Two methods for geometric morphometric analysis of trichodinids from killifishes.

In this study, we compared the efficacy of geometric morphometric techniques, including outlines and landmark-based approaches, to support the differentiation of Trichodina bellottii from three co-occurring killifish species. Both methods were able to differentiate trichodinids from different host species. However, discriminat analyses and MANOVA results based on landmarks had greater accuracy possibly because these analyses only provide information on certain points defined by the researcher, while the analyses based on outlines take into account points with less taxonomic information.

A Pilot Study of Gene Expression Modulation from Antioxidant System of Killifish Austrolebias charrua After Exposure to Roundup Transorb®.

Roundup Transorb® (RDT) is the most popular glyphosate-based herbicide (GHB) used in agriculture, and its impact extends to non-target organisms. The annual killifish Austrolebias charrua is an endangered species endemic to southern South America and inhabits temporary ponds. This study evaluates the effects of RDT concentrations (0.065 and 5 mg/L GAE) on A. charrua exposed for 96 h. Gene expression of cat, sod2, gstα, gclc, and ucp1 was evaluated on the liver and gills. Highlighting that even at low concentrations permitted by Brazilian legislation, the RDT can have adverse effects on A. charrua.

Fish (eggs) out of water: evolutionary divergence in terrestrial embryonic plasticity in Trinidadian killifish.

Externally laid eggs are often responsive to environmental cues; however, it is unclear how such plasticity evolves. In Trinidad, the killifish (Anablepsoides hartii) is found in communities with and without predators. Here, killifish inhabit shallower, ephemeral habitats in sites with predators. Such shifts may increase the exposure of eggs to air and lead to possible desiccation. We compared egg-hatching plasticity between communities by rearing eggs terrestrially on peat moss or in water. The timing of hatching did not differ between communities when eggs were reared in water. Eggs from sites with predators responded to terrestrial incubation by hatching significantly earlier compared with water-reared eggs. These responses were weaker in sites with no predators. Such divergent trends show that the presence of predators is associated with evolutionary shifts in hatching plasticity. Our results provide evidence for local adaptation in embryonic plasticity at the population scale.

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

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

Morphofunctional analysis of the gastrointestinal tract of an annual killifish: In search of structural traits related to the annual life history.

Annual killifishes have active and voracious rates in acquisition of food resources with display of behaviors that allow them to maintain high metabolic rates to deal with the harsh and unpredictable conditions prevalent in temporary pools where they inhabit. The objective of this research was to describe histologically and ultrastructurally, the digestive system of the annual killifish Millerichthys robustus to identify morphological traits related to its annual life history and digestive physiology. Also, we quantify food items along the intestine as a proxy for rates of digestion. Millerichthys has a short digestive system, associated with a carnivorous diet, with no evidence of a stomach. Instead, the presence of pharyngeal jaws with caniform teeth was documented, related to the breakdown of invertebrate exoskeletons, allowing prey fluids to be tasted by taste corpuscles related to selection of food items, and that digestive enzymes penetrate once in the intestine. The histological morphology of the intestine showed four different regions, associated with its digestive rates: (i) reception of food from the esophagus with intact pray; (ii) digestion with enzymes from the pancreas and liver/gallbladder of simple exoskeleton prey (Entognatha), and beginning of absorption; (iii) absorption of nutrients, and digestion of large-complex exoskeleton prey (Hexanauplia, Brachiopoda, and Ostracoda); and (iv) probable absorption of intact macromolecules. The second region of the intestine presented two anatomical loops and the highest thickness that may be related to reducing the speed of food transit, allowing for more efficient digestion given the large amount of food ingested by this species.

Sex-biased response of pollution biomarkers in fish: Insights from the killifish Poecilia vivipara.

Copper ions (Cu) are one of the most frequent trace-contaminants found in Brazilian waters and, although considered as an essential element, in high concentrations can accumulate and induce toxicity. Biomarkers are important tools that can be used to assess these impacts, but to be considered trustworthy, they have to be previously tested in target organisms through laboratory studies under controlled conditions. However, many of these experiments are conducted using only males, as it is believed that the hormonal variation of females can bias the results, increasing data variability. Notwithstanding, few studies have actually tested this hypothesis, highlighting the importance of considering and measuring the role of sex in ecotoxicological studies. The aim this study was to evaluate the influence of sex on biomarkers classically used in environmental monitoring programs using the fish Poecilia vivipara as model. For this, females and males were exposed for 96 h to two Cu concentrations (9 and 20 µg/L) and a control group. In liver and gills, Cu accumulation, total antioxidant capacity (TAC) and lipid peroxidation (LPO) were evaluated. In addition, samples of peripheral blood were used for neutrophil to lymphocyte ratio determination, a measure of the onset of secondary stress. Results show that Cu hepatic accumulation did not differ between females and males, but higher levels of this metal were observed in exposed animals compared to control fish. Additionally, interactive effects were observed for hepatic LPO, as males showed elevated oxidative damage in comparison to females. Moreover, Cu exposure elevated hepatic LPO relative to control only in males, but this increase in oxidative damage was not accompanied by changes in liver TAC. On the other hand, differences in branchial Cu accumulation and LPO were not observed. Conversely, control females showed elevated TAC in comparison to control males, but Cu exposure eliminated this difference. Cu exposure also induced an increase in the N:L ratio, indicating the presence of a secondary stress response unrelated to sex. Ultimately, the findings of this study demonstrate that sex can influence the response of biomarkers that are typically used in ecotoxicological investigations in a multifaceted manner. As a result, using animals from a singular sex in such studies may result in consequential outcomes, potentially leading to underestimation or overestimation of results.

Effect of a temperature gradient on the behaviour of an endangered Mexican topminnow and an invasive freshwater fish.

Climate change and biological invasions are two of the major threats to biodiversity. They could act synergistically to the detriment of natives as non-native species may be more plastic and resilient when facing changing environments. The twoline skiffia (Skiffia bilineata) is an endangered Mexican topminnow that cohabits with invasive guppies (Poecilia reticulata) in some areas in central Mexico. Guppies have been found to take advantage from associating with the twoline skiffia and are considered partially responsible for the decline of its populations. Refuge use and exploratory behaviours are trade-offs between being safe from the unknown and the opportunity to explore novel areas in search for better resources or to disperse. The aim of this study is to investigate how a change in temperature affects the refuge use and exploratory behaviours for both species. We found that temperature affects the refuge use of twoline skiffias, and the swimming activity of both species. Skiffias explored the rock more than guppies regardless of the temperature scenario. Also, smaller fish spent more time performing exploratory behaviours than bigger ones. Our study is the first to test the effect of temperature on the refuge use and exploratory behaviour of a goodeid species, and our results contribute to the idea that some natives could be more affected by climate change than some invaders.

Development stage-dependent oxidative stress responses to the exposure to roundup original© in a neotropical annual killifish.

Herbicides are the most commonly applied pesticides in Brazil, specifically those based on glyphosate, and are used for different crops, near the habitats of annual killifish. Annual killifish presents a short life cycle with generally restricted geographic distribution. In this context, we evaluated the effect of the Roundup Original© (65, 130 and 260 µg. L-1 of glyphosate) herbicide on different development stages (adult-young and senile) of the annual killifish (Cynopoecilus sp.). We quantified the oxidative balance markers (superoxide dismutase, catalase, glutathione S-transferase, lipid peroxidation levels, and total proteins). We observed that the senile individuals presented 2-fold higher lipid peroxidation levels associated with the maintenance of superoxide dismutase and catalase activity levels even after exposure to the herbicide. However, senile subjects were negatively impacted by the exposure to formulations containing glyphosate, and this was related to a loss of glutathione S-transferase activity. Our research demonstrated that the established physiological markers and this species look promising for toxicology studies.

How maternal age and environmental cues influence embryonic developmental pathways and diapause dynamics in a North American annual killifish.

BACKGROUND: Temporary pools are variable environments with seasonal drought/flood phases. Annual killifish have adapted to life in temporary pools by producing embryos that undergo diapause to traverse the dry phase. To fill existing knowledge gaps about embryo diapause regulation and evolution in annual killifishes, we test the effect of maternal age, incubation temperature, and incubation medium on diapause induction and length in Millerichthys robustus, the only North American fish species that has evolved an annual life history. RESULTS: All embryos at extreme temperatures follow a defined developmental pathway: skipping diapause at 30°C, and entering diapause at 18°C, both regardless of maternal age, and incubation medium. However, maternal age, and incubation medium influenced whether diapause is entered, and time arrested in diapause for embryos incubated at 25°C. At 25°C, five-week-old, and 52-week-old females produced more embryos that entered diapause than 26-week-old females. Also, embryos incubated in aqueous medium skipped diapause more frequently at this intermediate temperature. CONCLUSIONS: Millerichthys developmental dynamics associated with maternal age under intermediate range of temperatures are likely adapted to the particular patterns of flood/drought in North American temporary pools. Millerichthys also exhibits developmental patterns largely comparable with other annual fishes, probably due to common seasonal patterns in temporary pools.

Protogynous functional hermaphroditism in the North American annual killifish, Millerichthys robustus.

Sex change (sequential hermaphroditism) has evolved repeatedly in teleost fishes when demographic conditions mediate fundamentally different sex-specific returns for individuals of particular age and size. We investigated the conditions for potential sex change in an annual killifish (Millerichthys robustus) from temporary pools in Mexico. In natural populations, we detected adults with intersex colouration and gonads. Therefore, we experimentally tested whether this apparent sex change can be generated by manipulation of ecological and social conditions, rather than being caused by environmental disturbance. We demonstrated functional protogynous (female-to-male) sex change in 60% replicates, when groups of five females interacted and had a visual and olfactory cue of a male. Only one female changed sex in any given replicate. The sex change never occurred in isolated females. Protandrous (male-to-female) hermaphroditism was not recorded. We characterized gradual changes in behaviour, colouration and gonad structure during the sex change process. The first behavioural signs of sex change were observed after 23 days. Secondary males spawned successfully after 75 days. We discuss the adaptive potential of sex change in short-lived annual fishes through the seasonal decline of males, and during colonization of new habitats. This is the first observation of functional hermaphroditism in an annual killifish.

Saccocoelioides kirchneri n. sp. (Digenea: Haploporidae) from the killifish Cnesterodon decemmaculatus (Cyprinodontiformes: Poeciliidae) from Argentina, morphological and molecular description.

This paper presents a new haploporid digenean that expands the number of species of Saccoccoelioides to 27. The new species, Saccocoelioides kirchneri n. sp. was collected from the intestine of Cnesterodon decemmaculatus (Poeciliidae: Cyprinodontiformes) from Lago del Bosque, La Plata, Argentina. The new species possesses the diagnostic features for Saccocoelioides: a sac like ceca; the vitellarium confined in two irregular groups of follicles distributed between the ventral sucker and the anterior margin of the testis; and a uterus confined largely in the hind-body, but encroaching into the range of the ventral sucker. The new species is differentiated from the 26 congeners by the body size, pharynx size, ventral sucker size, posterior extent of ceca, posterior extent of uterus and egg size. S. kirchneri n. sp. also is supported by the molecular analysis.

Genome of the Rio Pearlfish (Nematolebias whitei), a bi-annual killifish model for Eco-Evo-Devo in extreme environments.

The Rio Pearlfish, Nematolebias whitei, is a bi-annual killifish species inhabiting seasonal pools in the Rio de Janeiro region of Brazil that dry twice per year. Embryos enter dormant diapause stages in the soil, waiting for the inundation of the habitat which triggers hatching and commencement of a new life cycle. Rio Pearlfish represents a convergent, independent origin of annualism from other emerging killifish model species. While some transcriptomic datasets are available for Rio Pearlfish, thus far, a sequenced genome has been unavailable. Here, we present a high quality, 1.2 Gb chromosome-level genome assembly, genome annotations, and a comparative genomic investigation of the Rio Pearlfish as representative of a vertebrate clade that evolved environmentally cued hatching. We show conservation of 3D genome structure across teleost fish evolution, developmental stages, tissues, and cell types. Our analysis of mobile DNA shows that Rio Pearlfish, like other annual killifishes, possesses an expanded transposable element profile with implications for rapid aging and adaptation to harsh conditions. We use the Rio Pearlfish genome to identify its hatching enzyme gene repertoire and the location of the hatching gland, a key first step in understanding the developmental genetic control of hatching. The Rio Pearlfish genome expands the comparative genomic toolkit available to study convergent origins of seasonal life histories, diapause, and rapid aging phenotypes. We present the first set of genomic resources for this emerging model organism, critical for future functional genetic, and multiomic explorations of "Eco-Evo-Devo" phenotypes of resilience and adaptation to extreme environments.

Genome sequencing and transcriptomic analysis of the Andean killifish Orestias ascotanensis reveals adaptation to high-altitude aquatic life.

Orestias ascotanensis (Cyprinodontidae) is a teleost pupfish endemic to springs feeding into the Ascotan saltpan in the Chilean Altiplano (3,700 m.a.s.l.) and represents an opportunity to study adaptations to high-altitude aquatic environments. We have de novo assembled the genome of O. ascotanensis at high coverage. Comparative analysis of the O. ascotanensis genome showed an overall process of contraction, including loss of genes related to G-protein signaling, chemotaxis and signal transduction, while there was expansion of gene families associated with microtubule-based movement and protein ubiquitination. We identified 818 genes under positive selection, many of which are involved in DNA repair. Additionally, we identified novel and conserved microRNAs expressed in O. ascotanensis and its closely-related species, Orestias gloriae. Our analysis suggests that positive selection and expansion of genes that preserve genome stability are a potential adaptive mechanism to cope with the increased solar UV radiation to which high-altitude animals are exposed to.

Against the Odds: Hybrid Zones between Mangrove Killifish Species with Different Mating Systems.

Different mating systems are expected to affect the extent and direction of hybridization. Due to the different levels of sexual conflict, the weak inbreeder/strong outbreeder (WISO) hypothesis predicts that gametes from self-incompatible (SI) species should outcompete gametes from self-compatible (SC) ones. However, other factors such as timing of selfing and unilateral incompatibilities may also play a role on the direction of hybridization. In addition, differential mating opportunities provided by different mating systems are also expected to affect the direction of introgression in hybrid zones involving outcrossers and selfers. Here, we explored these hypotheses with a unique case of recent hybridization between two mangrove killifish species with different mating systems, Kryptolebias ocellatus (obligately outcrossing) and K. hermaphroditus (predominantly self-fertilizing) in two hybrid zones in southeast Brazil. Hybridization rates were relatively high (~20%), representing the first example of natural hybridization between species with different mating systems in vertebrates. All F1 individuals were sired by the selfing species. Backcrossing was small, but mostly asymmetrical with the SI parental species, suggesting pattern commonly observed in plant hybrid zones with different mating systems. Our findings shed light on how contrasting mating systems may affect the direction and extent of gene flow between sympatric species, ultimately affecting the evolution and maintenance of hybrid zones.

Biomarkers of oxidative stress in the post-embryonic characterization of the neotropical annual killifish.

Annual killifish are among the most remarkable extremophile species with the shortest vertebrate life span. Few studies have reported on the oxidative balance throughout their life cycle and its association to the natural aging process of these neotropical animals in a natural environment. We standardized and analyzed physiological markers related to the redox balance of the annual killifish (Cynopoecilus fulgens) throughout the post-embryonic life cycle (enzyme activity of Superoxide Dismutase, Catalase, Glutathione Peroxidase, and Glutathione S-transferase, as well as the determination of the levels of Lipoperoxidation, Carbonylated Proteins, and Total Proteins). We tested the influence of environmental variables on these biomarkers. Individuals were collected, including juveniles, adults, and seniles, in three sampling units around the Parque Nacional da Lagoa do Peixe, located in the Coastal Plain of Rio Grande do Sul. We observed that males and females used different physiological strategies of their redox balance during their life cycle, and their oxidative balance was influenced by their reproductive period and environmental variables (water temperature, abundance of predators, abundance of another sympatric annual killifish species, and abundance of C. fulgens). The population of each temporary pond presented different physiological responses to the adaptation of their life cycle, and there was an influence of environmental component as a modulator of this cycle. Our study offers reference values that will be useful for comparison in future research with short-lived organisms.

Recognizing the enemy: do predator cues influence hatching in Neotropical annual killifish?

Annual fish species have evolved complex adaptations to survive in temporary wetlands. The main adaptation of these fish is the ability to produce embryos that survive dry periods. Embryo development of this fish can show variation at multiple levels influenced by many environmental factors, such as photoperiod and temperature. Predator cues are another factor that can influence the embryonic stage. One way in which annual fish could adapt to predators is by using risk-spreading strategies (through bet-hedging). Nonetheless, this strategy depends on the coevolutionary history between predators and preys and on the degree of environmental unpredictability, resulting in different responses across different species. This study investigated the influence of predator cues on the embryonic development and hatching of two Austrolebias species that inhabit ponds that present differences in hydroperiod and the risk of predator presence. The results confirmed a differentiated response between the two annual fish species tested, corroborating the modulation of hatching against the risk of predation by native predatory fish. The authors further showed that development times varied between the two annual fish species, regardless of the presence of predators. They highlight that the variation in embryonic development is strongly affected by different levels of hydroperiod unpredictability faced by the two species. To unravel finer-scale local adaptations in the annual fish embryo development, future studies should focus on a region with greater spatial gradient.

Agonistic interactions with asymmetric body size in two adult-age groups of the annual killifish Millerichthys robustus (Miller & Hubbs, 1974).

In this study, the author evaluated two adult age groups of the Mexican rivulus Millerichthys robustus with body size asymmetries to determine the strategies used by an annual killifish during agonistic interactions of different ontogenetic stages. To achieve this goal, the author first characterized the ethogram of agonistic interactions of M. robustus composed of seven behavioural units in males and five behavioural units in females. The author then analysed agonistic interaction strategies used by males and females with body size asymmetries in two groups of different adult ages that represent different ontogenetic stages: (a) just after sexual maturity was reached, at 5 weeks of age, and (b) near natural death, at 24 weeks of age. The agonistic behaviour patterns of M. robustus were compatible with the logic of mutual assessment. Large males had an advantage during their interactions in both age groups, winning all of the encounters. Nonetheless, there was more aggression in 5-week-old fish encounters. In addition, small 24-week-old fish were more aggressive than small 5-week-old fish. These changing strategies may be because of the cost-benefits required during a fight at each ontogenetic stage. In the female encounters, size did not predict winners, as both small and large fish won a similar number of encounters, and some contests remained unresolved regardless of age group. There was a tendency for small females of any age to risk more than males in fights to maintain reproductive fitness.

Phylogenetic relationships and ecological niche conservatism in killifish (Profundulidae) in Mesoamerica.

The family Profundulidae is a group of small-sized fish species distributed between southern Mexico and Honduras, where they are frequently the only fish representatives at higher elevations in the basins where they occur. We characterized their ecological niche using different methods and metrics drawn from niche modelling and by re-examining phylogenetic relationships of a recently published molecular phylogeny of this family to gain a better understanding of its biogeographic and evolutionary history. We assessed both lines of evidence from the perspective of niche conservatism to set a foundation for discussing hypotheses about the processes underlying the distribution and evolution of the group. In fish clades where the species composition is not clear, we examined whether niche classification could be informative to discriminate groups geographically and ecologically consistent with any of the different hypotheses of valid species. The characterization of the ecological niche was carried out using the Maxent algorithm under different parameterizations and the projection of the presence on the main components of the most relevant environmental coverage, and the niche comparison was calculated with two indices (D and I), both in environmental space and in that projected geographically. With the molecular data, a species tree was generated using the *BEAST method. The comparison of these data was calculated with an age-overlap correlation test. Based on the molecular phylogeny and on niche overlap analyses, we uncovered strong evidence to support the idea that ecologically similar species are not necessarily sister species. The correlation analysis for genetic distance and niche overlap was not significant (P > 0.05). In clades with taxonomic conflicts, we only identified Profundulus oaxacae as a geographically and ecologically distinct group from P. punctatus. All the evidence considered leads us to propose that Profundulidae do not show evidence of niche conservatism and that there are reasons to consider P. oaxacae as a valid species. Our study suggests that niche divergence is a driving evolutionary force that caused the diversification and speciation processes of the Profundulidae, along with the geological and climatic events that promoted the expansion or contraction of suitable environments.

Extensive hybridization and past introgression between divergent lineages in a quasi-clonal hermaphroditic fish: Ramifications for species concepts and taxonomy.

Extreme inbreeding is expected to reduce the incidence of hybridization, serving as a prezygotic barrier. Mangrove rivulus is a small killifish that reproduces predominantly by self-fertilization, producing highly homozygous lines throughout its geographic range. The Bahamas and Caribbean are inhabited by two highly diverged phylogeographic lineages of mangrove rivulus, Kryptolebias marmoratus and a 'Central clade' closely related to K. hermaphroditus from Brazil. The two lineages are largely allopatric, but recently were found in syntopy on San Salvador, Bahamas, where a single hybrid was reported. To better characterize the degree of hybridization and the possibility of secondary introgression, here we conducted a detailed genetic analysis of the contact zone on San Salvador. Two mixed populations were identified, one of which contained sexually mature hybrids. The distribution of heterozygosity at diagnostic microsatellite loci in hybrids showed that one of these hybrids was an immediate offspring from the K. marmoratus x Central clade cross, whereas the remaining five hybrids were products of reproduction by self-fertilization for 1-3 generations following the initial cross. Two hybrids had mitochondrial haplotypes of K. marmoratus and the remaining four hybrids had a haplotype of the Central clade, indicating that crosses go in both directions. In hybrids, alleles of parental lineages were represented in equal proportions suggesting lack of recent backcrossing to either of the parental lineages. However, sympatric populations of two lineages were less diverged than allopatric populations, consistent with introgression. Results are discussed in terms of applicability of the biological species concept for isogenic, effectively clonal, organisms.

More than meets the eye: syntopic and morphologically similar mangrove killifish species show different mating systems and patterns of genetic structure along the Brazilian coast.

Different mating systems can strongly affect the extent of genetic diversity and population structure among species. Given the increased effects of genetic drift on reduced population size, theory predicts that species undergoing self-fertilisation should have greater population structure than outcrossed species; however, demographic dynamics may affect this scenario. The mangrove killifish clade is composed of the two only known examples of self-fertilising species among vertebrates (Kryptolebias marmoratus and Kryptolebias hermaphroditus). A third species in this clade, Kryptolebias ocellatus, inhabits mangrove forests in southeast Brazil; however, its mating system and patterns of genetic structure have been rarely explored. Here, we examined the genetic structure and phylogeographic patterns of K. ocellatus along its distribution, using mitochondrial DNA and microsatellites to compare its patterns of genetic structure with the predominantly selfing and often-syntopic, K. hermaphroditus. Our results indicate that K. ocellatus reproduces mainly by outcrossing, with no current evidence of selfing, despite being an androdioecious species. Our results also reveal a stronger population subdivision in K. ocellatus compared to K. hermaphroditus, contrary to the theoretical predictions based on reproductive biology of the two species. Our findings indicate that, although morphologically similar, K. ocellatus and K. hermaphroditus had remarkably different evolutionary histories when colonising the same mangrove areas in southeastern Brazil, with other factors (e.g., time of colonisation, dispersal/establishment capacity) having more profound effects on the current population structuring of those species than differences in mating systems.