New occurrences of the endangered Notholebias minimus (Cyprinodontiformes: Rivulidae) in coastal plains of the State of Rio de Janeiro, Brazil: populations features and conservation

Notholebias minimus is an endangered annual killifish endemic to the coastal plains of the State of Rio de Janeiro, Brazil. This study aimed to present new occurrences in the Atlantic Forest biome, provide unprecedented population features (body and egg size, fecundity, sexual ratio, and length-weight relationship - LWR), and compare changes in land use and coverage between 1985 and 2021 in biotopes located inside and outside protected areas. Three new occurrence localities were found in shallow temporary wetlands with acidic pH (6.4 ± 0.2) and low concentrations of dissolved oxygen (2.0 ± 0.9 mg/L). Males and females total length ranged from 11.1 to 31 mm and 11 to 26 mm, respectively. Batch fecundity ranged from 18 to 40 oocytes (24.8 ± 8.8), corresponding to oocytes with sizes between 800-1,006 µm (905 ± 56). Males were significantly larger than females (W = 2193.5, p = 0.0067), but both sexes occurred in similar proportions (p = 0.472). LWR showed positive allometry (b = 3.18). Biotopes located within protected areas exhibited higher conservation. Our discoveries expand the knowledge about habitat and population features of N. minimus and reinforce the importance of establishing protected areas for the conservation of annual fish biotopes.

Notholebias minimus é um peixe anual ameaçado de extinção, endêmico das planícies costeiras do Estado do Rio de Janeiro, Brasil. Neste estudo, objetivamos apresentar novas ocorrências no bioma Mata Atlântica, fornecer características populacionais inéditas (tamanho do corpo e dos ovos, fecundidade, proporção sexual e relação peso-comprimento), e comparar mudanças no uso e cobertura do solo entre 1985 e 2021 em biótopos localizados dentro e fora de unidades de conservação. Registramos três novos locais em áreas úmidas temporárias rasas com pH ácido (6,4 ± 0,2) e baixas concentrações de oxigênio dissolvido (2,0 ± 0,9 mg/L). O comprimento total de machos e fêmeas variou de 11,1 a 31 mm e de 11 a 26 mm, respectivamente. A fecundidade do lote variou entre 18-40 oócitos (24,8 ± 8,8), correspondendo a diâmetros entre 800-1.006 µm (905 ± 56). Os machos foram significativamente maiores que as fêmeas (W = 2193,5; p = 0,0067), mas ocorreram em proporções similares (p = 0,472). A relação peso-comprimento detectou alometria positiva (b = 3,18). Biótopos localizados dentro de áreas protegidas exibiram maior preservação ambiental. Nossas descobertas ampliam o conhecimento sobre as características do habitat e da população de N. minimus e reforçam a importância do estabelecimento de áreas protegidas para a conservação dos biótopos dos peixes anuais.

Salinity and prey concentration on larviculture of killifish Hypsolebias radiseriatus (Cyprinodontiformes: Rivulidae)

The aim of this study was to investigate the tolerance of Hypsolebias radiseriatuslarvae to different salinities, and the effects of different prey concentrations and water salinities on the larviculture of this species. Salinity tolerance was tested by subjecting newly-hatched larvae to 96 hours of osmotic shock testing (experiment I) and gradual acclimatization (experiment II) of the following salinities: freshwater (control), 2, 4, 6 and 8 g of salt L-1. A third experiment (experiment III) evaluated three water salinities (S0-freshwater, S2-2 g of salt L-1and S4–4 g of salt L-1) and three initial daily prey concentrations (100, 300 and 500 artemia nauplii larva-1). In experiments I and II, survival was only influenced by the salinity of 8 g of salt L-1(p < 0.01). After 35 days, weight was only influenced by prey concentration (p < 0.05), with the highest value being with 500 artemia nauplii larva-1. The lowest survival was for 4 g of salt L-1and for 100 artemia nauplii larva-1. H. radiseriatuslarviculture can be carried out in salinity of up to 2 g of salt L-1and initial daily prey concentrations with 500 artemia nauplii larva-1.

Salinity and prey concentration on larviculture of killifish Hypsolebias radiseriatus (Cyprinodontiformes: Rivulidae)

The aim of this study was to investigate the tolerance of Hypsolebias radiseriatuslarvae to different salinities, and the effects of different prey concentrations and water salinities on the larviculture of this species. Salinity tolerance was tested by subjecting newly-hatched larvae to 96 hours of osmotic shock testing (experiment I) and gradual acclimatization (experiment II) of the following salinities: freshwater (control), 2, 4, 6 and 8 g of salt L-1. A third experiment (experiment III) evaluated three water salinities (S0-freshwater, S2-2 g of salt L-1and S4–4 g of salt L-1) and three initial daily prey concentrations (100, 300 and 500 artemia nauplii larva-1). In experiments I and II, survival was only influenced by the salinity of 8 g of salt L-1(p < 0.01). After 35 days, weight was only influenced by prey concentration (p < 0.05), with the highest value being with 500 artemia nauplii larva-1. The lowest survival was for 4 g of salt L-1and for 100 artemia nauplii larva-1. H. radiseriatuslarviculture can be carried out in salinity of up to 2 g of salt L-1and initial daily prey concentrations with 500 artemia nauplii larva-1.(AU)

The recapture of Leptopanchax opalescens (Aplocheiloidei: Rivulidae), a critically endangered seasonal killifish: habitat and aspects of population structure

Leptopanchax opalescens (Myers, 1942) is a small seasonal killifish endemic to coastal drainages in the state of Rio de Janeiro, Brazil. Leptopanchax opalescens was recaptured in temporary open vegetation pools (22°42.35S, 43°41.59W) in the hydrographic basin of the Guandu River, state of Rio de Janeiro, Brazil, after eight years without records. Information on habitat and population parameters (Capture per unit area (CPUA), size, sex ratio and length-weight relationship (LWR)) are given to provide subsidies for the conservation of this species. Specimens were captured during the rainy season (January-April 2020) in shallow pools (mean depth = 21 ± 12 s.d. cm), with relatively acidic pH (5.2 ± 0.8) and low concentrations of dissolved oxygen (3.5 ± 1 mg/L). The estimated CPUA of the population was 0.89 fish/m2. The total length (TL) of males ranged from 21.1 to 28.8 cm, and females ranged from 17.0 to 31.2 mm. Males were significantly larger (t-test = 2.04, p 0.05) than females, which were in greater proportion (2.83Female: 1Male, p 0.001). A positive allometric coefficient (b = 3.19) was found through length-weight relationship curve, indicating that individuals of the species gain proportionally more increment in weight than in length. Leptopanchax opalescens is a critically endangered species, and the results of this study contribute to the knowledge of the distribution, population structure and conservation of the species and its natural habitats.

The recapture of Leptopanchax opalescens (Aplocheiloidei: Rivulidae), a critically endangered seasonal killifish: habitat and aspects of population structure

Leptopanchax opalescens (Myers, 1942) is a small seasonal killifish endemic to coastal drainages in the state of Rio de Janeiro, Brazil. Leptopanchax opalescens was recaptured in temporary open vegetation pools (22°42.35S, 43°41.59W) in the hydrographic basin of the Guandu River, state of Rio de Janeiro, Brazil, after eight years without records. Information on habitat and population parameters (Capture per unit area (CPUA), size, sex ratio and length-weight relationship (LWR)) are given to provide subsidies for the conservation of this species. Specimens were captured during the rainy season (January-April 2020) in shallow pools (mean depth = 21 ± 12 s.d. cm), with relatively acidic pH (5.2 ± 0.8) and low concentrations of dissolved oxygen (3.5 ± 1 mg/L). The estimated CPUA of the population was 0.89 fish/m2. The total length (TL) of males ranged from 21.1 to 28.8 cm, and females ranged from 17.0 to 31.2 mm. Males were significantly larger (t-test = 2.04, p 0.05) than females, which were in greater proportion (2.83Female: 1Male, p 0.001). A positive allometric coefficient (b = 3.19) was found through length-weight relationship curve, indicating that individuals of the species gain proportionally more increment in weight than in length. Leptopanchax opalescens is a critically endangered species, and the results of this study contribute to the knowledge of the distribution, population structure and conservation of the species and its natural habitats.(AU)

Age-associated liver alterations in wild populations of Austrolebias minuano, a short-lived Neotropical annual killifish.

Aging processes have become an attractive field for researchers and annual fish have been used as biological models. However, the study on the changes in age-associated markers during the normal aging in wild populations of annual fish remains open. Austrolebias is a genus of Neotropical annual killifishes, distributed mainly in ephemeral pools across grassland floodplains of temperate South America and represent an emerging biological model for aging research, but studies investigating rapid aging and senescence in this genus of annual fish are almost non-existent. This study was undertaken to examine the changes in age-associated liver markers at the different developmental stages in wild populations of Austrolebias minuano. We demonstrate that A. minuano has a number of liver alterations of different severities throughout the life cycle, suggesting that these changes tend to increase with age. Our results revealed that > 70% of the analyzed livers presented alterations. Thus, our study should instigate new approaches on aging using Neotropical annual fish, and could be useful to improve the knowledge already provided by consecrated biological aging models as e.g. Nothobranchius killifishes.

Variación en el patrón de coloración de Anablepsoides hartii (Cyprinodontiformes: Cynolebiidae) en arroyos costeros de Venezuela / Variation in the coloration pattern of Anablepsoides hartii (Cyprinodontiformes: Cynolebiidae) in coastal streams of Venezuela

Resumen Anablepsoides hartii es un pez no anual de la familia Cynolebiidae. Históricamente, incongruencias en la identificación de dicha especie han surgido debido a su variación en el patrón de coloración. Por tal motivo, se describe e ilustra los diferentes tipos de patrones de coloración de esta especie. Se revisaron 336 ejemplares de 19 lotes preservados en alcohol al 70 %, depositado en las colecciones ictiológicas del Museo de Biología de la Universidad Central de Venezuela y el Museo de Ciencias Naturales en Guanare. Los ejemplares fueron separados por características distintivas de tamaño, sexo y localidad. Adicionalmente, se tomaron fotografías de cada individuo que fueron vectorizadas a un máximo nivel de detalle, separando claros y oscuros que delimitan los patrones con un mismo tono de coloración con el programa Corel Draw versión x7 en español. Anablepsoides hartii presenta cuatro (4) patrones de coloración. Los cromotipos son: 1.- masculino, 2.- femenino, 3.- intermedio y 4.- montano. Los dos primeros cromotipos son separados por caracteres sexuales, a su vez el cromotipo intermedio presenta caracteres tanto de machos como de hembras en un mismo individuo. Finalmente, el cromotipo montano se diferencia del resto por presentar la serie de hileras laterales incompleta seguido por un reticulado de medio cuerpo hasta la cola. Esta contribución permite aclarar las diferencias que existen en una especie morfológicamente variable.

Abstract Anablepsoides hartii is a non-annual fish of the family Cynolebiidae. Historically, this species has presented some inconsistencies for proper identification, due to a variation in the color pattern. The aim of this study was to describe and illustrate the different types of color patterns found in this species. We examined a total of 336 preserved specimens (19 lots) deposited in the ichthyological collections of the Museo de Biologia de la Universidad Central de Venezuela and the Museo de Ciencias Naturales in Guanare, Venezuela. The specimens were sorted by distinctive characteristics of size, sex and locality. Besides, we took photographs of each individual, vectorized them with detail, separating light and dark colorations and delimited color patterns of the same tone with the program Corel Draw version x7 (Spanish). My analysis showed that A. hartii presented four colors patterns. The color morphs were classified as: 1 male, 2 female, 3 intermediate and 4 montane. The first two color morphs were separated by sexual characters (sexual dimorphism); in turn, the intermediate color morph presented characters of both males and females in the same individual. Finally, the montane color morph differed from the previous ones by presenting a series of incomplete lateral rows, followed by a half-body reticulation to the tail. This analysis allowed the identification of the differences in this morphologically variable species. Rev. Biol. Trop. 66(1): 293-302. Epub 2018 March 01.

Sex determination in annual fishes: Searching for the master sex-determining gene in Austrolebias charrua (Cyprinodontiformes, Rivulidae).

Evolution of sex determination and differentiation in fishes involves a broad range of sex strategies (hermaphroditism, gonochorism, unisexuality, environmental and genetic sex determination). Annual fishes inhabit temporary ponds that dry out during the dry season when adults die. The embryos exhibit an atypical developmental pattern and remain buried in the bottom mud until the next rainy season. To elucidate genomic factors involved in the sex determination in annual fish, we explored the presence of a candidate sex-specific gene related to the cascade network in Austrolebias charrua. All phylogenetic analyses showed a high posterior probability of occurrence for a clade integrated by nuclear sequences (aprox. 900 bp) from both adults (male and female), with partial cDNA fragments of A. charrua from juveniles (male) and the dsx D. melanogaster gene. The expressed fragment was detected from blastula to adulthood stages showing a sexually dimorphic expression pattern. The isolated cDNA sequence is clearly related to dsx D. melanogaster gene and might be located near the top of the sex determination cascade in this species.

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.

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 influence of temperature on the embryonic development of the annual fish Cynopoecilus melanotaenia (Cyprinodontiformes, Rivulidae)

The present study aims to provide data about the time required for Cynopoecilus melanotaenia kept at different temperatures to complete embryonic development. This information can be valuable for optimizing laboratory culture and facilitating future use of this species as a test organism in toxicity tests. Temperature effects on hatching rate are presented as well as information related to embryonic development stages. Eggs were observed daily, from start to finish of embryonic development. Thirteen developmental stages were described. Eggs were kept at two constant temperatures (20°C and 25°C) and at a variable ambient temperature (16-25°C - mean = 21°C, sd = 1.95), to determine developmental rate (velocity) at each temperature. A shorter incubation period was necessary to complete development at 25° ± 1°C. However, all embryos kept at this temperature hatched with morphological defects, which prevented their survival. No significant difference in developmental time period (p = 0.05) was observed at the 20°C and 16°-25°C (mean = 21°C, sd = 1.95) temperatures.

O presente trabalho visa estabelecer informações referentes ao tempo requerido para embriões de Cynopoecilus melanotaenia, mantidos em diferentes temperaturas, completarem seu desenvolvimento embrionário. Esses dados são importantes na otimização de cultivos dessa espécie em laboratório e podem facilitar seu futuro uso como organismos-teste em testes de toxicidade. O efeito da temperatura na taxa de eclosão e informações sobre os estágios de desenvolvimento embrionário são apresentados. Os ovos foram observados diariamente, do início até o final do desenvolvimento embrionário. Foram descritos 13 estágios de desenvolvimento. Os ovos foram mantidos a duas temperaturas constantes (20°C e 25°C) e a uma temperatura variável (16-25°C - média = 21°C, desvio = 1,95), a fim de determinar a velocidade de desenvolvimento para cada temperatura. Foi necessário menor período de incubação para completar o desenvolvimento a 25° ± 1°C. Entretanto, todos os embriões mantidos a essa temperatura eclodiram, apresentando malformações morfológicas que inviabilizavam sua sobrevivência. Não foi observada diferença estatisticamente significativa (p = 0,05) no tempo necessário para completar o desenvolvimento embrionário nos embriões mantidos nas temperaturas de 20°C e 16°-25°C (média = 21°C, desvio = 1,95).

The influence of temperature on the embryonic development of the annual fish Cynopoecilus melanotaenia (Cyprinodontiformes, Rivulidae)

The present study aims to provide data about the time required for Cynopoecilus melanotaenia kept at different temperatures to complete embryonic development. This information can be valuable for optimizing laboratory culture and facilitating future use of this species as a test organism in toxicity tests. Temperature effects on hatching rate are presented as well as information related to embryonic development stages. Eggs were observed daily, from start to finish of embryonic development. Thirteen developmental stages were described. Eggs were kept at two constant temperatures (20°C and 25°C) and at a variable ambient temperature (16-25°C - mean = 21°C, sd = 1.95), to determine developmental rate (velocity) at each temperature. A shorter incubation period was necessary to complete development at 25° ± 1°C. However, all embryos kept at this temperature hatched with morphological defects, which prevented their survival. No significant difference in developmental time period (p = 0.05) was observed at the 20°C and 16°-25°C (mean = 21°C, sd = 1.95) temperatures.

O presente trabalho visa estabelecer informações referentes ao tempo requerido para embriões de Cynopoecilus melanotaenia, mantidos em diferentes temperaturas, completarem seu desenvolvimento embrionário. Esses dados são importantes na otimização de cultivos dessa espécie em laboratório e podem facilitar seu futuro uso como organismos-teste em testes de toxicidade. O efeito da temperatura na taxa de eclosão e informações sobre os estágios de desenvolvimento embrionário são apresentados. Os ovos foram observados diariamente, do início até o final do desenvolvimento embrionário. Foram descritos 13 estágios de desenvolvimento. Os ovos foram mantidos a duas temperaturas constantes (20°C e 25°C) e a uma temperatura variável (16-25°C - média = 21°C, desvio = 1,95), a fim de determinar a velocidade de desenvolvimento para cada temperatura. Foi necessário menor período de incubação para completar o desenvolvimento a 25° ± 1°C. Entretanto, todos os embriões mantidos a essa temperatura eclodiram, apresentando malformações morfológicas que inviabilizavam sua sobrevivência. Não foi observada diferença estatisticamente significativa (p = 0,05) no tempo necessário para completar o desenvolvimento embrionário nos embriões mantidos nas temperaturas de 20°C e 16°-25°C (média = 21°C, desvio = 1,95).