basihyal cartilage about 50-60% of the basihyal length. The recognition of this new species belonging to the A. alexandri group endemic of the middle course of Rio Camaquã and the recent discovery of Austrolebias bagual in the same general region reinforces the claim of the middle Rio Camaquã basin as an area of endemism for annual fishes and as such, a priority area for conservation in southern Brazil.

Metaplasia in swim bladder epithelium of the endangered annual fish Austrolebias nigrofasciatus (Cyprinodontiformes: Rivulidae) results in inadequate swimming and delayed growth

Laboratory breeding of annual fish is often impaired when fish fail to inflate the swim bladder after hatching. These fish, which are known as belly-sliders, cannot swim in the water column, moving mainly on the bottom of water. Therefore, we wanted to understand the factors underlying failed inflation of swim bladder in the annual fish Austrolebias nigrofasciatus and determine the implications of this condition on the growth of juveniles. To identify how the initial filling of the gaseous vesicle is compromised, and to determine the implications of this condition on juvenile growth, we analysed the histology of the swim bladder of both normal swimming fish and belly-sliders during their initial growth phase (first month of life). In addition to slower growth rate compared with fish swimming normally, we found that belly-sliders have metaplasia characterized by epithelium peeling of the swim bladder and the presence of haemorrhage. Based on our findings, it can be concluded that metaplasia of the swim bladder is recurrent in annual fish bred in the laboratory, which explains the large number of unviable larvae. However, its causes remain unknown.(AU)

A criação de peixes anuais é frequentemente comprometida pela ocorrência de peixes que não inflam a vesícula gasosa após a eclosão. Estes peixes não têm capacidade de se manter e nadar na coluna d'água e se locomovem no fundo da água, por isso são chamados belly-sliders. Portanto, objetivamos entender os fatores subjacentes à inflação falhada da bexiga natatória no peixe anual Austrolebias nigrofasciatus e determinar as implicações dessa condição no crescimento de juvenis. Para identificar de que forma o enchimento inicial da vesícula gasosa é comprometido, e determinar as implicações desta condição no crescimento dos juvenis, analisamos histologicamente a vesícula gasosa de peixes com natação normal e de belly-sliders durante o crescimento inicial (primeiro mês de vida). Verificamos que os belly-sliders apresentam metaplasia com descamação do epitélio da vesícula gasosa e presença de hemorragia, além de apresentar crescimento mais lento em relação aos peixes com natação normal. Concluímos que a incidência desta patologia é um problema recorrente e de grande relevância para a manutenção de peixes anuais em laboratório, sendo responsável por uma grande quantidade de juvenis inviáveis, no entanto suas causas permanecem desconhecidas.(AU)

Review of the family Rivulidae (Cyprinodontiformes, Aplocheiloidei) and a molecular and morphological phylogeny of the annual fish genus Austrolebias Costa 1998

The family Rivulidae is the fourth most diverse clade of Neotropical fishes. Together with some genera of the related African family Nothobranchiidae, many rivulids exhibit a characteristic annual life cycle, with diapausing eggs and delayed embryonic development, which allows them to survive in the challenging seasonal ponds that they inhabit. Rivulidae also includes two species known as the only the self-fertilizing vertebrates and some species with internal fertilization. The first goal of this article is to review the systematics of the family considering phylogenetic relationships and synapomorphies of subfamilial clades, thus unifying information that is dispersed throughout the literature. From this revision, it is clear that phylogenetic relationships within Rivulidae are poorly resolved, especially in one of the large clades that compose it, the subfamily Rivulinae, where conflicting hypotheses of relationships of non-annual and annual genera are evident. The second goal of this work is to present an updated phylogenetic hypothesis (based on mitochondrial, nuclear, and morphological information) for one of the most speciose genus of Rivulidae, Austrolebias. Our results confirm the monophyly of the genus and of some subgeneric clades already diagnosed, but propose new relationships among them and their species composition, particularly in the subgenus Acrolebias.

a familia Rivulidae es el cuarto clado más diverso dentro de los peces Neotropicales. Junto con algunos géneros de la familia Nothobranchiidae, muchos rivulidos presentan un característico ciclo de vida anual, con huevos resistentes a la desecación y embriones con diapausas que les permiten sobrevivir en los ambientes estacionales donde habitan. Los Rivulidae presentan también dos especies consideradas como los únicos vertebrados hermafroditas suficientes y algunas especies con inseminación interna. El primer objetivo de este artículo es actualizar la sistemática de la familia considerando las relaciones filogenéticas y las sinapomorfías de los clados que la componen, reuniendo información que se encuentra dispersa en la literatura. De esta revisión surge que las relaciones filogenéticas dentro de Rivulidae están todavía sin resolver, especialmente en uno de los grandes clados que la componen, la subfamilia Rivulinae, donde relaciones conflictivas entre géneros anuales y no anuales son evidentes. El segundo objetivo de este trabajo es presentar una hipótesis filogenética, basada en datos morfológicos, mitocondriales y nucleares, de uno de los géneros más diversos de la familia, el género Austrolebias. Nuestros resultados confirman la monofilia del género y de algunos clados subgenéricos previamente definidos, y propone nuevas relaciones entre ellos, particularmente de las especies del subgénero Acrolebias(AU)

Discovery of endangered annual killifish Austrolebias cheradophilus (Aplocheiloidei: Rivulidae) in Brazil, with comments on habitat, population structure and conservation status

Austrolebias genus comprises about 40 small annual killifishes endemic to South America and their highest diversity occurs in southern Brazil and Uruguay, especially in drainages of Patos-Mirim system. Austrolebias species are severely threatened with extinction because their life cycle and restricted ranges. Low dispersal ability and the extensive loss and fragmentation of freshwater wetlands contribute to this threat. Accurate information on the geographic distribution and ecology of the species, vital to plan conservation and management strategies, are scarce. In order to provide basic knowledge for annual fish conservation this paper reports the presence of Austrolebias cheradophilus and present data about its population structure (CPUA, size, sex ratio, length-weight relationships and condition factor) and conservation status in southern Brazil. The estimated CPUA of populations was 0.86 fish/m2. Standard length (SL) of males ranged between 32.14 and 49.17 mm and for females between 25.11 and 41.6 mm. There were no differences in SL between the sexes (t-test = - 1.678; P = 0.105), and Chi-squared test demonstrated marginal differences in proportions of sexes (2.25:1; χ2 = 3.846; P = 0.07). Allometric coefficient of the LWR was slightly hyperallometric (b = 3.08) and K of the specimens ranged from 1.84 to 2.42 (mean ± S.E. = 2.12 ± 0.04). Populations have low density and their biotopes are under critical threat, mainly due to suppression by agriculture, pastures for livestock and increase housing. The species is considered "Critically Endangered" in Brazil, furthermore is strongly recommended its inclusion on lists of endangered fauna and ensures the protection of their remaining habitats. O gênero Austrolebias compreende cerca de 40 espécies de peixes anuais endêmicos da América do Sul, cuja maior diversidade está concentrada no sul do Brasil e Uruguai, especialmente nas drenagens do sistema hidrográfico Patos-Mirim. A combinação de seu ciclo de vida peculiar com suas restritas distribuições, baixa capacidade de dispersão e ampla fragmentação das áreas úmidas fazem com que suas espécies sejam consideradas ameaçadas de extinção. Entretanto, informações sobre sua distribuição e ecologia são escassas. Visando fornecer informações básicas para a conservação, este estudo registra a presença de Austrolebias cheradophilus no sul do Brasil e apresenta dados sobre sua estrutura populacional (CPUA, tamanho, razão sexual, relação peso-comprimento e fator de condição) e estado de conservação. O CPUA total das populações foi de 0,86 peixe/m2. Não foram encontradas diferenças no tamanho padrão entre os sexos (t-test = - 1,678; P = 0,105) e o teste do Qui-quadrado apresentou um valor marginalmente significativo para a proporção dos sexos (2,25:1; χ2 = 3.846; P = 0.07). O coeficiente de "b" calculado com base na relação peso-comprimento indica um padrão de crescimento levemente alométrico positivo (b = 3,08). O fator de condição dos indivíduos variou entre 1,84 e 2,42 (média ± S.E. = 2,12 ± 0,04). As populações identificadas têm baixa densidade e estão sob ameaça crítica, principalmente devido à supressão do habitat pela agricultura, pecuária e aumento da urbanização. A espécie foi considerada "Criticamente Ameaçada", portanto recomenda-se sua inclusão em listas de fauna ameaçada e a urgente proteção dos seus habitats.

Growth of Critically Endangered annual fish Austrolebias wolterstorffi (Cyprinodontiformes: Rivulidae) at different temperatures

Given the importance of knowledge of the biology of endangered species for the planning of conservation and management efforts, the aim of this study was to investigate the influence of temperature on the growth of Austrolebias wolterstorffi (Ahl, 1924). To clarify the thermal influence on the growth of the species, temperatures of 16, 20, 24 , and 28°C were tested in triplicate. The present study showed that a water temperature of 28°C is detrimental to the growth of the species. Among the other tested temperatures, it was found that the optimum temperature for growth decreases as an individual ages and is slightly lower for females. It was demonstrated here that males reach a higher weight and length, and grow faster than females, reaching sexual maturity earlier. To optimize the growth of this species in captivity, the ideal temperature of the water during the initial life period is 24°C, until after puberty when the temperature should decrease to about 21°C.

Devido à importância do conhecimento da biologia das espécies ameaçadas de extinção para o planejamento de ações de conservação e manejo, o objetivo deste estudo foi investigar a influência da temperatura sobre o crescimento de Austrolebias wolterstorffi (Ahl, 1924). Para verificar a influência da temperatura no crescimento da espécie, as temperaturas de 16, 20, 24 e 28° C foram testadas em triplicata. O presente estudo mostrou que a temperatura da água de 28° C é prejudicial para o crescimento da espécie. Entre as outras temperaturas testadas, verificou-se que a temperatura ótima para o crescimento diminui ao longo da vida e é ligeiramente menor para as fêmeas. Foi demonstrado aqui que os machos alcançam um maior peso e comprimento e crescem mais rápido do que as fêmeas, atingindo a maturidade sexual anteriormente. Para otimizar o crescimento da espécie em cativeiro, a temperatura ideal da água durante o período inicial de vida é de 24° C, já após a puberdade, a temperatura deverá diminuir para cerca de 21°C.

Effect of temperature on growth of the threatened annual fish Austrolebias nigrofasciatus Costa & Cheffe 2001 / Efeito da temperatura no crescimento do peixe anual ameaçado Austrolebias nigrofasciatus Costa & Cheffe 2001

This study evaluated the effect of temperature on growth of Austrolebias nigrofasciatus, an endemic and threatened annual killifish species of the Patos-Mirim lagoon system in Southern Brazil. In order to verify the effect of temperature on initial growth of A. nigrofasciatus, eggs stored in the laboratory were hatched and juveniles reared for eight weeks at 16 and 22 ºC. The standard length of newly hatched fishes was 4.67 ± 0.25 mm and after eight weeks they reached 23.68 ± 3.73 and 22.68 ± 5.36 mm, respectively at 16 and 22 ºC. However, initial growth of fish reared at 22 ºC was faster and they reached sexual dimorphism at an earlier age compared to those reared at 16 ºC. Final length of females reared at 22 ºC was 23.00 ± 2.83 mm, they were significantly larger than those reared at 16 ºC (17.91 ± 2.47 mm). Males were significantly larger than the females at 16 ºC, but there was no difference for growth between sexes of fish reared at 22 ºC. The sex ratios were 1:0.6 and 1:1.1 (M:F) at 16 ºC and 22 ºC, respectively, suggesting temperature determination of phenotypic sex. Considering the results, it appears that juveniles to be developed in captivity should be kept at 22 ºC during the first six weeks of life, thus ensuring a higher growth rate until puberty.

Este estudo avaliou o efeito da temperatura no crescimento de Austrolebias nigrofasciatus, uma espécie de peixe anual endêmica e ameaçada do sistema lagunar Patos-Mirim no extremo sul do Brasil. Para avaliar o efeito da temperatura no crescimento inicial e na diferenciação sexual de A. nigrofasciatus, ovos mantidos em laboratório foram eclodidos e os peixes foram mantidos por oito semanas a temperaturas de 16 e 22 ºC. O comprimento padrão dos peixes recém eclodidos foi de 4,67 ± 0,25 mm e ao final de oito semanas atingiram 23,68 ± 3,73 e 22,68 ± 5,36 mm, respectivamente para 16 e 22 ºC. O crescimento inicial foi maior a 22 ºC e os peixes apresentaram precocidade na maturação sexual quando comparados a 16 ºC. As fêmeas criadas a 22 ºC atingiram maior comprimento (23,00 ± 2,83 mm) em relação aquelas mantidas em 16 ºC (17,91 ± 2,47 mm). Machos foram significativamente maiores que as fêmeas a 16 ºC, mas não foi constatada diferença de comprimento entre os sexos dos peixes mantidos a 22 ºC. A relação sexual foi de 1:0.6 e 1:1.1 (M:F) a 16 ºC e 22 ºC, respectivamente, sugerindo uma possível influência da temperatura na diferenciação sexual fenotípica. Considerado os resultados obtidos concluímos que juvenis de A. nigrofasciatus devem ser criados a 22 ºC durante os primeiros meses de idade, garantindo assim uma maior taxa de crescimento até a puberdade.

The opportunistic feeding and reproduction strategies of the annual fish Cynopoecilus melanotaenia (Cyprinodontiformes: Rivulidae) inhabiting ephemeral habitats on southern Brazil

A maioria dos peixes da família Rivulidae são popularmente conhecidos como anuais por completarem todo seu ciclo biológico em pequenos corpos de água temporários que secam obrigatoriamente em determinados períodos do ano causando a morte dos indivíduos adultos. Possuem características biológicas peculiares como pequeno porte, maturação sexual precoce, reprodução contínua, um elaborado padrão de corte e uma grande capacidade reprodutiva entre os peixes. Os rivulídeos se encontram amplamente distribuídos nas Américas do Norte, Central e Sul. Este trabalho analisou a dieta e a biologia reprodutiva de Cynopoecilus melanotaenia. Foram coletados 263 exemplares e a análise de 233 conteúdos gastrointestinais revelou uma dieta invertívora composta principalmente por microcrustáceos (Cladocera, Amphipoda e Ostracoda) e insetos imaturos (Chaoboridae, Culicidae, Syrphidae, mas principalmente larvas de Chironomidae). Foi registrada a lepidofagia na dieta dos machos. A fecundidade foi estimada pela análise de 59 pares de ovários maduros e variou entre 2 a 157 ovócitos (média, 19 ± 26[DP]). A espécie apresentou desova parcelada, uma estratégia para aumentar a chance de sobrevivência a depleções prolongadas. Este trabalho apresenta a primeira investigação sobre a biologia reprodutiva de C. melanotaenia. Os resultados encontrados sugerem a estratégia reprodutiva oportunista de C. melanotaenia e fornecem novas informações biológicas que podem contribuir para pesquisas de conservação e manutenção da espécie em seu ambiente natural.

Most Rivulidae fishes are popularly known as annual fishes which live in ephemeral environments such as pools, that obligatorily dry out seasonally causing the death of adult individuals. They have unique biological characteristics such as small body size, early sexual maturation, continuous reproduction, an elaborated courtship behavior, and a great reproductive capacity among fishes. The rivulids are widely distributed in North, Central and South America. In this study, the diet and reproductive biology of Cynopoecilus melanotaenia was analyzed. A total of 263 specimens were collected and the analysis of 233 gastrointestinal contents revealed an invertivorous diet composed mainly of small crustaceans (Cladocera, Amphipoda, and Ostracoda) and immature insects (Chaoboridae, Culicidae, Syrphidae, but mainly Chironomidae larvae). Lepidophagy on male’s diet was also registered. Fecundity was estimated by analyzing 59 pairs of mature ovaries and ranged from 2 to 157 oocytes (mean, 19 ± 26[SD]). The species has fractional spawning, a strategy to increase the chance of survival to prolonged depletions. This study is the first to investigate the reproductive biology of C. melanotaenia. The results confirmed the opportunistic character of the rivulid C. melanotaenia and provided unreported reproductive information that may aid conservation of the species.