Skin ionocyte density of amphibious killifishes is shaped by phenotypic plasticity and constitutive interspecific differences.

When amphibious fishes are on land, gill function is reduced or eliminated and the skin is hypothesized to act as a surrogate site of ionoregulation. Skin ionocytes are present in many fishes, particularly those with amphibious life histories. We used nine closely related killifishes spanning a range of amphibiousness to first test the hypothesis that amphibious killifishes have evolved constitutively increased skin ionocyte density to promote ionoregulation on land. We found that skin ionocyte densities were constitutively higher in five of seven amphibious species examined relative to exclusively water-breathing species when fish were prevented from leaving water, strongly supporting our hypothesis. Next, to examine the scope for plasticity, we tested the hypothesis that skin ionocyte density in amphibious fishes would respond plastically to air-exposure to promote ionoregulation in terrestrial environments. We found that air-exposure induced plasticity in skin ionocyte density only in the two species classified as highly amphibious, but not in moderately amphibious species. Specifically, skin ionocyte density significantly increased in Anablepsoides hartii (168%) and Kryptolebias marmoratus (37%) following a continuous air-exposure, and only in K. marmoratus (43%) following fluctuating air-exposure. Collectively, our data suggest that highly amphibious killifishes have evolved both increased skin ionocyte density as well as skin that is more responsive to air-exposure compared to exclusively water-breathing and less amphibious species. Our findings are consistent with the idea that gaining the capacity for cutaneous ionoregulation is a key evolutionary step that enables amphibious fishes to survive on land.

Retention of larval skin traits in adult amphibious killifishes: a cross-species investigation.

The gills are the primary site of exchange in fishes. However, during early life-stages or in amphibious fishes, ionoregulation and gas-exchange may be primarily cutaneous. Given the similarities between larval and amphibious fishes, we hypothesized that cutaneous larval traits are continuously expressed in amphibious fishes across all life-stages to enable the skin to be a major site of exchange on land. Alternatively, we hypothesized that cutaneous larval traits disappear in juvenile stages and are re-expressed in amphibious species in later life-stages. We surveyed six species spanning a range of amphibiousness and characterized cutaneous ionocytes and neuroepithelial cells (NECs) as representative larval skin traits at up to five stages of development. We found that skin ionocyte density remained lower and constant in exclusively water-breathing, relative to amphibious species across development, whereas in amphibious species ionocyte density generally increased. Additionally, adults of the most amphibious species had the highest cutaneous ionocyte densities. Surprisingly, cutaneous NECs were only identified in the skin of one amphibious species (Kryptolebias marmoratus), suggesting that cutaneous NECs are not a ubiquitous larval or amphibious skin trait, at least among the species we studied. Our data broadly supports the continuous-expression hypothesis, as three of four amphibious experimental species expressed cutaneous ionocytes in all examined life-stages. Further, the increasing density of cutaneous ionocytes across development in amphibious species probably facilitates the prolonged occupation of terrestrial habitats.

Effects of crowding stress on the hypothalamo-pituitary-interrenal axis of the self-fertilizing fish, Kryptolebias marmoratus.

We tested whether crowding stress affects the hypothalamo-pituitary-interrenal (HPI) axis of the self-fertilizing fish, Kryptolebias marmoratus, which is known to be aggressive in the laboratory conditions but sometimes found as a group from a single land crab burrow in the wild. The projection of corticotropin-releasing hormone (CRH) neurons to the adrenocorticotropic hormone (ACTH) cells in the pituitary was confirmed by dual-label immunohistochemistry; CRH-immunoreactive (ir) fibers originating from cell bodies located in the lateral tuberal nucleus (NLT) of the hypothalamus were observed to project to ACTH-ir cells in the rostral pars distalis of the pituitary. Then, fish were reared solitary or in pairs for 14 days, and the number of CRH-ir cell bodies in the NLT of the hypothalamus and cortisol levels in the body without head region were compared. The number of CRH-ir cell bodies and cortisol levels were significantly higher in paired fish. These results indicate that crowding stress affects the HPI axis in K. marmoratus which thrive in small burrows with limited water volume.

Fish-Released Kairomones Affect Mosquito Oviposition and Larval Life History.

Several species of mosquitoes respond to the presence of kairomones released by larvivorous predators during oviposition habitat selection and larval development. These responses may differ among mosquito species and do not always correlate with larval survival. This study examined the responses of the mosquito Culiseta longiareolata Macquart (Diptera: Culicidae) to kairomones released by three species of fish, Gambusia affinis (Cyprinodontiformes: Poeciliidae), Aphanius mento (Cyprinodontiformes: Cyprinodontidae) and Garra rufa (Cypriniformes: Cyprinidae) during oviposition. In addition, the study examined the effects of kairomones released by Gm. affinis on larval development and survival. Results show that ovipositing female avoided cues from the two larvivorous fish species but not the algivorous Gr. rufa. In addition, developing larvae metamorphosed slower and showed increased mortality when exposed to fish-released kairomones. Culiseta longiareolata larvae are known as dominant competitors, and the straightforward responses of both larvae and adult female to fish-released kairomones may be explained by the lack of additional sources of larval stress other than the presence of predators.

Widespread alterations to hypothalamic-pituitary-gonadal (HPG) axis signaling underlie high temperature reproductive inhibition in the eurythermal sheepshead minnow (Cyprinodon variegatus).

Fish experiencing abnormally high or prolonged elevations in temperature can exhibit impaired reproduction, even for species adapted to warm water environments. Such high temperature inhibition of reproduction has been linked to diminished gonadal steroidogenesis, but the mechanisms whereby hypothalamic-pituitary-gonadal (HPG) axis signaling is impacted by high temperature are not fully understood. Here, we characterized differences in HPG status in adult sheepshead minnow (Cyprinodon variegatus), a eurythermal salt marsh and estuarine species of eastern North America, exposed for 14 d to temperatures of 27 °C or 37 °C. Males and females at 37 °C had lower gonadosomatic index (GSI) values compared to fish at 27 °C, and females at 37 °C had fewer spawning capable eggs and lower circulating 17ß-estradiol (E2). Gene transcripts encoding gonadotropin-inhibitory hormone (gnih) and gonadotropin-releasing hormone-3 (gnrh3) were higher in relative abundance in the hypothalamus of both sexes at 37 °C. While pituitary mRNAs for the ß-subunits of follicle-stimulating hormone (fshß) and luteinizing hormone (lhß) were lowered only in males at 37 °C, Fsh and Lh receptor mRNA levels in the gonads were at lower relative levels in both the ovary and testis of fish at 37 °C. Females at 37 °C also showed reduced ovarian mRNA levels for steroid acute regulatory protein (star), P450 side-chain cleavage enzyme (cyp11a1), 3ß-hydroxysteroid dehydrogenase (3ßhsd), 17ß-hydroxysteroid dehydrogenase (hsd17ß3), and ovarian aromatase (cyp19a1a). Females at the higher 37 °C temperature also had a lower liver expression of mRNAs encoding estrogen receptor α (esr1) and several vitellogenin and choriogenin genes, but elevated mRNA levels for hepatic sex hormone-binding globulin (shbg). Our results substantiate prior findings that exposure of fish to high temperature can inhibit gonadal steroidogenesis and oogenesis, and point to declines in reproductive performance emerging from alterations at several levels of HPG axis signaling including increased hypothalamic Gnih expression, depressed gonadal steroidogenesis, and reduced egg yolk and egg envelope protein production in the liver.

Multi-stressor Effects of Ultraviolet Light, Temperature, and Salinity on Louisiana Sweet Crude Oil Toxicity in Larval Estuarine Organisms.

When oil is spilled into the environment its toxicity is affected by abiotic conditions. The cumulative and interactive stressors of chemical contaminants and environmental factors are especially relevant in estuaries where tidal fluctuations cause wide variability in salinity, temperature, and ultraviolet (UV) light penetration, which is an important modifying factor for polycyclic aromatic hydrocarbon (PAH) toxicity. Characterizing the interactions of multiple stressors on oil toxicity will improve prediction of environmental impacts under various spill scenarios. This study examined changes in crude oil toxicity with temperature, salinity, and UV light. Oil exposures included high-energy, water-accommodated fractions (HEWAFs) and thin oil sheens. Larval (24-48 h post hatch) estuarine species representing different trophic levels and habitats were evaluated. Mean 96 h LC50 values for oil prepared as a HEWAF and tested under standard conditions (20 ppt, 25 °C, No-UV) were 62.5 µg/L tPAH50 (mud snails), 198.5 µg/L (grass shrimp), and 774.5 µg/L (sheepshead minnows). Thin oil sheen 96 h LC50 values were 5.3 µg/L tPAH50 (mud snails), 14.7 µg/L (grass shrimp), and 22.0 µg/L (sheepshead minnows) under standard conditions. UV light significantly increased the toxicity of oil in all species tested. Oil toxicity also was greater under elevated temperature and lower salinity. Multi-stressor (oil combined with either increased temperature, decreased salinity, or both) LC50 values were reduced to 3 µg/L tPAH50 for HEWAFs and < 1.0 µg/L tPAH50 for thin oil sheens. Environmental conditions at the time of an oil spill will significantly influence oil toxicity and organismal response and should be taken into consideration in toxicity testing and oil spill damage assessments.

The pharmaceutical prednisone affects sheepshead minnow (Cyprinodon variegatus) metabolism and swimming performance.

High usage of the synthetic glucocorticoids (GCs) has led to significant presence of this pharmaceutical group in surface waters where it can affect non-target organisms such as fish. Assessment of a fish's metabolism and swimming performance provide reliable sub-lethal measures of effects of GCs on oxygen-requiring processes and ability to swim. In this study, we determined time-dependent (7, 14 and 21 days) effects of the synthetic GC prednisone (1 µg L-1) on sheepshead minnow (SHM) (Cyprinodon variegatus). Standard (SMR), routine (RMR) and maximum (MMR) metabolic rate, metabolic scope (MS), excess post-exercise oxygen consumption (EPOC), cost of transport (COT) and critical swimming speed (Ucrit) were determined. Twenty-one days exposure to prednisone resulted in significantly higher SMR, RMR, MMR, MS, EPOC and COT compared with 7d and 14d prednisone fish. However, Ucrit was not significantly different between prednisone and solvent control exposed fish (within 7d, 14d, 21d groups). SMR, RMR and MMR were lower in the 7d and 14d prednisone exposed fish compared with their solvent control groups. In contrast, SMR, RMR and MMR were all significantly higher in the 21d prednisone exposed fish compared with solvent control. EPOC was significantly higher in 14d prednisone exposed fish and trending higher in 21d and 7d prednisone exposed fish compared with their solvent controls. EPOC was significantly higher in 21d compared with 7d prednisone exposed fish. A significantly higher COT was seen in the 21d compared with 7d and 14d prednisone fish. Collectively, this study showed time-dependent effects of prednisone on SHM metabolism and swimming performance.

Changes in regeneration-responsive enhancers shape regenerative capacities in vertebrates.

Vertebrates vary in their ability to regenerate, and the genetic mechanisms underlying such disparity remain elusive. Comparative epigenomic profiling and single-cell sequencing of two related teleost fish uncovered species-specific and evolutionarily conserved genomic responses to regeneration. The conserved response revealed several regeneration-responsive enhancers (RREs), including an element upstream to inhibin beta A (inhba), a known effector of vertebrate regeneration. This element activated expression in regenerating transgenic fish, and its genomic deletion perturbed caudal fin regeneration and abrogated cardiac regeneration altogether. The enhancer is present in mammals, shares functionally essential activator protein 1 (AP-1)-binding motifs, and responds to injury, but it cannot rescue regeneration in fish. This work suggests that changes in AP-1-enriched RREs are likely a crucial source of loss of regenerative capacities in vertebrates.

Early-life exposure to weathered, unweathered and dispersed oil has persisting effects on ecologically relevant behaviors in sheepshead minnow.

The Deepwater Horizon oil spill released 3.19 million barrels of crude oil into the Gulf of Mexico, making it the largest oil spill in U.S. history. Weathering and the application of dispersants can alter the solubility of compounds within crude oil, thus modifying the acute toxicity of the crude oil to aquatic life. The primary aim of our study was to determine the lasting impact of early-life stage sheepshead minnow (Cyprinodon variegatus variegatus) exposure to weathered, unweathered and dispersed crude oil on prey capture, male aggression, novel object interaction and global DNA methylation. Embryos were exposed from 1 to 10 dpf to water accommodations of crude oil and were raised to adulthood in artificial seawater. Our results suggest exposure to crude oil did not result in lasting impairment of complex behavioral responses of male sheepshead minnow. Exposure to dispersed weathered oil, however, decreased border dwelling in response to a novel object (i.e. decreased anxiety). Principal component analysis revealed that exposure to weathered oil had no overarching effect, but that unweathered crude oil increased variability in exploratory behaviors but decreased variability in anxiety-associated behaviors. Further work is needed to understand the effects of oil exposure on fish behavior and the potential ecological impact of subtle behavioral changes in fishes.

The metabolism and swimming performance of sheepshead minnows (Cyprinodon variegatus) following thermal acclimation or acute thermal exposure.

Ectothermic animals are especially susceptible to temperature change, considering that their metabolism and core temperature are linked to the environmental temperature. As global water temperatures continue to increase, so does the need to understand the capacity of organisms to tolerate change. Sheepshead minnows (Cyprinodon variegatus) are the most eurythermic fish species known to date and can tolerate a wide range of environmental temperatures from - 1.9 to 43.0 °C. But little is known about the physiological adjustments that occur when these fish are subjected to acute thermal challenges and long-term thermal acclimation. Minnows were acclimated to 10, 21, or 32 °C for 4 weeks or acutely exposed to 10 and 32 °C and then assessed for swimming performance [maximum sustained swimming velocity (Ucrit), optimum swimming velocity (Uopt)] and metabolic endpoints (extrapolated standard and maximum metabolic rate [SMR, MMR), absolute aerobic scope (AS), and cost of transport (COT)]. Our findings show that the duration of thermal exposure (acute vs. acclimation) did not influence swimming performance. Rather, swimming performance was influenced by the exposure temperature. Swimming performance was statistically similar in fish exposed to 21 or 32 °C (approximately 7.0 BL s-1), but was drastically reduced in fish exposed to 10 °C (approximately 2.0 BL s-1), resulting in a left-skewed performance curve. There was no difference in metabolic end points between fish acutely exposed or acclimated to 10 °C. However, a different pattern was observed in fish exposed to 32 °C. MMR was similar between acutely exposed or acclimated fish, but acclimated fish had a 50% reduction in extrapolated SMR, which increased AS by 25%. However, this enhanced AS was not associated with changes in swimming performance, which opposes the oxygen-capacity limited thermal tolerance concept. Our findings suggest that sheepshead minnows may utilize two distinct acclimation strategies, resulting in different swimming performance and metabolic patterns observed between 10 and 32 °C exposures.

Thermal stability vs. variability: Insights in oxidative stress from a eurytolerant fish.

Climate change will increase the frequency, intensity, and duration of heatwaves. This thermal volatility will challenge to the oxidative homeostasis of aquatic ectotherms through many temperature-dependent environmental factors. In this study, we examined the effects of chronic exposure of sheepshead minnows (Cyprinodon variegatus) to multiple thermal regimes on the oxidative physiology of white muscle in these eurytolerant fish. The thermal treatments included stable (15 °C and 30 °C) and cycling regimes (between 21 and 29 °C at 6, 8 and 10-h intervals). The effect of these thermal treatments on oxidative stress during an acute thermal challenge (12 h at 32 °C) was also examined. Enzymatic activity of catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx), scavenging capacities of hydroxyl and peroxyl radicals, and lipid peroxidation (LPO) damage were quantified. We found no differences between or across treatments in any of the enzymatic antioxidants or LPO damage. We found that peroxyl radical scavenging was greatest at the peak of the 8- and 10-h thermal cycles. Peroxyl scavenging after an acute thermal challenge was greater than before the challenge for the steady 15 °C and 8-h cycle treatments, greater before the acute challenge for the steady 30 °C and 6-h cycle, and equivalent in the 10-h cycle. These findings demonstrate that even the most tolerant of marine ectotherms must engage oxidative defenses when presented with thermal variability and heighten concerns about the impact of climate change on less tolerant species.

Oral shelling within an adaptive radiation of pupfishes: Testing the adaptive function of a novel nasal protrusion and behavioural preference.

Dietary specialization on hard prey items, such as mollusks and crustaceans, is commonly observed in a diverse array of fish species. Many fish consume these types of prey by crushing the shell to consume the soft tissue within, but a few fishes extricate the soft tissue without breaking the shell using a method known as oral shelling. Oral shelling involves pulling a mollusc from its shell and it may be a way to subvert an otherwise insurmountable shell defence. However, the biomechanical requirements and potential adaptations for oral shelling are unknown. Here, we test the hypothesis that a novel nasal protrusion is an adaptation for oral shelling in the durophagous pupfish (Cyprinodon brontotheroides). We first demonstrate oral shelling in this species and then predict that a larger nasal protrusion would allow pupfish to consume larger snails. Durophagous pupfish are found within an endemic radiation of pupfish on San Salvador Island, Bahamas. We took advantage of closely related sympatric species and outgroups to test: (a) whether durophagous pupfish shell and consume more snails than other species, (b) if F1 and F2 durophagous hybrids consume similar amounts of snails as purebred durophagous pupfish, and (c) if nasal protrusion size in parental and hybrid populations increases the maximum size of consumed snails. We found that durophagous pupfish and their hybrids consumed the most snails, but did not find a strong association between nasal protrusion size and maximum snail size consumed within the parental or F2 hybrid population, suggesting that the size of their novel nasal protrusion does not provide a major benefit in oral shelling. Instead, we suggest that the nasal protrusion may increase feeding efficiency, act as a sensory organ, or is a sexually selected trait, and that a strong feeding preference may be most important for oral shelling.

Spatial and temporal expression pattern of sex-related genes in ovo-testis of the self-fertilizing mangrove killifish (Kryptolebias marmoratus).

In vertebrates, sex determination and differentiation comprehend a fine balance between female and male factors, leading the bipotential anlage to develop towards ovary or testis, respectively. Nevertheless, the mangrove killifish, (Kryptolebias marmoratus) a simultaneous hermaphroditic species, could overcome those antagonistic pathways and evolved to develop and maintain reproductively active ovarian and testicular tissues in the same organ. Morphological and mRNA localization analyzes of developing and adult gonads demonstrate that genes related to testis (dmrt1 and amh) and ovary differentiation (foxl2 and sox9a) follow the same expression pattern observed in gonochoristic species, thus functioning as two independent organs. In addition, Amh expression patterns make it a strong candidate for initiation of the formation and maintenance of the testicular tissue in the hermaphroditic gonad. Differently from described so far, foxl3 seems to have an important role in oogenesis as well as spermatogenesis and gonadal structure.

Oxidative stress resistance in a short-lived Neotropical annual killifish.

Oxidative stress plays an important role in the evolution of aging and life history. High investments in life-history traits and environmental conditions can be associated with increased oxidative stress and aging process. However, to date, most studies that investigated variations in oxidative status were performed with long-lived vertebrates. Studies with short-lived vertebrates in wild are nonexistent. Annual killifishes have the shortest lifespans among vertebrates and inhabit temporary ponds subject to large variations in environmental conditions. In this sense, we investigated whether the high investment in growth and reproduction in a short-lived vertebrate and the large variations in environment has any cost in susceptibility to oxidative stress. We assessed the seasonal variation and the environmental correlates of four different oxidative status markers (lipid peroxidation and activity of the antioxidant enzymes Superoxide Dismutase, Catalase and Glutathione S-Transferase) along the life cycle of wild individuals of the Neotropical annual fish Austrolebias minuano. Males showed reduction in all biomarkers (except proteins) along their life cycle, while females showed increased oxidative stress only in the growth period. In addition, we showed that water physicochemical parameters, habitat structure and presence of co-occurring killifish species influenced the seasonal variation of the biomarkers. A. minuano showed an efficient antioxidant system for most part of their life cycle (mainly in males), suggesting a well-developed oxidative stress regulation system. We also show that annual fish mortality (mainly in males) apparently is not related to oxidative stress. Thus, environmental factors should drive annual fish aging and mortality.

Hormones, developmental plasticity, and adaptive evolution: Endocrine flexibility as a catalyst for 'plasticity-first' phenotypic divergence.

Explaining how populations adapt to environments is among the foremost objectives of evolutionary theory. Over generations, natural selection impels the phenotypic distribution of a population based on individual variation in phenotype and fitness. However, environmental conditions can also shape how individuals develop within their lifetime to influence which phenotypes are expressed in a population. It has been proposed that such environmentally-initiated phenotypic variation - also termed developmental plasticity - may enable adaptive evolution under some scenarios. As dynamic regulators of development and phenotypic expression, hormones are important physiological mediators of developmental plasticity. Patterns of hormone secretion, hormone transport, and the sensitivity of tissues to hormones can each be altered by environmental conditions, and understanding how endocrine regulation shapes phenotypic development in an ecologically-relevant context has much to contribute toward clarifying the role of plasticity in evolutionary adaptation. This article explores how the environmental sensitivity of endocrine regulation may facilitate 'plasticity-first' evolution by generating phenotypic variants that precede adaptation to altered or novel environments. Predictions arising from 'plasticity-first' evolution are examined in the context of thyroid hormone mediation of morphological plasticity in Cyprinodon pupfishes from the Death Valley region of California and Nevada, USA. This clade of extremophile fishes diversified morphologically over the last ~20,000 years, and observations that some populations experienced contemporary phenotypic differentiation under recent habitat change provide evidence that hormone-mediate plasticity preceded genetic assimilation of morphology in one of the region's species: the Devils Hole pupfish, Cyprinodon diabolis. This example illustrates how conceptualizing hormones not only as regulators of homeostasis, but also as developmental intermediaries between environment conditions and phenotypic variation at the individual-, population-, and species-levels can enrich our understanding of endocrine regulation both as a facilitator of phenotypic change under shifting environments, and as important proximate mechanisms that may initiate 'plasticity-first' evolutionary adaptation.

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.

Hypoxia and reduced salinity exacerbate the effects of oil exposure on sheepshead minnow (Cyprinodon variegatus) reproduction.

Estuaries of the northern Gulf of Mexico are dynamic environments, with fluctuations in salinity and dissolved oxygen, including areas of seasonal hypoxia. Fish that reside and reproduce in these estuaries, including sheepshead minnow (Cyprinodon variegatus; SHM), were at significant risk of oil exposure following the Deepwater Horizon oil spill. It is poorly understood how differences in environmental conditions during oil exposure impact its toxicity. The present study investigated the effects of crude oil high-energy water accommodated fraction (HEWAF) on SHM reproduction in three environmental scenarios (normoxic, hypoxic, and hypoxic with low salinity) to determine if differences in salinity (brackish vs low salinity) and dissolved oxygen (normoxia vs hypoxia) could exacerbate the effects of HEWAF-derived polycyclic aromatic hydrocarbons (PAHs). We observed that HEWAF exposure significantly increased liver somatic index of SHM compared to control, but this effect was not exacerbated by hypoxia or low salinity. HEWAF exposure also significantly decreased egg production and egg fertilization rate, but only in the hypoxic and hypoxic with low salinity scenarios. A significant correlation existed between body burdens of PAHs and reproductive endpoints, providing substantial evidence that oil exposure reduced reproductive capacity in SHM, across a range of environmental conditions. These data suggest that oil spill risk assessments that fail to consider other environmental stressors (i.e. hypoxia and salinity) may be underestimating risk.

Ovarian structure, folliculogenesis and oogenesis of the annual killifish Millerichthys robustus (Cyprinodontiformes: Cynolebiidae).

Cellular aspects of oocyte development of the Mexican rivulus Millerichthys robustus were morphologically described in order to analyze ovarian function and the cellular recruitment dynamics associating it with life history strategies of annual killifishes. Millerichthys is an iteroparous batch spawner with continuous oocyte recruitment and indeterminate fecundity with asynchronous development of the follicles. It has two ovaries of cystovarian type, with a central lumen, which communicates with the outside through the caudal region of the ovary, that is, the gonoduct. From the walls of the ovary, irregular lamellae composed of germinal epithelium and vascularized stroma project. Oogenesis starts with oogonial proliferation, found alone or in nests within the germinal epithelium. The oogonia come into meiosis becoming oocytes and advancing to the chromatin nucleolus stage and to early primary growth stage. Folliculogenesis is completed in the primary growth stage and cortical alveoli step. Follicles moves toward the stroma, but they continue to be attached to the germinal epithelium through the basement membrane until ovulation. The inclusion of fluid yolk in the follicles during the secondary growth stage was observed. During ovulation, the follicle collapsed, the oocyte was released into the lumen, and the constitutive elements of the post-ovulatory follicle complex remained in the stroma.

Comparison of steroidogenic gene expression in mummichog (Fundulus heteroclitus) testis tissue following exposure to aromatizable or non-aromatizable androgens.

Androgens are a recognized class of endocrine disrupting compounds with the ability to impact reproductive status in aquatic organisms. The current study utilized in vitro exposure of mummichog (Fundulus heteroclitus) testis tissue to either the aromatizable androgen 17α-methyltestosterone (MT) or the non-aromatizable androgen 5α-dihydrotestosterone (DHT) over the course of 24 h to determine if there were differential effects on steroidogenic gene expression. Testis tissue was exposed to androgen concentrations of 10-12 M, 10-9 M and 10-6 M for 6, 12, 18 or 24 h, after which a suite of steroidogenic genes, including steroidogenic acute regulatory protein, 3ß-hydroxysteroid dehydrogenase (3ßhsd) and cytochrome P450 17A1 (cyp17a1), were quantified using real-time polymerase chain reaction. Both androgens affected steroidogenic gene expression, with most alterations occurring at the 24-hour time point. The gene with the highest fold-change, and shortest interval to expression alteration, was 3ßhsd for both androgens. Potential differences between the two model androgens were observed in increased expression of cyp17a1 and 11ß-hydroxysteroid dehydrogenase (11ßhsd), which were only altered after exposure to DHT and in expression levels of cytochrome P450 11A1 (cyp11a1), which was upregulated by MT but not altered by DHT. Results from this study show both androgens interact at the gonadal level of the hypothalamus-pituitary-gonadal axis and may possess some distinct gene expression impacts. These data strengthen the current research initiatives of establishing in vitro test systems that allow toxic potential of untested chemicals to be predicted from molecular perturbations.