Radiotagging a long-distance migratory Characiform fish: reproduction after surgery, tag losses, and effects in weight

Although biotelemetry studies bring useful information, tagging is a highly invasive procedure. For this reason, we assess if intracoelomic tagging alters survivorship, weight gain and/or reproductive capacity of a neotropical migratory fish. In August 2016, 60 fish were equally and randomly distributed among 3 treatments: Control (anesthesia), sham surgery (anesthesia and surgery), and true surgery (anesthesia, surgery and tagging). Surveys for weight and tag expulsion were performed periodically from surgery through spawning (November/2016), with a final survey occurring in May/2017. Mortality was higher in true surgery (25% died) and for males. Twenty percent expelled tags, and initial weight loss followed by gain were verified for all treatments. Females of all treatments spawned, and fecundity and fertility were not different. Then, future studies should consider the losses due to death and tag expulsion when defining the sample number. We also encourage further investigations about differences in mortality between sex and weight variation, as well as any other factor that leads to increase in mortality. This is the first study that investigated tagging effects in reproduction of neotropical fishes, and since the methods and tags used are not species-specific, we infer that similar effects could occur in other species.(AU)

Embora estudos de biotelemetria gerem informações úteis, processos de marcação são invasivos. Por isso, avaliamos se a marcação intracelomática altera sobrevivência, ganho de peso e/ou capacidade reprodutiva de um peixe migrador neotropical. Em agosto de 2016, 60 peixes foram distribuídos igualmente e aleatoriamente entre os tratamentos controle (anestesia), cirurgia-falsa (anestesia e cirurgia) e cirurgia-verdadeira (anestesia, cirurgia e marcação). Vistorias para pesagem e observação de expulsão de marcas foram realizadas periodicamente da cirurgia até a desova (novembro/2016), com uma vistoria final em maio/2017. A mortalidade foi maior nos peixes marcados (25% morreram) e entre machos. Vinte por cento expulsaram as marcas e, para todos os tratamentos, foi observado perda de peso inicial com posterior recuperação. Fêmeas de todos os tratamentos desovaram; fecundidade e fertilidade não foram diferentes. Novos estudos devem considerar as perdas de marcas por mortalidade e rejeição na definição do número amostral. Também recomendamos estudos que investiguem a mortalidade diferencial entre sexos e variação de peso, bem como fatores causadores de mortalidade. Este é o primeiro estudo que investigou efeitos da marcação na reprodução de peixes neotropicais e, uma vez que métodos e marcas utilizadas não são espécie-específicos, inferimos que efeitos similares podem ocorrer em outras espécies.(AU)

Effects of 17ß-estradiol on early gonadal development and expression of genes implicated in sexual differentiation of a South American teleost, Astyanax altiparanae.

Gonadal sex differentiation in teleost fish shows greater plasticity as compared to other vertebrates, as it can be influenced by a variety of factors such as exogenous sex steroids. Exogenous estrogens, such as 17ß-estradiol (E2), can induce feminization when administered during early embryonic development. However, the mechanisms underlying the E2-induced feminization are not fully understood, especially in Neotropical species. Therefore, the aim of this study was to evaluate the effects of E2 administration on the phenotypic sex characteristics, histological assessment of the gonads, and the expression of selected genes in Astyanax altiparanae exposed to dietary E2 prior to gonadal differentiation. At 4 days post-hatch (dph), groups of 30-40 undifferentiated larvae were fed with a diet containing varying amounts of E2 for 28 days, and fish were sampled at 90 dph. Previous studies revealed that ovary formation in A. altiparanae occurred at 58 dph, whereas the first sign of testis formation was found at 73 dph. In relation to the control, E2 exposure increased the proportion of phenotypic females in 120% and 148.4% for 4 and 6 mg E2/Kg, respectively. However, histological analysis revealed that treatments did not affect gonadal sex ratio between males and females, but induced intersex (testis-ova) in the group treated with 6 mg E2/Kg food. Treatment with E2 also altered gonadal transcript levels of a selected number of genes implicated in sexual differentiation. Males overexpressed dmrt1, sox9 and amh following E2 treatment as compared to control. Females showed increased mRNA levels of dmrt1 and sox9, which might be related to the down-regulation of cyp19a1a after E2 exposure. In summary, E2 exposure during early gonadal development affected male secondary characteristics without changing the gonadal sex ratio, and altered expression of genes implicated in sexual differentiation.

Dark world rises: The emergence of cavefish as a model for the study of evolution, development, behavior, and disease.

A central question in biology is how naturally occurring genetic variation accounts for morphological and behavioral diversity within a species. The Mexican tetra, Astyanax mexicanus, has been studied for nearly a century as a model for investigating trait evolution. In March of 2019, researchers representing laboratories from around the world met at the Sixth Astyanax International Meeting in Santiago de Querétaro, Mexico. The meeting highlighted the expanding applications of cavefish to investigations of diverse aspects of basic biology, including development, evolution, and disease-based applications. A broad range of integrative approaches are being applied in this system, including the application of state-of-the-art functional genetic assays, brain imaging, and genome sequencing. These advances position cavefish as a model organism for addressing fundamental questions about the genetics and evolution underlying the impressive trait diversity among individual populations within this species.

Repeated evolution of eye loss in Mexican cavefish: Evidence of similar developmental mechanisms in independently evolved populations.

Evolution in similar environments often leads to convergence of behavioral and anatomical traits. A classic example of convergent trait evolution is the reduced traits that characterize many cave animals: reduction or loss of pigmentation and eyes. While these traits have evolved many times, relatively little is known about whether these traits repeatedly evolve through the same or different molecular and developmental mechanisms. The small freshwater fish, Astyanax mexicanus, provides an opportunity to investigate the repeated evolution of cave traits. A. mexicanus exists as two forms, a sighted, surface-dwelling form and at least 29 populations of a blind, cave-dwelling form that initially develops eyes that subsequently degenerate. We compared eye morphology and the expression of eye regulatory genes in developing surface fish and two independently evolved cavefish populations, Pachón and Molino. We found that many of the previously described molecular and morphological alterations that occur during eye development in Pachón cavefish are also found in Molino cavefish. However, for many of these traits, the Molino cavefish have a less severe phenotype than Pachón cavefish. Further, cave-cave hybrid fish have larger eyes and lenses during early development compared with fish from either parental population, suggesting that some different changes underlie eye loss in these two populations. Together, these data support the hypothesis that these two cavefish populations evolved eye loss independently, yet through some of the same developmental and molecular mechanisms.

Dual roles of the retinal pigment epithelium and lens in cavefish eye degeneration.

Astyanax mexicanus consists of two forms, a sighted surface dwelling form (surface fish) and a blind cave-dwelling form (cavefish). Embryonic eyes are initially formed in cavefish but they are subsequently arrested in growth and degenerate during larval development. Previous lens transplantation studies have shown that the lens plays a central role in cavefish eye loss. However, several lines of evidence suggest that additional factors, such as the retinal pigment epithelium (RPE), which is morphologically altered in cavefish, could also be involved in the eye regression process. To explore the role of the RPE in cavefish eye degeneration, we generated an albino eyed (AE) strain by artificial selection for hybrid individuals with large eyes and a depigmented RPE. The AE strain exhibited an RPE lacking pigment granules and showed reduced expression of the RPE specific enzyme retinol isomerase, allowing eye development to be studied by lens ablation in an RPE background resembling cavefish. We found that lens ablation in the AE strain had stronger negative effects on eye growth than in surface fish, suggesting that an intact RPE is required for normal eye development. We also found that the AE strain develops a cartilaginous sclera lacking boney ossicles, a trait similar to cavefish. Extrapolation of the results to cavefish suggests that the RPE and lens have dual roles in eye degeneration, and that deficiencies in the RPE may be associated with evolutionary changes in scleral ossification.

Does soil color affect fish evolution? Differences in color change rate between lineages of the sailfin tetra

Several organisms match their skin color to the prevalent background color, granting crypsis against predators. The rate at which body color changes occur varies among organisms as a result of physiological constraints and adaptation to variation in contrasts between objects and the environmental background. Faster darkening of body color is favored in environments that show higher amounts of contrast between common objects and the prevailing background. Soil types in Amazon forest streams (igarapés) create distinct environments with respect to the amount of contrast, a result of the amount of sand and clay, which offers different contrasts against dead leaves. Here, we investigated differences in the rates of color change among populations of the sailfin tetra (Crenuchus spilurus) that represent lineages that live in regions of different soil types. Populations inserted into blackwaters (sandy soil) showed higher rates of color darkening in response to exposure to a dark environment composed by dead leaves. We propose that natural selection stemming from predation can favor faster color change rate in environments where there is higher variability of contrasts between leaf litter and soil, which is common in most blackwater streams.(AU)

Diversos organismos combinam sua coloração corporal com a cor de fundo predominante no ambiente, conferindo cripticidade contra predadores. A taxa na qual as mudanças de coloração corpórea ocorrem varia entre os organismos como resultado de restrições fisiológicas e adaptação à variação de contrastes entre objetos e o ambiente. O escurecimento mais rápido da cor do corpo é favorecido em ambientes que mostram maiores quantidades de contraste entre objetos comuns e o fundo predominante. Tipos de solo em igarapés da floresta amazônica criam ambientes distintos em relação à quantidade de contraste, resultado da quantidade de areia e argila, que oferece diferentes contrastes contra folhiço submerso. Nós investigamos as diferenças nas taxas de mudança de cor entre populações do tetra-colibri Crenuchus spilurus que representam linhagens que vivem em regiões de diferentes tipos de solo. Populações inseridas em águas pretas (solo arenoso) apresentaram maiores taxas de escurecimento da cor em resposta à exposição ao ambiente escuro de folhiço submerso. Nós propomos que a seleção natural decorrente da predação pode favorecer uma taxa de mudança de cor mais rápida em ambientes onde há maior variabilidade de contrastes entre o folhiço submerso e o solo, o que é comum na maioria dos igarapés de águas pretas.(AU)

Reproductive biology of pequira Bryconamericus stramineus (Eigenmann, 1908) in Funil Reservoir-MG, Brazil / Biologia reprodutiva da Pequira Bryconamericus stramineus (Eigenmann, 1908) no reservatório de Funil-MG, Brasil

Abstract In order to evaluate aspects of reproductive biology of the "pequira" ( Bryconamericus stramineus) in the elevator of the Funil Dam - MG, fish capture was carried out from November 2008 to January 2009 and 317 individuals were collected. The mean standard length (SL) of the population was 4.96 cm and mean weight 1.80 g. The females had SL of 5.0 cm, while males had a SL of 4.6 cm. A sex ratio of 2.20: 1 (females: male) was observed. Our results show that 73% of the individuals analyzed were considered adults. The species presented low fecundity, mean of 470.9 oocytes per female and a mean diameter of 221.08 μm, with an increase in oocyte diameter over the evaluation period. The length of the first gonadal maturation was estimated at 5.0 cm. The results obtained in this work suggest that the reproductive cycle of the species occurs in the analyzed period. Although this species does not have migratory reproductive habits, the presence of adults in the reproductive stage was observed in the transposition area, which suggests a search for new environments for spawning.

Resumo Com o objetivo de avaliar aspectos da biologia reprodutiva da pequira Bryconamericus stramineus no elevador da represa do Funil - MG foram realizadas capturas entre os meses de novembro de 2008 a janeiro de 2009, sendo coletados 317 indivíduos. O comprimento padrão (CP) médio da população foi de 4,96 cm e peso médio de 1,80 g. As fêmeas apresentaram CP de 5,0 cm, enquanto que os machos obtiveram um CP de 4,6 cm. Foi observada proporção sexual de 2,20:1(fêmeas:macho). Dos indivíduos analisados, 73% foram considerados adultos. A espécie apresentou baixa fecundidade, com média de 470,9 ovócitos por fêmea e diâmetro médio de 221,08 μm, ocorrendo aumento no diâmetro de ovócitos com o decorrer do tempo no período avaliado. O comprimento de primeira maturação gonadal foi estimado em 5,0 cm. Os resultados obtidos neste trabalho sugerem que o ciclo reprodutivo da espécie ocorre no período analisado. Embora esta espécie não tenha hábitos reprodutivos migratórios, a presença de adultos no estágio reprodutivo foi observada na área de transposição, o que sugere a busca de novos ambientes para a reprodução.

Digestibilidade aparente do farelo de palmiste em tambaqui (Colossoma macropomum) / Apparent digestibility of palm kernel meal to tambaqui (Colossoma macropomum)

O objetivo deste trabalho foi determinar a digestibilidade do farelo de palmiste (Elaeis guineensis) para o tambaqui (Colossoma macropomum), em duas classes de peso: 1 (210 alevinos de 4,45±1,18g) e 2 (54 juvenis de 115,91±4,01g). Os coeficientes de digestibilidade aparente (CDA) da matéria seca, proteína bruta e energia bruta do farelo de palmiste foram avaliados pela metodologia de substituição da dieta referência, utilizando-se 0,1% de óxido crômico como indicador externo. Os dados foram analisados pelo teste t de Student, a 5% de probabilidade. Os CDAs da matéria seca, proteína bruta e energia bruta do ingrediente foram iguais (P>0,05) nas classes de peso avaliadas. Os CDAs observados nas classes 1 e 2, respectivamente, foram: matéria seca (17,52% e 20,75%), proteína bruta (62,83% e 63,75%) e energia bruta (14,16% e 22,43%). A capacidade do tambaqui para digerir os nutrientes do farelo de palmiste não foi influenciada pelo peso corporal, e o aproveitamento satisfatório da proteína (63,29%) faz desse ingrediente uma potencial fonte alternativa de proteína em dietas para a espécie.(AU)

The objective of this work was to determine the digestibility of palm kernel meal (Elaeis guineensis) in tambaqui (Colossoma macropomum), in two weight classes: 1 (210 fingerlings of 4.45±1.18g) and 2 (54 juveniles of 115.91±4.01g). The apparent digestibility coefficients (ADC) of dry matter, crude protein and crude energy of the palm kernel meal were evaluated by the substitution of the reference diet methodology, using 0.1% chromic oxide as an external indicator. Data were analyzed by Student's t-test at 5% probability. The dry matter, crude protein and crude energy ADCs of the ingredient were the same (P> 0.05) in the weight classes evaluated. The ADCs observed in classes 1 and 2, respectively, were: dry matter (17.52% and 20.75%), crude protein (62.83% and 63.75%) and crude energy (14.16% and 22.43%). The ability of tambaqui to digest the nutrients of palm kernel meal was not influenced by body weight, and satisfactory protein utilization (63.29%) makes this ingredient a potential alternative source of protein in diets for the species.(AU)

Evolution of acoustic communication in blind cavefish.

Acoustic communication allows the exchange of information within specific contexts and during specific behaviors. The blind, cave-adapted and the sighted, river-dwelling morphs of the species Astyanax mexicanus have evolved in markedly different environments. During their evolution in darkness, cavefish underwent a series of morphological, physiological and behavioral changes, allowing the study of adaptation to drastic environmental change. Here we discover that Astyanax is a sonic species, in the laboratory and in the wild, with sound production depending on the social contexts and the type of morph. We characterize one sound, the "Sharp Click", as a visually-triggered sound produced by dominant surface fish during agonistic behaviors and as a chemosensory-, food odor-triggered sound produced by cavefish during foraging. Sharp Clicks also elicit different reactions in the two morphs in play-back experiments. Our results demonstrate that acoustic communication does exist and has evolved in cavefish, accompanying the evolution of its behaviors.

In-Frame Indel Mutations in the Genome of the Blind Mexican Cavefish, Astyanax mexicanus.

Organisms living in the subterranean biome evolve extreme characteristics including vision loss and sensory expansion. Despite prior work linking certain genes to Mendelian traits, the genetic basis for complex cave-associated traits remains unknown. Moreover, it is unclear if certain forms of genetic variation (e.g., indels, copy number variants) are more common in regressive evolution. Progress in this area has been limited by a lack of suitable natural model systems and genomic resources. In recent years, the Mexican tetra, Astyanax mexicanus, has advanced as a model for cave biology and regressive evolution. Here, we present the results of a genome-wide screen for in-frame indels using alignments of RNA-sequencing reads to the draft cavefish genome. Mutations were discovered in three genes associated with blood physiology (mlf1, plg, and wdr1), two genes associated with growth factor signaling (ghrb, rnf126), one gene linked to collagen defects (mia3), and one gene which may have a global epigenetic impact on gene expression (mki67). With one exception, polymorphisms were shared between Pachón and Tinaja cavefish lineages, and different from the surface-dwelling lineage. We confirmed the presence of mutations using direct Sanger sequencing and discovered remarkably similar developmental expression in both morphs despite substantial coding sequence alterations. Further, three mutated genes mapped near previously established quantitative trait loci associated with jaw size, condition factor, lens size, and neuromast variation. This work reveals previously unappreciated traits evolving in this species under environmental pressures (e.g., blood physiology) and provides insight to genetic changes underlying convergence of organisms evolving in complete darkness.

Water temperature and acid pH influence the cytotoxic and genotoxic effects of aluminum in the freshwater teleost Astyanax altiparanae (Teleostei: Characidae).

The toxicity of metals, including aluminum (Al), can be potentiated by temperature and acid pH, a concern in view of the current global warming scenario. The aim of this study was to evaluate the bioconcentration of Al in the testes and semen of Astyanax altiparanae and the potential of this metal, at different environmental temperatures and acid pH, to cause cytotoxicity and genotocixity in erythrocytes and spermatozoa. A. altiparanae males were divided into nine experimental groups: at each of three different water temperatures (20, 25 and 30 °C), the fish were exposed to a neutral pH, an acid pH and acidic water containing Al (0.5 mg.L-1). The fish were subjected to subacute, semi-static exposure and sampled at 24 and 96 h. After each exposure period the comet assay (blood and semen) and micronucleus test (blood) were performed. Bioconcentration of Al was evaluated in the testes and semen. Exposure time and temperature influenced the Al bioconcentration pattern in the testes. Al concentration in the semen was higher in fish exposed at 20 and 25 °C (24 h). The DNA fragmentation score for the semen and blood was higher in fish exposed to Al at 20 (24 h) and 30 °C (96 h). The frequency of nuclear abnormalities in erythrocytes was higher in the group exposed to Al at 30 °C (96 h). It was concluded that Al bioconcentrates in the testes and semen of A. altiparanae at different temperatures and is potentially cytotoxic and genotoxic to erythrocytes and spermatozoa in this species.

Differential Expression of dmrt1 in Astyanax scabripinnis (Teleostei, Characidade) Is Correlated with B Chromosome Occurrence.

Astyanax is an abundant fish genus in South America. Some species of this group are characterized by the presence of B chromosomes and absence of morphologically differentiated sex chromosomes. In this study, we used quantitative real-time polymerase chain reaction to characterize mRNA expression of dmrt1 in Astyanax scabripinnis gonads. Maturing gonads of males with the B chromosome overexpressed dmrt1. Our findings suggest that B chromosomes may have an adaptive role in A. scabripinnis sex determination and maintenance.

A complete enzymatic capacity for long-chain polyunsaturated fatty acid biosynthesis is present in the Amazonian teleost tambaqui, Colossoma macropomum.

In vertebrates, the essential fatty acids (FA) that satisfy the dietary requirements for a given species depend upon its desaturation and elongation capabilities to convert the C18 polyunsaturated fatty acids (PUFA), namely linoleic acid (LA, 18:2n-6) and α-linolenic acid (ALA, 18:3n-3), into the biologically active long-chain (C20-24) polyunsaturated fatty acids (LC-PUFA), including arachidonic acid (ARA, 20:4n-6), eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3). Recent studies have established that tambaqui (Colossoma macropomum), an important aquaculture-produced species in Brazil, is a herbivorous fish that can fulfil its essential FA requirements with dietary provision C18 PUFA LA and ALA, although the molecular mechanisms underpinning such ability remained unclear. The present study aimed at cloning and functionally characterizing genes encoding key desaturase and elongase enzymes, namely fads2, elovl5 and elovl2, involved in the LC-PUFA biosynthetic pathways in tambaqui. First, a fads2-like desaturase was isolated from tambaqui. When expressed in yeast, the tambaqui Fads2 showed Δ6, Δ5 and Δ8 desaturase capacities within the same enzyme, enabling all desaturation reactions required for ARA, EPA and DHA biosynthesis. Moreover, tambaqui possesses two elongases that are bona fide orthologs of elovl5 and elovl2. Their functional characterization confirmed that they can operate towards a variety of PUFA substrates with chain lengths ranging from 18 to 22 carbons. Overall our results provide compelling evidence that demonstrates that all the desaturase and elongase activities required to convert LA and ALA into ARA, EPA and DHA are present in tambaqui within the three genes studied herein, i.e. fads2, elovl5 and elovl2.

Reproductive biology of pequira Bryconamericus stramineus (Eigenmann, 1908) in Funil Reservoir-MG, Brazil.

In order to evaluate aspects of reproductive biology of the "pequira" ( Bryconamericus stramineus) in the elevator of the Funil Dam - MG, fish capture was carried out from November 2008 to January 2009 and 317 individuals were collected. The mean standard length (SL) of the population was 4.96 cm and mean weight 1.80 g. The females had SL of 5.0 cm, while males had a SL of 4.6 cm. A sex ratio of 2.20: 1 (females: male) was observed. Our results show that 73% of the individuals analyzed were considered adults. The species presented low fecundity, mean of 470.9 oocytes per female and a mean diameter of 221.08 µm, with an increase in oocyte diameter over the evaluation period. The length of the first gonadal maturation was estimated at 5.0 cm. The results obtained in this work suggest that the reproductive cycle of the species occurs in the analyzed period. Although this species does not have migratory reproductive habits, the presence of adults in the reproductive stage was observed in the transposition area, which suggests a search for new environments for spawning.

Geographic variation of Moenkhausia bonita (Characiformes: Characidae) in the rio de la Plata basin, with distributional comments on M. intermedia

Moenkhausia bonita occurs in numerous additional localities from the Bermejo, Paraná, Paraguay, and Uruguay river basins. Given that this finding greatly expands the distributional range of M. bonita, we carried out an intraspecific comparison, using multivariate methods for 18 morphometric and eight meristic characters taken from a comprehensive sample of 536 specimens. All localities were distributed in four major geographic groups as follows: Bermejo, Paraná, Paraguay, and Uruguay. Results of the morphometric comparisons showed significant differences among the studied groups except between the Paraguay and Uruguay groups. Statistical differences in meristic values were found for most between-group comparisons, especially in those resulting from discriminant canonical analyses (DCA). Specimens from the Bermejo basin were the most distinct group in most morphological comparisons. However, the overall subtle differences found in body morphology likely reflect intraspecific variation within M. bonita and seem to be mainly influenced by spatial and environmental features of drainages. As M. bonita was previously identified as M. intermedia in the río de La Plata basin, distributional comments on the latter species in that basin are provided.(AU)

Moenkhausia bonita es registrada en numerosas localidades adicionales de las cuencas de los ríos Bermejo, Paraná, Paraguay, y Uruguay. Dado que estos hallazgos expanden ampliamente el rango distribucional de M. bonita, nosotros llevamos a cabo una comparación intraespecífica, usando métodos multivariados para 18 características morfométricas y 8 merísticas que fueron tomados en una muestra exhaustiva de 536 especímenes. Todas las localidades fueron repartidas en cuatro grupos principales como sigue: Bermejo, Paraná, Paraguay y Uruguay. Los resultados de las comparaciones morfométricas mostraron diferencias significativas a través de los grupos bajo estudio, excepto entre los grupos Paraguay y Uruguay. Diferencias estadísticas fueron encontrados en la mayoría de las comparaciones entre los grupos, especialmente en aquellas obtenidas de los análisis discriminantes canónicos (ADC). Los especímenes de la cuenca del Bermejo fueron encontrados como el grupo más divergente en la mayoría de las comparaciones morfológicas. No obstante, estas leves diferencias encontradas en la morfología del cuerpo son consideradas dentro de la variación intraespecífica de M. bonita y parecen estar influidas por características ambientales y espaciales de los drenajes. Dado que M. bonita fue previamente identificada como M. intermedia en la cuenca del río de La Plata, comentarios distribucionales sobre esta última especie en esta cuenca son presentados.(AU)

Toxicity of copper oxide nanoparticles to Neotropical species Ceriodaphnia silvestrii and Hyphessobrycon eques.

The increase of production and consumption of copper oxide nanostructures in several areas contributes to their release into aquatic ecosystems. Toxic effects of copper oxide nanoparticles (CuO NPs), in particular, on tropical aquatic organisms are still unknown, representing a risk for biota. In this study, the effects of rod-shaped CuO NPs on the Neotropical species Ceriodaphnia silvestrii and Hyphessobrycon eques were investigated. We also compared the toxicity of CuO NPs and CuCl2 on these species to investigate the contribution of particles and cupper ions to the CuO NPs toxicity. Considering the low copper ions release from CuO NPs (<1%), our results revealed that the toxicity of CuO NPs to C. silvestrii and H. eques was mainly induced by the NPs. The 48 h EC50 for C. silvestrii was 12.6 ±â€¯0.7 µg Cu L-1 and for H. eques the 96 h LC50 was 211.4 ±â€¯57.5 µg Cu L-1 of CuO NPs. There was significant decrease in reproduction, feeding inhibition and increase in reactive oxidative species (ROS) generation in C. silvestrii exposed to CuO NPs. In fish H. eques, sublethal exposure to CuO NPs caused an increase in ROS generation in gill cells and an increase in cells number that were in early apoptotic and necrotic stages. Our results showed that CuO NPs caused toxic effects to C. silvestrii and H. eques and ROS play an important role in the toxicity pathway observed. Data also indicated that C. silvestrii was among the most sensitive species for CuO NPs. Based on predicted environmental concentration in water bodies, CuO NPs pose potential ecological risks for C. silvestrii and H. eques and other tropical freshwater organisms.

Facial bone fragmentation in blind cavefish arises through two unusual ossification processes.

The precise mechanisms underlying cranial bone development, evolution and patterning remain incompletely characterised. This poses a challenge to understanding the etiologies of craniofacial malformations evolving in nature. Capitalising on natural variation, "evolutionary model systems" provide unique opportunities to identify underlying causes of aberrant phenotypes as a complement to studies in traditional systems. Mexican blind cavefish are a prime evolutionary model for cranial disorders since they frequently exhibit extreme alterations to the skull and lateral asymmetries. These aberrations occur in stark contrast to the normal cranial architectures of closely related surface-dwelling fish, providing a powerful comparative paradigm for understanding cranial bone formation. Using a longitudinal and in vivo analytical approach, we discovered two unusual ossification processes in cavefish that underlie the development of 'fragmented' and asymmetric cranial bones. The first mechanism involves the sporadic appearance of independent bony elements that fail to fuse together later in development. The second mechanism involves the "carving" of channels in the mature bone, a novel form of post-ossification remodeling. In the extreme cave environment, these novel mechanisms may have evolved to augment sensory input, and may indirectly result in a trade-off between sensory expansion and cranial bone development.

Metal accumulation and expression of genes encoding for metallothionein and copper transporters in a chronically exposed wild population of the fish Hyphessobrycon luetkenii.

In the present study, metal (As, Cd, Cu, Fe, Mn, Pb and Zn) accumulation and expression of genes involved in metal metabolism (metallothioneins, ATP7A and CTR1) were evaluated in gills and muscle of the fish Hyphessobrycon luetkenii living in the João Dias creek, a site historically (~1870-1996) contaminated with a metal mixture associated with copper mining (Minas do Camaquã, southern Brazil). Fish were collected in a metal impacted site of the João Dias creek and kept in a cage at this site (PP fish) or translocated to a non-metal impacted reach of this creek (PC fish). Gill metal concentrations and metallothionein gene expression were lower in PC fish than in PP fish at any experimental time (24, 48 and 72 h). In muscle, no significant changes were observed. These findings indicate that metal accumulated in gills of wild fish chronically exposed to the metal mixture are more easily excreted than those accumulated in the muscle. In this case, expression of gene encoding for metallothionein is shown to play a key role in the regulation of metal accumulation in gills of H. luetkenii living in an area historically contaminated with a metal mixture associated with copper mining.

An epigenetic mechanism for cavefish eye degeneration.

Coding and non-coding mutations in DNA contribute significantly to phenotypic variability during evolution. However, less is known about the role of epigenetics in this process. Although previous studies have identified eye development genes associated with the loss-of-eyes phenotype in the Pachón blind cave morph of the Mexican tetra Astyanax mexicanus, no inactivating mutations have been found in any of these genes. Here, we show that excess DNA methylation-based epigenetic silencing promotes eye degeneration in blind cave A. mexicanus. By performing parallel analyses in A. mexicanus cave and surface morphs, and in the zebrafish Danio rerio, we have discovered that DNA methylation mediates eye-specific gene repression and globally regulates early eye development. The most significantly hypermethylated and downregulated genes in the cave morph are also linked to human eye disorders, suggesting that the function of these genes is conserved across vertebrates. Our results show that changes in DNA methylation-based gene repression can serve as an important molecular mechanism generating phenotypic diversity during development and evolution.