Reproductive biology of the electric lanternfish Electrona risso (Myctophidae) and the bigscale fishes Melamphaes polylepis and Scopelogadus mizolepis (Melamphaidae).

This study was the first to investigate the key reproductive traits of the electric lantern fish Electrona risso (Myctophidae, n = 918) and the bigscale fishes (Melamphaidae) Melamphaes polylepis (n = 260) and Scopelogadus mizolepis (n = 649). Specimens of these mesopelagic species were collected in March and April 2015 in the eastern Central Atlantic (0-24° N, 20-26° W). Sex ratio was not significantly different from 1:1 in E. risso and M. polylepis but significantly skewed toward female dominance in S. mizolepis. Reproductive phases were determined macroscopically and by histological analyses on selected individuals. Female length at 50% maturity (L50 ) was 55.1 mm standard length (LS ) in E. risso, with an observed female maximum length (Lmax ) of 81.2 mm LS . M. polylepis females had an L50 of 40.2 mm LS and an Lmax of 86.7 mm LS . S. mizolepis had an L50 of 46 mm LS and an Lmax of 97.9 mm LS . The three species show histological features of iteroparity, but the E. risso population appears to occur in two year-classes and experience only one spawning season per lifetime in the study region. All three species are batch-spawners. A batch fecundity of 2668 eggs was estimated from one E. risso individual, with a relative batch fecundity of 369 eggs g-1 gonad-free body mass. M. polylepis had a batch fecundity of 1027 eggs and a relative batch fecundity of 149 eggs g-1 (n = 3). S. polylepis had a batch fecundity of 1545 eggs and a relative batch fecundity of 215 eggs g-1 (n = 21). The median gonado-somatic index during the actively spawning phase of E. risso was 4.5, significantly lower than that of M. polylepis (7.5) and S. mizolepis (7.1). No regressing or regenerating phases were observed in this study. Batch-spawning in all three species is suggested to be advantageous to cope with intra-annual variability in food supply and other risks for offspring survival. With what appears to be in effect a (facultative) semelparous strategy in combination with a short life span in E. risso, interannual differences would have a great effect on population dynamics of this species. Knowledge is still lacking on temporal aspects of reproduction such as the duration of the spawning season and the frequency of spawning, as well as age and growth.

Investigating the Ovarian Microstructure in the Genera Helicolenus and Scorpaena (Teleostei, Sub-Order Scorpaenoidei) with Implications for Ovarian Dynamics and Spawning.

The sub-order Scorpenoidei appears to be particularly interesting due to the presence of intermediate stages between oviparity and viviparity in several species. The present study aims to describe the ovarian morphology, using a histological and histochemical approach, in four ovuliparous species belonging to Scorpaena genus compared with a zygoparous species, H. dactylopterus, focusing also on the assessment of the ovarian dynamics in the populations of such species in Sardinia waters (central-western Mediterranean). Ovarian sections of all species were examined using light microscopy. All species showed a specialized ovary, cystovarian type II-3, strictly related to the production of gelatinous matrices surrounding the eggs. Some microscopic peculiarities in the oogenesis process were found: thin zona pellucida, small and low cortical alveoli, and a specialized ovarian wall during the spawning period. All species analyzed were batch-spawners with an asynchronous ovarian organization. A continuous recruitment of oocytes and the occurrence of de novo vitellogenesis was also observed. During the spawning period, low atresia intensity was detected, while a marked increase in this intensity found in the ovaries at the end of spawning season. Our observations may support an indeterminate fecundity type for these species.

Common dolphinfish, Coryphaena hippurus, spawn at their easternmost Atlantic range adjacent to the Mediterranean Sea.

This study provides sound evidence of the spawning activity of common dolphinfish (Coryphaena hippurus) in the Gulf of Cadiz (southwest Spain) during summer months. Along with waters off Côte d'Ivoire, this area adjacent to the Mediterranean Sea is the easternmost Atlantic spawning ground documented for the species. All the fish analysed (5 males and 15 females) were at spawning stage, except the smallest female (61.5 cm in fork length), which might not have yet reached sexual maturation. The oceanographic conditions during the samplings were similar to those occurring during the peak spawning season in the Gulf of Mexico. The study results are consistent with recent genetic studies indicating differentiation between Mediterranean and Atlantic dolphinfish populations, with the Strait of Gibraltar representing a boundary between two distinct reproductive units. The present results may contribute to improve the understanding of the dynamics of dolphinfish population and the management of eastern Atlantic and Mediterranean stocks.

Sensitivity to cadmium of the endangered freshwater pearl mussel Margaritifera margaritifera from the Dronne River (France): experimental exposure.

Margaritifera margaritifera is a critically endangered species in Europe. Among the causes explaining its decline, metal pollution had never been deeply studied. Thus, an ecotoxicological investigation was developed on this species which comes from the Dronne River (South-West of France). Cadmium (Cd) exposure of mussels at 2 and 5 µg/L for 7 days was conducted to test their vulnerability to this metal, and also the potential endocrine disruption power of Cd. Morphometric analyses, gonad histological observations, metal bioaccumulation, metallothionein (MTs) production, measures of malondialdehyde (MDA), and finally quantitative relative expression analysis of genes involved in various metabolic functions were performed.The main results showed Cd accumulation increasing in a dose-dependent manner, especially in the gills. The same trend was observed for gene expression relative to oxidative stress. Histological analysis of the gonads highlighted a predominance of hermaphrodite individuals, but after 7 days of exposure to Cd, the percentage of female was largely increased compared with controls, from 17 to 33%. These results demonstrate the endocrine disruption effect of Cd on freshwater pearl mussels.The pearl mussel Margaritifera margaritifera is sensitive to cadmium since the metallothioneins are poorly induced, gene expression reveals oxidative stress, and gonads tend to be feminized.

Reproductive biology of Parona signata (Actinopterygii: Carangidae), a valuable economic resource, in the coastal area of Mar del Plata, Buenos Aires, Argentina

The reproductive biology and life cycle of Parona leatherjacket, Parona signata, present in Mar del Plata (38º 00'S 57º 33'W) coast, was studied. Samples were obtained monthly since January 2018 to February 2019 from the artisanal fishermen and the commercial fleet of Mar del Plata. A histological analysis was carried out and the main biologic-reproductive parameters were estimated: fecundity, oocyte frequency distribution and gonadosomatic index (GSI). Both the macroscopic and microscopic analyses showed reproductive activity in March and November. Mature females were recorded, which showed hydrated oocytes, as was evidenced by the histological procedures. Both, the histological and the oocyte diameter distribution analyses showed the presence of all oocyte maturation stages in ovaries in active-spawning subphase, indicating that P. signata is a multiple spawner with indeterminate annual fecundity. Batch fecundity ranged between 36,426 and 126,035 hydrated oocytes/ female. Relative fecundity ranged between 42 and 150 oocytes/ g female ovary free.(AU)

Se estudió la biología reproductiva y el ciclo de vida de la palometa pintada, Parona signata en Mar del Plata (38°00'S 57°33'O). Las muestras fueron obtenidas mensualmente desde enero de 2018, hasta febrero de 2019, provenientes de la pesca artesanal y/o de la flota comercial de la localidad Mar del Plata. Además, se llevó a cabo un análisis histológico y se estimaron los principales parámetros reproductivos tales como: la fecundidad, análisis de la distribución de la frecuencia de diámetros ovocitarios e índice gonadosomático (GSI) mensual. Tanto el análisis macroscópico como el microscópico permitieron afirmar que en los meses de marzo y noviembre existe actividad reproductiva. Se encontraron hembras en puesta, las cuales presentaron ovocitos hidratados en los cortes histológicos. Ambos, los análisis histológicos y de distribución de la frecuencia de diámetros oocitarios mostraron la presencia de todas las fases de maduración oocitaria en ovarios correspondientes a la sub-fase de desove activo, indicando que P. signata es un desovante parcial con fecundidad anual indeterminada. La fecundidad parcial osciló entre 36.426 y 126.035 oocitos hidratados/hembra. La fecundidad relativa varió entre 42 y 150 ovocitos por gramo de hembra libre de ovario.(AU)

Gonadal macrophage aggregates in fish: A preliminary quantitative study in red mullet.

A histological study was conducted in red mullet, Mullus barbatus, collected from two sites characterized by different anthropogenic impacts. The aim of the study was to assess sex-, size-, season- and site-related variation in gonadal macrophage aggregate (MA) size, number, and relative area. Gonadal MAs were most abundant in males than in females. The number of MA was significantly higher in males from the most impacted site in October, with larger individuals showing more MA than smaller ones. MAs were always found in ripe testes, whereas they occurred only in regressing ovaries. These preliminary findings suggest that the presence of ovarian MA in red mullet is most likely related to ovary regression after spawning, whereas the presence of testicular MA is not necessarily associated to gonad regression, and may vary with season, size, and water quality.

Reproductive Biology of Johnius taiwanensis (Perciformes: Sciaenidae) in Fujian Waters, Southern China.

Johnius taiwanensis is a newly described species from the Family Sciaenidae (Perciformes). The species is commonly found in shallow coastal waters along both sides of the Taiwan Strait, on the west sides of Zhejiang, Fujian, Guangdong and Hong Kong and east side of Taiwan, and has been misidentified for decades. We studied the reproductive biology of J. taiwanensis from Fujian coastal waters, southern China, using gonadosomatic index (GSI) and gonad histology analyses. Monthly sampling from July 2016 to October 2017 was conducted and a total of 638 specimens were collected, ranging from 7.3 to 19.0 cm standard length (SL). Gonad histology suggested that the spawning activity of J. taiwanensis females and males lasted from April to October, and the peak spawning months for females was July to September. Mature females and males were 12.5 and 11.8 cm SL, respectively, while the estimated sizes at 50% maturity were 12.0 cm and 10.9 cm SL, respectively. Vitellogenic stage oocytes (O3) and post-ovulatory follicles (POF) or hydrated oocytes (HO) were observed, and POF and O3 in ovaries indicated that J. taiwanensis spawns multiple times each spawning season. HO or both HO and POF were observed in ovaries collected from one same location in May 2017 and August 2016.

Development of secondary sexual characters and their relationship to ontogeny and seasonal reproductive period in Hyphessobrycon igneus (Ostariophysi: Characiformes).

Sexual dimorphism in size, anal-fin shape and coloration of Hyphessobrycon igneus, Characidae, were examined. Males were more frequent at larger body sizes, confirming body size as a sexually dimorphic trait. Anal-fin shape and the colour of all fins were the same for females and juveniles, differing only in adult males. Likewise, only adult males had bony hooks on fin rays; larger and more sexually mature males had the most numerous and developed hooks and hooks were most developed in degree and number during peak reproductive periods. Fin hooks regressed in number and developmental degree after the reproductive period, but restarted development with the beginning of the new reproductive period without completely disappearing. Results show that bony hooks have a development and regression cycle related to reproductive seasonality.

Development and recovery of histopathological alterations in the gonads of zebrafish (Danio rerio) after single and combined exposure to endocrine disruptors (17α-ethinylestradiol and fadrozole).

Exposure of wildlife to endocrine disrupting chemicals (EDCs) is not necessarily continuous. Due to seasonal changes and variable industrial and agricultural activities it often occurs intermittently. Thus, it is possible that aquatic organisms may be more affected by periodic peak exposure than by chronic exposure. Therefore, an experimental scenario including an exposure from 2h to 90 days post-fertilization (dpf) and a subsequent recovery period until 150 dpf was chosen to assess the potential reversibility of the effects of sex steroids on sexual and gonad development of zebrafish (Danio rerio). The aim of this study was to investigate the persistence of the endocrine effects of an estrogen (EE2-17α-ethinylestradiol, 4ng/L), an inhibitor of estrogen synthesis (Fad-fadrozole, 50µg/L) or their binary mixture (Mix-EE2+ Fad, 4ng/L+50µg/L). Afterwards, a semi-quantitative histological assessment was used to investigate histopathological changes on gonad differentiation and development. The data showed that fadrozole, alone or in combination with EE2, permanently disrupts the sexual development, inducing masculinization and causing severe pathological alterations in testis, such as intersex associated to the enlargement of sperm ducts, interstitial changes, asynchronous development and detachment of basal membrane. After exposures to both EDCs and their mixture, the gonad histopathology revealed interstitial proteinaceous fluid deposits and, in ovaries, there were atretic oocytes, and presumably degenerative mineralization. On the other hand, the gonadal changes induced by EE2 alone seem to be partially reversible when the exposure regime changed to a recovery period. In addition, EE2 enhanced zebrafish growth in both genders, with male fish presenting signs of early obesity such as the presence of adipocytes in testis. Moreover, sex ratio was slightly skewed toward females, at 90 and 105 dpf, in zebrafish exposed to EE2. The data further indicate that long-term studies on impacts of single EDCs and their mixtures with recovery periods are crucial to reveal the possibility of sex reversal and pathological changes of gonads that can adversely affect breeding.

Prochloraz causes irreversible masculinization of zebrafish (Danio rerio).

The aim of the present study was to investigate the persistence of endocrine effects by prochloraz, a fungicide known to have multiple effects on the endocrine system of vertebrates. Since discontinuous exposure is particularly relevant in aquatic ecosystems, an exposure scenario with an exposure phase and a subsequent recovery period was chosen to assess the potential for reversibility of effects by prochloraz on the sexual development of zebrafish (Danio rerio). Zebrafish were exposed to different concentrations of prochloraz (10-300 µg/L) until 60 days post hatch (dph), which includes the period of sexual differentiation. For the subsequent 40 days, fish were either held in clean water for depuration or under further continuous exposure. Histological investigations of the gonads revealed persistent effects on sexual differentiation. The sex ratio was skewed towards males and significantly more intersex individuals were found after exposure to prochloraz at 60 dph. No intersex fish, but masculinized sex ratios were still present after the depuration period, documenting that prochloraz irreversibly affects the sexual development of zebrafish.

Persistence of endocrine disruption in zebrafish (Danio rerio) after discontinued exposure to the androgen 17ß-trenbolone.

The aim of the present study was to investigate the effects of the androgenic endocrine disruptor 17ß-trenbolone on the sexual development of zebrafish (Danio rerio) with special emphasis on the question of whether adverse outcomes of developmental exposure are reversible or persistent. An exposure scenario including a recovery phase was chosen to assess the potential reversibility of androgenic effects. Zebrafish were exposed to environmentally relevant concentrations of 17ß-trenbolone (1 ng/L-30 ng/L) from fertilization until completion of gonad sexual differentiation (60 d posthatch). Thereafter, exposure was either followed by 40 d of recovery in clean water or continued until 100 d posthatch, the age when zebrafish start being able to reproduce. Fish exposed for 100 d to 10 ng/L or 30 ng/L 17ß-trenbolone were masculinized at different biological effect levels, as evidenced from a concentration-dependent shift of the sex ratio toward males as well as a significantly increased maturity of testes. Gonad morphological masculinization occurred in parallel with decreased vitellogenin concentrations in both sexes. Changes of brain aromatase (cyp19b) mRNA expression showed no consistent trend with respect to either exposure duration or concentration. Gonad morphological masculinization as well as the decrease of vitellogenin persisted after depuration over 40 d in clean water. This lack of recovery suggests that androgenic effects on sexual development of zebrafish are irreversible.

Reversibility of endocrine disruption in zebrafish (Danio rerio) after discontinued exposure to the estrogen 17α-ethinylestradiol.

The aim of the present study was to investigate the persistence of the feminizing effects of discontinued 17α-ethinylestradiol (EE2) exposure on zebrafish (Danio rerio). An exposure scenario covering the sensitive phase of sexual differentiation, as well as final gonad maturation was chosen to examine the estrogenic effects on sexual development of zebrafish. Two exposure scenarios were compared: continuous exposure to environmentally relevant concentrations (0.1-10 ng/L EE2) up to 100 days post-hatch (dph) and developmental exposure up to 60 dph, followed by 40 days of depuration in clean water. The persistence of effects was investigated at different biological organization levels from mRNA to population-relevant endpoints to cover a broad range of important parameters. EE2 had a strong feminizing and inhibiting effect on the sexual development of zebrafish. Brain aromatase (cyp19b) mRNA expression showed no clear response, but vitellogenin levels were significantly elevated, gonad maturation and body growth were inhibited in both genders, and sex ratios were skewed towards females and undifferentiated individuals. To a large extent, all of these effects were reversed after 40 days of recovery, leading to the conclusion that exposure to the estrogen EE2 results in very strong, but reversible underdevelopment and feminization of zebrafish. The present study is the first to show this reversibility at different levels of organization, which gives better insight into the mechanistic basis of estrogenic effects in zebrafish.

Ecología reproductiva de Geobatrachus walkeri (Anura: Srabomantidae) / Reproductive ecology of Geobatrachus walkeri (Anura: Strabomantidae)

Geobatrachus walkeri is a frog that belongs to a monotypic genus and is endemic to the Sierra Nevada de Santa Marta, Colombia. The species inhabits different microhabitats between 2 000 to 3 500m altitude, including the leaf litter of a pine plantation. To understand its reproductive ecology, we conducted eight frog samplings, covering the rainy and dry seasons, and two habitat types (secondary native forest and pine plantation) during 2010-2011. For this study, we also included data obtained from five previous similar samplings undertaken during 2008-2009. The pine leaf litter was the main microhabitat where frogs were found; we heard choruses of six-ten calling males during all sampled months, and observed the frogs having diurnal and nocturnal activity. Regardless of the year of study, the population consisted of neonates recruited several times of the year, a large number of juveniles with a wide range of body sizes, and fewer adults with a narrower range of body size. The histological analyses of the gonads showed that the size at maturity was near 18mm SVL for males and females, and those adult males and females were reproductive active during all sampling months, suggesting a continuous reproductive activity pattern. However, during the dry season, the seminiferous tubules showed a drastically diminished spermatic epithelium although containing abundant luminal spermatozoa, which suggest a reduction in the sperm production at the end of this season. Similarly, frogs of all age categories were significantly more abundant during the early dry season, whereas were significantly less abundant with the advancement of the dryness suggesting that the intensity of the dry season could temporally stop the activity and reproduction of this population.

Geobatrachus walkeri es una rana que pertenece a un género monotípico y es endémica de la Sierra Nevada de Santa Marta, Colombia. La especie habita en diferentes microhábitats entre los 2 000 y 3 500m de altitud, incluyendo la hojarasca de una plantación de pinos. Para entender su ecología reproductiva se realizaron ocho muestreos de individuos, que abarcaron las estaciones de lluvias y de sequía y dos tipos de hábitats (bosque secundario y plantación de pino) durante 2010-2011. También se incluyeron los datos de cinco muestreos similares obtenidos previamente durante el período 2008-2009. El principal microhábitat donde las ranas se encontraron fue la hoja-rasca de pino, allí escuchamos coros de seis-diez machos durante todos los meses muestreados, y observamos que las ranas tienen actividad diurna y nocturna. Sin importar el año de estudio, la población se compone de neonatos reclutados en varios meses del año, un gran número de juveniles con un amplio rango de tamaños corporales, y un menor número de adultos con un rango más reducido de tamaño corporal. Los análisis histológicos de las gónadas mostraron que el tamaño a la madurez se adquiere cerca de los 18mm LRC para machos y hembras, y que los machos y hembras adultos fueron reproductivos durante todo el muestreo, lo que sugiere un patrón continuo de actividad reproductiva. Sin embargo, durante la estación seca, los túbulos seminíferos presentan un epitelio espermático drásticamente disminuido, aunque contienen abundantes espermatozoides luminales, lo que sugiere una reducción en la producción de espermatozoides en el final de la estación seca. Del mismo modo, las ranas de todas las categorías de edad fueron significativamente más abundantes durante el inicio de la estación seca, mientras que fueron significativamente menos abundantes con el avance de la sequedad, lo que sugiere que una mayor intensidad de la temporada de sequía podría detener temporalmente la actividad y la reproducción en esta población.