Life in the fastlane? A comparative analysis of gene expression profiles across annual, semi-annual, and non-annual killifishes (Cyprinodontiformes: Nothobranchiidae).

The Turquoise Killifish is an important vertebrate for the study of aging and age-related diseases due to its short lifespan. Within Nothobranchiidae, species possess annual, semi-annual, or non-annual life-histories. We took a comparative approach and examined gene expression profiles (QuantSeq) from 62 individuals from eleven nothobranchid species that span three life-histories. Our results show significant differences in differentially expressed genes (DEGs) across life-histories with non-annuals and semi-annuals being most similar, and annuals being the most distinct. At finer scales, we recovered significant differences in DEGs for DNA repair genes and show that non-annual and semi-annuals share similar gene expression profiles, while annuals are distinct. Most of the GO terms enriched in annuals are related to metabolic processes. However, GO terms, including translation, protein transport, and DNA replication initiation also are enriched in annuals. Non-annuals are enriched in Notch signaling pathway genes and downregulated in the canonical Wnt signaling pathway compared to annual species, which suggests that non-annuals have stronger regulation in cellular processes. This study provides support for congruency in DEGs involved in these life-histories and provides strong evidence that a particular set of candidate genes may be worthy of study to investigate their role in the aging process.

Effects of Bisphenol A on Male Gonadal Tissue of Viviparous Fish: Histopathological Evidence in Goodea atripinnis / Efectos del Bisfenol A en tejido Gonadal Masculino de Peces Vivíparos: Evidencia Histopatológica en Goodea atripinnis

SUMMARY: BPA is a multifunctional endocrine disruptor with ubiquitous presence in aquatic ecosystems. The Mexican Central Plateau is an area severely impacted by pollution, inhabited by endemic viviparous fish. However, efforts to understand the effects of BPA on native species such as Goodea atripinnis are non-existent. This study focused on providing in vivo evidence of alterations in the testes of G. atripinnis males due to acute exposure to BPA at test concentrations of 1 mg/L, 10 mg/L, and 50 mg/L for 96 h. BPA exposition 1 mg/L and 10 mg/L showed degeneration and disorganization in germinal tissue. Furthermore, there was a notable decrease in sperm within the seminiferous tubules of males exposed to 10 mg/L of BPA. In all treatments, somatic cells had alterations by connective tissue thickening and an increase in collagen fibers. Additionally, inflammation and bleeding occurred in the testes of males exposed to 1 and 10 mg/L BPA. The alterations in the testes of G. atripinnis are related to BPA toxicity, which can lead to apoptosis in germ cells increasing connective tissue. Finally, even though the changes produced by BPA became evident in acute exposure (96 h), its effects are probably irreversible, compromising the reproduction of G. atripinnis.

El BPA es un disruptor endocrino multifuncional con presencia ubicua en los ecosistemas acuáticos. La Meseta Central mexicana habitada por peces vivíparos endémicos, es una zona severamente impactada por la contaminación. Sin embargo, los esfuerzos por comprender los efectos del BPA en especies nativas como Goodea atripinnis son inexistentes. Este estudio se centró en proporcionar evidencia in vivo de alteraciones en los testículos de machos de G. atripinnis debido a la exposición aguda al BPA en concentraciones de prueba de 1 mg/L, 10 mg/L y 50 mg/L durante 96 h. La exposición a BPA 1 mg/L y 10 mg/L mostró degeneración y desorganización en el tejido germinal. Además, hubo una disminución notable de los espermatozoides dentro de los túbulos seminíferos de machos expuestos a 10 mg/L de BPA. En todos los tratamientos las células somáticas presentaron alteraciones por engrosamiento del tejido conectivo y aumento de las fibras de colágeno. Además, se produjo inflamación y sangrado en los testículos de machos expuestos a 1 y 10 mg/L de BPA. Las alteraciones en los testículos de G. atripinnis están relacionadas con la toxicidad del BPA, lo que puede provocar apoptosis en las células germinales aumentando el tejido conectivo. Finalmente, si bien los cambios producidos por el BPA se hicieron evidentes en la exposición aguda (96 h), sus efectos probablemente sean irreversibles, comprometiendo la reproducción de G. atripinnis.

Swordtail fish hybrids reveal that genome evolution is surprisingly predictable after initial hybridization.

Over the past 2 decades, biologists have come to appreciate that hybridization, or genetic exchange between distinct lineages, is remarkably common-not just in particular lineages but in taxonomic groups across the tree of life. As a result, the genomes of many modern species harbor regions inherited from related species. This observation has raised fundamental questions about the degree to which the genomic outcomes of hybridization are repeatable and the degree to which natural selection drives such repeatability. However, a lack of appropriate systems to answer these questions has limited empirical progress in this area. Here, we leverage independently formed hybrid populations between the swordtail fish Xiphophorus birchmanni and X. cortezi to address this fundamental question. We find that local ancestry in one hybrid population is remarkably predictive of local ancestry in another, demographically independent hybrid population. Applying newly developed methods, we can attribute much of this repeatability to strong selection in the earliest generations after initial hybridization. We complement these analyses with time-series data that demonstrates that ancestry at regions under selection has remained stable over the past approximately 40 generations of evolution. Finally, we compare our results to the well-studied X. birchmanni × X. malinche hybrid populations and conclude that deeper evolutionary divergence has resulted in stronger selection and higher repeatability in patterns of local ancestry in hybrids between X. birchmanni and X. cortezi.

Phylogenomic analyses of all species of swordtail fishes (genus Xiphophorus) show that hybridization preceded speciation.

Hybridization has been recognized to play important roles in evolution, however studies of the genetic consequence are still lagging behind in vertebrates due to the lack of appropriate experimental systems. Fish of the genus Xiphophorus are proposed to have evolved with multiple ancient and ongoing hybridization events. They have served as an informative research model in evolutionary biology and in biomedical research on human disease for more than a century. Here, we provide the complete genomic resource including annotations for all described 26 Xiphophorus species and three undescribed taxa and resolve all uncertain phylogenetic relationships. We investigate the molecular evolution of genes related to cancers such as melanoma and for the genetic control of puberty timing, focusing on genes that are predicted to be involved in pre-and postzygotic isolation and thus affect hybridization. We discovered dramatic size-variation of some gene families. These persisted despite reticulate evolution, rapid speciation and short divergence time. Finally, we clarify the hybridization history in the entire genus settling disputed hybridization history of two Southern swordtails. Our comparative genomic analyses revealed hybridization ancestries that are manifested in the mosaic fused genomes and show that hybridization often preceded speciation.

Hormonal dynamics of matrotrophy vs. lecithotrophy in live-bearing fish reproduction.

We explored the relationship between gestational states, fecundity, and steroid hormone levels in three species of live-bearing fish with different maternal provisioning strategies. We studied two lecithotrophic species, Gambusia affinis and Xiphophorus couchianus, where embryos feed exclusively on yolk stored in the eggs, and one matrotrophic species, Heterandria formosa, which actively transfers nutrients to embryos through a follicular placenta. We measured water-borne cortisol, estradiol, and progesterone along with brood size (fecundity) and gestational stage(s). We examined the physiological costs of both maternal provisioning modes. Matrotrophy likely imposes energetic demands due to active nutrient transfer, while lecithotrophy may incur costs from carrying many large embryos. We hypothesized that fecundity, gestational stage, and hormones would covary differently in lecithotrophic vs. matrotrophic species. We found no relationships between hormones and fecundity or gestational stage in any species. However, in H. formosa, we found a positive relationship between estradiol levels and female mass, and a negative relationship between progesterone levels and female mass indicating a change in the circulating levels of both hormones as females grow. We observed differences in average hormone levels among species: the matrotrophic species had higher progesterone and lower estradiol compared to lecithotrophic species. Higher estradiol in lecithotrophic species may relate to egg yolk formation, while placental structures could play a role in progesterone production in matrotrophic species. Elevated cortisol in H. formosa suggests either higher energetic costs or a preparative role for reproduction. Our findings highlight progesterone's importance in maintaining gestation in matrotrophic species, like other placental species.

Phallusia nigra-mediated vanadium removal from brine: Assessment and optimization.

The rejected brines from desalination plants contain significant amounts of heavy metals. In this study, we evaluated the effectiveness of Phallusia nigra Savigny, 1816 (P. nigra) in removing vanadium from the rejected brines of desalination plants through the bioaccumulation process. Initial assessments revealed a remarkably high accumulation rate of vanadium in P. nigra with a bioaccumulation factor exceeding 4.7 × 104 in the tunic and 5.1 × 105 in the mantle body. Acclimation experiments demonstrated that P. nigra could survive salinities up to 56 practical salinity units (psu), temperatures of ≤32 °C, and pH of 6.5-8.5. We employed the L-16 Taguchi approach in experimental design to optimize environmental conditions for vanadium removal by P.nigra. Our results indicated that temperature has the most significant effect on increasing vanadium bioaccumulation in P. nigra, followed by salinity and pH. Under optimal conditions, the vanadium concentration reached 1892.30 ppm in the entire body of P. nigra compared to 350 ppm in natural conditions. Considering that, a high concentration of vanadium is toxic to the environment and the conventional methods of its removal from brine are costly and include the use of chemicals that pollute the environment, therefore, vanadium removal from brine using P. nigra can be considered a cost-effective and environmentally friendly method in the future, as opposed to some chemical methods.

Pharmaceutical Pollution Alters the Structure of Freshwater Communities and Hinders Their Recovery from a Fish Predator.

Freshwater ecosystems are under threat from rising pharmaceutical pollution. While such pollutants are known to elicit biological effects on organisms, we have limited knowledge on how these effects might cascade through food-webs, disrupt ecological processes, and shape freshwater communities. In this study, we used a mesocosm experiment to explore how the community impacts of a top-order predator, the eastern mosquitofish (Gambusia holbrooki), are mediated by exposure to environmentally relevant low (measured concentration: ∼10 ng/L) and high concentrations (∼110 ng/L) of the pervasive pharmaceutical pollutant fluoxetine. We found no evidence that exposure to fluoxetine altered the consumptive effects of mosquitofish on zooplankton. However, once mosquitofish were removed from the mesocosms, zooplankton abundance recovered to a greater extent in control mesocosms compared to both low and high fluoxetine-exposed mesocosms. By the end of the experiment, this resulted in fundamental differences in community structure between the control and fluoxetine-treated mesocosms. Specifically, the control mesocosms were characterized by higher zooplankton abundances and lower algal biomass, whereas mesocosms exposed to either low or high concentrations of fluoxetine had lower zooplankton abundances and higher algal biomass. Our results suggest that fluoxetine, even at very low concentrations, can alter aquatic communities and hinder their recovery from disturbances.

Different patterns of coupled predator-prey dynamics when the same species interact in different locations.

Individual predator and prey species exhibit coupled population dynamics in simple laboratory systems and simple natural communities. It is unclear how often such pairwise coupling occurs in more complex communities, in which an individual predator species might feed on several prey species and an individual prey species might be attacked by several predators. To examine this problem, we applied multivariate autoregressive state-space (MARSS) models to 5-year time-series of monthly surveys of a predatory fish, the eastern mosquitofish (Gambusia holbrooki), and its littoral zone prey species, the least killifish (Heterandria formosa), in three locations in north Florida. The MARSS models were consistent with coupled predator-prey dynamics at two of the three locations. In one of these two locations, the estimated densities of the two species displayed classic predator-prey oscillations. In the third location, there was a positive effect of killifish density on mosquitofish density but no detectable effect of mosquitofish density on killifish density. In all three locations, increased submergent vegetation cover was associated with increased prey density but not increased predator density. Eigenvalues analyses for the joint predator-prey dynamics indicated that one of the cyclic locations had more stable dynamics than the other locations. The three different patterns demonstrate that the dynamics of a pairwise predator-prey interaction emerge not only from the characteristics of the prey and the predator, but also those of the habitat and trophic web in which the predator and prey are embedded.

Habitat structural complexity predicts cognitive performance and behaviour in western mosquitofish.

Urban stream syndrome alters stream habitat complexity. We define habitat complexity as the degree of variation in physical habitat structure, with increasing variation equating to higher complexity. Habitat complexity affects species composition and shapes animal ecology, physiology, behaviour and cognition. We used a delayed detour test to measure whether cognitive processes (motor self-regulation) and behaviour (risk-taking) of female Western mosquitofish, Gambusia affinis, varied with habitat structural complexity (low, moderate and high) that was quantified visually for nine populations. We predicted that motor self-regulation and risk-taking behaviour would increase with increasing habitat complexity, yet we found support for the opposite. Lower complexity habitats offer less refuge potentially leading to higher predation pressure and selecting for greater risk-taking by fish with higher motor self-regulation. Our findings provide insight into how habitat complexity can shape cognitive processes and behaviour and offers a broader understanding of why some species may tolerate conditions of urbanized environments.

Patrolling the area, not ousting intruders, relates to reproductive success for territorial male Leon Springs pupfish, Cyprinodon bovinus.

We considered the relationship between the benefits and costs of territorial defense in a wild population of the Leon Springs pupfish, Cyprinodon bovinus. We defined benefit as the number of eggs deposited on an artificial substratum placed within the defender's territory. Costs included two defensive behaviors. First, males frequently "patrolled" their territories, swimming back-and-forth across their area. Second, males chased intruding Pecos gambusia (Gambusia nobilis) as well as small male and female conspecific C. bovinus from their territories. Both of these species prey on the territorial defenders' eggs; additionally, small male C. bovinus will attempt to "steal" spawns from the territorial defender by spawning with females in the territory. Our analyses revealed that only patrol frequency was related to the reproductive benefit of the territory. Neither chases against gambusia nor conspecifics were predicted by egg numbers on the breeding substrata. We speculate that the frequency of patrolling is an indicator of territorial value and note the qualitative differences in chasing behavior against the different species of intruder.

Microbiome and epigenetic variation in wild fish with low genetic diversity.

Non-genetic sources of phenotypic variation, such as the epigenome and the microbiome, could be important contributors to adaptive variation for species with low genetic diversity. However, little is known about the complex interaction between these factors and the genetic diversity of the host, particularly in wild populations. Here, we examine the skin microbiome composition of two closely-related mangrove killifish species with different mating systems (self-fertilising and outcrossing) under sympatric and allopatric conditions. This allows us to partition the influence of the genotype and the environment on their microbiome and (previously described) epigenetic profiles. We find the diversity and community composition of the skin microbiome are strongly shaped by the environment and, to a lesser extent, by species-specific influences. Heterozygosity and microbiome alpha diversity, but not epigenetic variation, are associated with the fluctuating asymmetry of traits related to performance (vision) and behaviour (aggression). Our study identifies that a proportion of the epigenetic diversity and microbiome differentiation is unrelated to genetic variation, and we find evidence for an associative relationship between microbiome and epigenetic diversity in these wild populations. This suggests that both mechanisms could potentially contribute to variation in species with low genetic diversity.

Evidence for gene flow and trait reversal during radiation of Mexican Goodeid fish.

Understanding the phylogeographic history of a group and identifying the factors contributing to speciation is an important challenge in evolutionary biology. The Goodeinae are a group of live-bearing fishes endemic to Mexico. Here, we develop genomic resources for species within the Goodeinae and use phylogenomic approaches to characterise their evolutionary history. We sequenced, assembled and annotated the genomes of four Goodeinae species, including Ataeniobius toweri, the only matrotrophic live-bearing fish without a trophotaenia in the group. We estimated timings of species divergence and examined the extent and timing of introgression between the species to assess if this may have occurred during an early radiation, or in more recent episodes of secondary contact. We used branch-site models to detect genome-wide positive selection across Goodeinae, and we specifically asked whether this differs in A. toweri, where loss of placental viviparity has recently occurred. We found evidence of gene flow between geographically isolated species, suggesting vicariant speciation was supplemented by limited post-speciation gene flow, and gene flow may explain previous uncertainties about Goodeid phylogeny. Genes under positive selection in the group are likely to be associated with the switch to live-bearing. Overall, our studies suggest that both volcanism-driven vicariance and changes in reproductive mode influenced radiation in the Goodeinae.

Genomes of the Orestias pupfish from the Andean Altiplano shed light on their evolutionary history and phylogenetic relationships within Cyprinodontiformes.

BACKGROUND: To unravel the evolutionary history of a complex group, a comprehensive reconstruction of its phylogenetic relationships is crucial. This requires meticulous taxon sampling and careful consideration of multiple characters to ensure a complete and accurate reconstruction. The phylogenetic position of the Orestias genus has been estimated partly on unavailable or incomplete information. As a consequence, it was assigned to the family Cyprindontidae, relating this Andean fish to other geographically distant genera distributed in the Mediterranean, Middle East and North and Central America. In this study, using complete genome sequencing, we aim to clarify the phylogenetic position of Orestias within the Cyprinodontiformes order. RESULTS: We sequenced the genome of three Orestias species from the Andean Altiplano. Our analysis revealed that the small genome size in this genus (~ 0.7 Gb) was caused by a contraction in transposable element (TE) content, particularly in DNA elements and short interspersed nuclear elements (SINEs). Using predicted gene sequences, we generated a phylogenetic tree of Cyprinodontiformes using 902 orthologs extracted from all 32 available genomes as well as three outgroup species. We complemented this analysis with a phylogenetic reconstruction and time calibration considering 12 molecular markers (eight nuclear and four mitochondrial genes) and a stratified taxon sampling to consider 198 species of nearly all families and genera of this order. Overall, our results show that phylogenetic closeness is directly related to geographical distance. Importantly, we found that Orestias is not part of the Cyprinodontidae family, and that it is more closely related to the South American fish fauna, being the Fluviphylacidae the closest sister group. CONCLUSIONS: The evolutionary history of the Orestias genus is linked to the South American ichthyofauna and it should no longer be considered a member of the Cyprinodontidae family. Instead, we submit that Orestias belongs to the Orestiidae family, as suggested by Freyhof et al. (2017), and that it is the sister group of the Fluviphylacidae family, distributed in the Amazonian and Orinoco basins. These two groups likely diverged during the Late Eocene concomitant with hydrogeological changes in the South American landscape.

Strength of female mate preferences in temperature manipulation study supports the signal reliability hypothesis.

Both sexually selected traits and mate preferences for these traits can be context dependent, yet how variation in preferred traits could select for context dependent preferences has rarely been examined. The signal reliability hypothesis predicts that mate preferences vary across contexts (e.g., environments) in relation to the reliability of the information preferred traits provide in those contexts. Extensive variation in copy number of mc4r B alleles on the Y-chromosome that associates with male size in Xiphophorus multilineatus allowed us to use a split-sibling design to determine if male size is more likely to provide information about male genotype (i.e., dam) when males were reared in a warm as compared to a cold environment. We then examined strength of preference for male size by females reared in the same two environments. We found that males were larger in the cold environment, but male size was more variable across dams in the warm environment, and therefore male size would be a more reliable indicator of dam (i.e., genetics) in the warm environment. Females reared in the warm environment had stronger mate preferences based on male size than cold reared females, with a significant influence of dam on strength of preference. Therefore, strength of female preference for male size was influenced by the temperature in which they were reared, with the direction of the difference across treatments supporting the signal reliability hypothesis. Understanding how the reliability of male traits can select for contextual variation in the strength of the female mate preferences will further our discovery of adaptive mate preferences. For example, a relationship between the strength of a female's mate preference and their growth rates was detected in the context where females had a preference based on male size, supporting a hypothesis from previous work with this species of disassortative mating in relation to growth rates to mitigate a documented growth-mortality tradeoff.

Warming, stochastic diel thermal fluctuations affect physiological performance and gill plasticity in an amphibious mangrove fish.

Natural temperature variation in many marine ecosystems is stochastic and unpredictable, and climate change models indicate that this thermal irregularity is likely to increase. Temperature acclimation may be more challenging when conditions are highly variable and stochastic, and there is a need for empirical physiological data in these thermal environments. Using the hermaphroditic, amphibious mangrove rivulus (Kryptolebias marmoratus), we hypothesized that compared with regular, warming diel thermal fluctuations, stochastic warm fluctuations would negatively affect physiological performance. To test this, we acclimated fish to: (1) non-stochastic and (2) stochastic thermal fluctuations with a similar thermal load (27-35°C), and (3) a stable/consistent control temperature at the low end of the cycle (27°C). We determined that fecundity was reduced in both cycles, with reproduction ceasing in stochastic thermal environments. Fish acclimated to non-stochastic thermal cycles had growth rates lower than those of control fish. Exposure to warm, fluctuating cycles did not affect emersion temperature, and only regular diel cycles modestly increased critical thermal tolerance. We predicted that warm diel cycling temperatures would increase gill surface area. Notably, fish acclimated to either thermal cycle had a reduced gill surface area and increased intralamellar cell mass when compared with control fish. This decreased gill surface area with warming contrasts with what is observed for exclusively aquatic fish and suggests a preparatory gill response for emersion in these amphibious fish. Collectively, our data reveal the importance of considering stochastic thermal variability when studying the effects of temperature on fishes.

Detection of relatedness in chemical alarm cues by a selfing vertebrate depends on population and the life stage producing the alarm cue.

Aquatic prey have impressive abilities to extract information from a variety of chemical cues. For example, they can use the alarm cues released by wounded individuals during a predator attack to learn about predation risk, and they can also distinguish kin from non-kin individuals during interactions. However, it remains unclear whether animals can combine this information on predation risk with kin recognition of the particular individuals under threat. To examine how the relatedness of the individuals in alarm cue affects behaviour we used the self-fertilizing hermaphroditic mangrove rivulus (Kryptolebias marmoratus), in which lineages produce genetically identical offspring through selfing. We explored this in two populations that differ in their level of outcrossing. We measured activity before and after exposure to alarm cue made from individuals (either adults or embryos) from their own lineage or an unrelated lineage from the same population. Fish responded weakly to embryo alarm cues, but tended to reduce their activity more when the alarm cues were from an unrelated lineage compared to alarm cues from their own lineage, particularly in fish from the outcrossing population. In contrast, there was no effect of cue relatedness on the response to adult alarm cues but there was a strong population effect. Specifically, individuals from the outcrossing population tended to react more strongly to alarm cues compared to individuals from the predominantly selfing population. We discuss the potential roles of the major histocompatibility complex in cue detection, differences between adult vs embryo alarm cues in terms of concentration and information, and underlying differences among populations and genetic lineages in their production and detection of chemical cues. Whether this kin recognition offers adaptive benefits or is simply a consequence of being able to detect relatedness in living individuals would be an exciting area for future research.

Season, size, and sex: factors influencing monogenean prevalence and intensity on Gambusia affinis in New Zealand.

A number of studies have been conducted on monogenean seasonality, though primarily in continental regions with wide annual temperatures ranges. We investigated seasonal changes in the prevalence and intensity of Salsuginus seculus infesting sexually dimorphic western mosquitofish (Gambusia affinis) in New Zealand. This represents the first examination of seasonality for this species globally, and the first seasonal assessment of any monogenean population in New Zealand, a temperate country with a mild oceanic climate. Prevalence and intensity of S. seculus with respect to fish size and sex was also examined. Prevalence of S. seculus changed temporally, peaking in summer, and was strongly positively correlated with algal concentrations. This relationship may be associated with increasing food levels, leading to an increase in fish courting and mating, resulting in high numbers and close physical associations of G. affinis individuals, facilitating transmission of the monogeneans. Thus, biotic factors may be important in determining temporal changes in S. seculus prevalence in New Zealand. Female G. affinis had a significantly higher prevalence and mean intensity of S. seculus than males. Longer fish had a higher mean intensity and prevalence of S. seculus. Female G. affinis likely host disproportionately more monogeneans as they are larger than males. Alternatively, females may have a compromised immune response during reproductive periods. Overall, seasonal change was observed in S. seculus prevalence and intensity under New Zealand's mild climatic conditions, and the larger female G. affinis in this dimorphic species supported a greater prevalence and intensity of infestation than males.

Transposable Element Expression Profiles in Premalignant Pigment Cell Lesions and Melanoma of Xiphophorus.

Transposable elements (TEs) are characterized by their ability to change their genomic position. Through insertion or recombination leading to deletions and other chromosomal aberrations, they can cause genetic instability. The extent to which they thereby exert regulatory influence on cellular functions is unclear. To better characterize TEs in processes such as carcinogenesis, we used the well-established Xiphophorus melanoma model. By transcriptome sequencing, we show that an increasing total number in transposons correlates with progression of malignancy in melanoma samples from Xiphophorus interspecific hybrids. Further, by comparing the presence of TEs in the parental genomes of Xiphophorus maculatus and Xiphophorus hellerii, we could show that even in closely related species, genomic location and spectrum of TEs are considerably different.

Combined effects of binary mixtures of 17ß-estradiol and testosterone in western mosquitofish (Gambusia affinis) after full life-cycle exposure.

Estrogens and androgens are typical steroid hormones and often occur together in contaminated aquatic environments, but their mixed effects in aquatic organisms have been less well reported. In this study, the endocrine disrupting effects of binary mixtures of 17ß-estradiol (E2) and testosterone (T) in western mosquitofish (Gambusia affinis) were assessed by analyzing the sex ratio, secondary sex characteristics, gonadal histology, and transcriptional expression of target genes related to the hypothalamic-pituitary-gonadal (HPG) axis in G. affinis (from embryos) continuously exposed to E2 (50 ng/L), T (T1: 50 ng/L; T2: 200 ng/L), and mixtures of both (E2 + T1: 50 + 50 ng/L; E2 + T2: 50 + 200 ng/L) for 119 d. The results showed that exposure to E2 + T1 and E2 + T2 reduced the length ratio of ray 4/6 ratio in male G. affinis, suggesting feminized phenomenon in male G. affinis. Furthermore, 16.7-38.5 % of female G. affinis showed masculinized anal fins and hemal spines when exposed to T alone and in combination with E2. Importantly, the transcriptional levels of certain target genes related to the HPG axis were significantly altered in G. affinis following exposure to E2 and T alone and in combinations. Moreover, exposure to E2 and T in combinations can lead to combined effects (such as synergistic and antagonistic effects) on the transcriptional levels of some genes. These results collectively suggest that exposure to environmentally relevant concentrations of E2 and T alone and in mixtures can impact the endocrine system of G. affinis, and may pose potential risks in aquatic systems.