Endocrine disruption caused by the aquatic exposure to aluminum and manganese in Astyanax altiparanae (Teleostei: Characidae) females during the final ovarian maturation.

Aluminum (Al) and manganese (Mn) can be toxic to aquatic biota and cause endocrine disruption in fish, affecting reproduction. This study evaluates the physiological responses of the ray-finned teleost fish Astyanax altiparanae vitellogenic females after acute exposure (96 h) to Al and Mn (alone and combined) in acid pH followed by the same period of exposure to metal-free water in neutral pH. The aim of this second period of exposure was to assess the recovery capacity from the toxic effects these metals. Five experimental groups were established: a control in neutral pH (Ctrl), and acidic pH (Ac), aluminum (Al), manganese (Mn), and Al + Mn groups, maintaining the acidic pH in the groups to which metals were added. The following biological parameters were evaluated: metal tissue concentration, relative fecundity (RF: absolute fecundity/body mass). Plasma levels of cortisol (proxy for stress) and 17α hydroxyprogesterone (17α-OHP), and gene expression of pituitary lhß mRNA (proxies for final maturation) were measured to evaluate endocrine disruption. In the synchronic exposure, the presence of Mn potentiated the accumulation of Al in gills. The females from acidic pH and Al groups showed a reduced RF. Exposure to Al and Mn triggered an endocrine disruption response, evidenced by a decrease in the plasma concentration of 17α-OHP and cortisol. Despite this anti-steroidogenic effect, no changes occurred in the pituitary gene expression of lhß. The endocrine changes and the metal accumulation were temporary, while the impacts on RF under the experimental conditions suggest permanent impairment in the reproduction of this species.

Modulation of immunity and hepatic antioxidant defense by corticosteroids in pacu (Piaractus mesopotamicus).

We investigated the impact of both the oral administration of hydrocortisone (HC) and an acute stressor on stress, innate immune responses and antioxidant system/oxidative stress responses of juvenile Piaractus mesopotamicus. Fish were either 1) given a commercial feed (C), 2) given a feed supplemented with 400 mg/kg HC, or 3) fed a commercial feed, chased for 2 min and exposed to air for 4 min (S). After initial sampling, fish C and HC were fed and sampled 1, 3, 6, 24 and 72 h post-feeding. Fish S were fed at the same time as the other groups, exposed to a stressor, and sampled 1, 3, 6, 24 and 72 h after. Exposure to the stressor increased circulating glucose and cortisol levels (at 1 and 3 h, respectively), while oral HC increased circulating cortisol at 1 h and glucose at 3 h. The stressor activated respiratory activity of leukocytes (RAL) at 3 h and reduced it at 6 h. HC did not activate RAL, but it did impair it at 6 h. The serum hemolytic activity of the complement system (HAC50) was impaired by the stressor at 1 and 3 h and by HC at 1 h. Regarding the antioxidant system, exposure to the stressor reduced glutathione peroxidase (GPx) and catalase (CAT) activity and decreased concentrations of reduced glutathione (GSH) in the liver up to 6 h. HC only impaired GPx. Additionally, stress induced the accumulation of melano-macrophage (MM) and melano-macrophage centers (MMC), which are biomarkers of oxidative stress, in the spleen. Differences in biomarkers in fish given cortisol and exposed to stress indicate that exogenous hormone was unable to precisely reproduce stress responses.

Vibration attraction response is a plastic trait in blind Mexican tetra (Astyanax mexicanus), variable within subpopulations inhabiting the same cave.

Animals evolve their sensory systems and foraging behaviours to adapt and colonize new and challenging habitats such as the dark cave environment. Vibration attraction behaviour (VAB) gives fish the ability to locate the source of a water disturbance in the darkness. VAB evolved in the blind Mexican cave tetra, Astyanax mexicanus. VAB is triggered in cavefish by vibration stimuli peaking at 35 Hz, which is within the main spectrum of water fluctuations produced by many prey crustaceans and insects. VAB has a genetic component and is correlated to an increased number of head mechanosensory neuromasts in the eye orbital region when compared to surface fish. Previous competitive prey capture assays have supported the advantage of VAB for foraging in the dark. Despite its putative adaptive function, VAB has been described as absent in some Astyanax cave populations (Tinaja and Molino) but present in others (Pachón, Piedras, Toro and Sabinos). Here we have tested the occurrence of VAB in the field and in multiple cave populations using a vibrating device in natural pools. Our results confirmed the presence of VAB in caves such as Pachón, Toro and Sabinos but showed that VAB is also present in the Tinaja and Molino cave populations, previously reported as VAB-negative in laboratory experiments. Thus, VAB is available throughout the range of hypogean A. mexicanus. However, and most notably, within a given cave the levels of VAB were highly variable among different pools. Fish at one pool may express no VAB, while fish at another nearby pool of the same cave may actively show VAB. While a variety of environmental conditions may foster this diversity, we found that individuals inhabiting pools with a high abundance of organic matter have reduced expression of VAB. In contrast, in pools with little organic debris where fish probably depend more on hunting than on scavenging, VAB is enhanced. Our results suggest that expression of VAB is a plastic trait whose variability can depend on local conditions. Such plasticity may be required within and among caves where high environmental variability between pools results in a diverse availability of food.

Daily variation feeding of Astyanax lacustris (Lütken 1875) in a subtropical river

This study aimed to know the daily variation of Astyanax lacustris (Lütken 1875) feeding in a lotic environment. Fish were caught with a net for three days every three hours in the summer to capture ten individuals per hour in a stretch of the Ijuí River, Middle Uruguay River, Brazil. The captured specimens had their stomachs removed and the content analyzed and separated into seven food categories with the aid of stereomicroscope. For the analysis of food items were used the frequency of occurrence methods, volumetric method and applied the Alimentary Index (IAi). Two hundred stomachs were analyzed, of which 95% had food content. The most abundant items were algae and autochthonous insects. Astyanax lacustris feeds throughout the day especially early in the morning (9 hours) and reduces its feeding at night (24 hours and 3 hours). It was also verified variation of AI of different food items throughout the day. It is concluded that A. lacustris feeds throughout the day, especially in the daytime and that throughout the 24 hours it varies its diet due to photoperiod and food availability.

Ecological impacts of pesticides on Astyanax jacuhiensis (Characiformes: Characidae) from the Uruguay river, Brazil.

Brazilian freshwater ecosystems are continuously exposed to pesticides and domestic sewage. The Uruguay River was chosen for this study because of its international importance, as it flows through Brazil, Argentina, and Uruguay. It receives contaminants such as pesticides and domestic residues. Thus, the aim of this study to assess the accumulation of pesticides in muscle of the fish Astyanax jacuhiensis, its biochemical responses, and the presence of pesticides in water. In total, seven pesticides were registered in water from both river sites. Eight pesticides were detected in fish muscle. The biochemical responses showed that brain lipid peroxidation (LPO) and protein carbonyl (PC) in A. jacuhiensis were higher in the summer. Muscle showed the highest LPO levels in the spring and the highest PC in the summer. Liver LPO and PC levels were higher in the spring and summer. In the gills, the PC was higher in the spring and the LPO in the spring and winter. In the brain and in the gills, glutathione-S-transferase activity was high in the summer and autumn. Catalase activity was lower during the winter and spring. Non-protein thiol (NPSH) levels were lower in the brain in the winter and spring. Muscle tissue showed lower NPSH in the winter (site 1). Liver NPSH showed increased levels in liver in the spring and winter (site 2). The biochemical results clearly is related to pesticides and/or to the presence of other contaminants in the water such as metals or domestic sewage. The accumulation of pesticides in fish muscle added evidence that pesticides have been used in the area surrounding the Uruguay River. In conclusion, the biomarkers assayed in the present study could be used in future investigations considering other sampling sites along Uruguay River.

Morphological malleability of the lateral line allows for surface fish (Astyanax mexicanus) adaptation to cave environments.

The lateral line is the primary modality fish use to create a hydrodynamic image of their environment. These images contribute to a variety of behaviors, from rheotaxis to escape responses. Here we discern the contributions of visual and lateral line modalities in hunting behavior of larvae that have developed under different photic conditions. In particular, cave animals have a hypertrophied sense of mechanosensation, and we studied the common animal model cavefish Astyanax mexicanus and its closest related surface relative. We raised larvae in a diurnal light-dark regimen and in complete darkness. We then examined the distribution of neuromasts in their lateral lines, and their hunting performance in light and dark conditions, with and without the contribution of the lateral line. We report that all larva depend on the lateral line for success in hunting and that surface fish raised in the dark have a greater dependency on the lateral line.

Molecular characterization of different preproGnRHs in Astyanax altiparanae (Characiformes): Effects of GnRH on female reproduction.

Gonadotropin-releasing hormone (GnRH) is a key molecule in the initiation of the hypothalamic-pituitary-gonadal axis. Thus, knowledge about GnRH may contribute to the effectiveness of species reproduction. Using a Neotropical tetra Astyanax altiparanae as a fish model species, the GnRH forms were characterized at the molecular level and the role of injected GnRHs in vivo was evaluated. The full-length complementary DNA (cDNA) sequences of preproGnRH2 (612 bp) and preproGnRH3 (407 bp) of A. altiparanae were obtained, and the GnRH1 form was not detected. The cDNA sequences of preproGnRH2 and preproGnRH3 were found to be conserved, but a change in the amino acid at position 8 of the GnRH3 decapeptide of A. altiparanae was observed. All the injected GnRHs stimulated lhß messenger RNA (mRNA) expression but not fshß mRNA expression, and only GnRH2 was able to increase maturation-inducing steroid (MIS) levels and possibly stimulate oocyte release. Furthermore, only GnRH2 was able to start the entire reproductive hormonal cascade and induce spawning.

Acute exposure to hyperosmotic conditions reduces sperm activation by urine in the yellowtail tetra Astyanax altiparanae, a freshwater teleost fish / Exposição aguda a condições hipereosmoticas reduz ativação do sêmen por urina em lambari do-rabo-amarelo (Astyanax altiparanae), um teleósteo de água doce

In freshwater fish with external fertilization, sperm sampling can be contaminated with urine, which triggers motility and gives rise to decreased fertilization success. The maintenance of freshwater fish in hyperosmotic conditions may reduce urine production and improve sperm quality. Thus, the aim of this work was to verify if acute exposure to various NaCl concentrations improves sperm quality in the yellowtail tetra Astyanax altiparanae. Spermiation was induced using a single dose of carp pituitary gland (5 mg kg-1) and the males were maintained at various NaCl concentrations: NaCl 0.00% (control), NaCl 0.45% (hypoosmotic), NaCl 0.9% (isosmotic) and NaCl 1.0% (hyperosmotic) for 6 h at 26 °C. Sperm was collected and verified for activation by urine and motility traits. At 0.00%, 0.45%, and 0.90%, the sperm was motile just after sampling, indicating activation by urine. Surprisingly, at hyperosmotic conditions, no activation was observed. Other sperm and motility parameters did not show any statistical differences, including sperm viability (P = 0.7083), concentration (P = 0.9030), total motility (P = 0.6149), VCL (curvilinear velocity; P = 0.1216), VAP (average path velocity; P = 0.1231) and VSL (straight-line velocity; P = 0.1340). Our results indicate that acute maintenance at hyperosmotic conditions eliminates sperm activation by urine and maintains sperm quality. Such a new procedure is interesting for both basic and applied sciences, including reproductive practice in fish.(AU)

Em peixes de água doce com fertilização externa, a amostragem de espermatozoides pode ser contaminada pela urina, o que desencadeia motilidade e gera menor sucesso na fertilização. A manutenção de peixes de água doce em condições hiperosmóticas pode reduzir a produção de urina e melhorar a qualidade do esperma. Assim, o presente trabalho foi delineado para verificar se a exposição aguda a várias concentrações de NaCl melhora a qualidade do esperma no tetra-amarelo Astyanax altiparanae. A espermiação foi induzida usando uma dose única de hipófise da carpa (5 mg kg-1) e os machos foram mantidos em várias concentrações de NaCl: NaCl 0,00% (controle), NaCl 0,45% (hipoosmótico), NaCl 0,9% (isosmótico) e NaCl 1,0% (hiperosmótico) por seis horas a 26 °C. O esperma foi colhido e verificado quanto à ativação por urina e traços de motilidade. Em 0,00%, 0,45%, 0,90% os espermatozóides eram móveis logo após a amostragem, indicando ativação pela urina. Surpreendentemente, em condições hiperosmóticas, nenhuma ativação foi observada. Outros parâmetros espermáticos e de motilidade não mostraram diferenças estatísticas, incluindo viabilidade espermática (P = 0,7083), concentração (P = 0,9030), motilidade total (P = 0,6149), VCL (Velocidade Curvilinear; P = 0,1216), VMD (Velocidade Média de Deslocamento; P = 0,1230) e VLR (Velocidade em linha Reta; P = 0,1340). Nossos resultados indicam que a manutenção aguda em condições hiperosmóticas elimina a ativação do esperma pela urina e mantém a qualidade do esperma. Esse novo procedimento é interessante para as ciências básicas e aplicadas, incluindo a prática reprodutiva em peixes.(AU)

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)

The use of evoked potentials to determine sensory sub-modality contributions to acoustic and hydrodynamic sensing.

Both the lateral line and the inner ear contribute to near-field dipole source detection in fish. The precise roles these two sensory modalities provide in extracting information about the flow field remain of interest. In this study, evoked potentials (EP, 30-200 Hz) for blind Mexican cavefish were measured in response to a dipole source. Greatest sensitivity was observed at the lower and upper ends of the tested frequency range. To evaluate the relative contributions of the lateral line and inner ear, we measured the effects of neomycin on EP response characteristics at 40 Hz, and used the vital dye DASPEI to verify neuromast ablation. Neomycin increased the latency of the EP response up until 60 min post-treatment. DASPEI results confirmed that neuromast hair cell death was significant in treated fish over this timeframe. These results indicate that the inner ear, whether it is sound pressure or particle motion detection, makes a significant contribution to the dipole-induced EP in blind cavefish at near-field low frequencies where the lateral line contribution would be expected to be strongest. The results from this study imply that under some circumstances, lateral line function could be complemented by the inner ear.

Maternally regulated gastrulation as a source of variation contributing to cavefish forebrain evolution.

Sequential developmental events, starting from the moment of fertilization, are crucial for the acquisition of animal body plan. Subtle modifications in such early events are likely to have major impacts in later morphogenesis, bringing along morphological diversification. Here, comparing the blind cave and the surface morphotypes of Astyanax mexicanus fish, we found heterochronies during gastrulation that produce organizer and axial mesoderm tissues with different properties (including differences in the expression of dkk1b) that may have contributed to cavefish brain evolution. These variations observed during gastrulation depend fully on maternal factors. The developmental evolution of retinal morphogenesis and hypothalamic patterning are among those traits that retained significant maternal influence at larval stages. Transcriptomic analysis of fertilized eggs from both morphotypes and reciprocal F1 hybrids showed a strong and specific maternal signature. Our work strongly suggests that maternal effect genes and developmental heterochronies that occur during gastrulation have impacted morphological brain change during cavefish evolution.

Effect of photoperiod on ovarian maturation in Cheirodon interruptus (Teleostei: Characidae) / Efeito do fotoperíodo na maturação dos ovários em Cheirodon interruptus (Teleostei: Characidae)

Abstract Cheirodon interruptus is a ubiquitous small characid that inhabits the Pampean region and commonly used as bait. Its vast market is supplied from the wild population causing a significant environmental impact. In this study, we assess the effect of photoperiod on ovarian maturation in order to evaluate its potential as a tool to manipulate reproduction under artificial conditions. Four treatments in triplicate (light: dark, L: D) were tested: 24L: 0D, 12L: 12D, 0L: 24D and a progressive photoperiod corresponding to the daily photoperiod increments in wild during winter-spring transition, accelerated three times. The experiment was conducted for 45 days. Gonadosomatic index (GSI), oocyte mean diameter, vitellogenic oocyte proportions, plasma estradiol concentrations (E2), condition factor (Kn) and standard length were estimated. Values of mean GSI, oocyte diameter, vitellogenic oocyte proportions and E2 concentration were maximum in the progressive treatment indicating vitellogenesis stimulation. In turn the same parameters were minimum in the 24L: 0D, revealing the vitellogenesis inhibition. This study showed that photoperiodic regime play an important role in the onset of ovarian maturation in C. interruptus.

Resumo Cheirodon interruptus é um pequeno caracídeo amplamente distribuído na região Pampeana e comumente usado como isca. Seu vasto mercado se abastece de populações silvestres causando um impacto ambiental significativo. Neste estudo, investigamos o efeito do fotoperíodo no amadurecimento do ovário para avaliar o potencial deste fator como ferramenta para manipular a reprodução em condições artificiais. Quatro tratamentos foram testados por triplicado (luz: escuro, L: D): 24L: 0D, 12L: 12D, 0L: 24D e um fotoperíodo progressivo que simula as mudanças das horas luz na natureza e na transição de inverno a primavera, acelerado três vezes. O experimento se realizou durante 45 dias. O índice gonadossomático (GSI), o diâmetro médio dos ovócitos, a proporção de ovócitos vitelogênicos, as concentrações plasmáticas de estradiol (E2), o fator de condição (Kn) e o comprimento padrão foram estimados. Os valores médios de GSI, o diâmetro dos ovócitos, as proporções de ovócitos vitelogênicos e a concentração de E2 foram maximizados no fotoperíodo progressivo, indicando estimulação de vitelogênesis. Por outro lado, esses mesmos parâmetros resultaram mínimos no tratamento 24L: 0D, revelando um efeito inibidor da vitelogênesis. Este estudo mostrou que as variações de fotoperíodo exercem um papel importante no início vitelogênesis reprodutiva em C. interruptus .

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.

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.

Neurotoxicity of organophosphate pesticides could reduce the ability of fish to escape predation under low doses of exposure.

Biomarkers are frequently used in ecotoxicology as they allow to study toxicant effects happening at low concentrations of exposure. However, most sublethal studies only evaluate cellular biomarkers which lack evident ecological relevance. We used a multibiomarker approach to estimate the toxic effects of ethoprophos, an organophosphate insecticide commonly used in banana plantations, on the tropical fish Astyanax aeneus (Characidae). We measured biomarkers at sub-individual (cellular) and individual (metabolism, behavior) levels and examined relationships among these responses. A sublethal exposure to ethoprophos caused a significant (54%) reduction of brain Cholinesterase (ChE) activity, reflecting the pesticide's high neurotoxicity. However, other biomarkers like oxidative stress, biotransformation reactions, and resting metabolic rate were not affected. Exposure to ethoprophos modified antipredator behaviors such as escape response and detection avoidance (light/dark preference): exposed fish escaped slower from a simulated attack and preferred brighter areas in a novel tank. The relationship between ChE activity and reaction time suggests that pesticide-induced ChE inhibition reduces escape ability in fish. Our results provide evidence that impacts of organophosphate pesticides on fish ecological fitness can occur even with short exposures at very low concentrations.

Behavioral Tracking and Neuromast Imaging of Mexican Cavefish.

Cave-dwelling animals have evolved a series of morphological and behavioral traits to adapt to their perpetually dark and food-sparse environments. Among these traits, foraging behavior is one of the useful windows into functional advantages of behavioral trait evolution. Presented herein are updated methods for analyzing vibration attraction behavior (VAB: an adaptive foraging behavior) and imaging of associated mechanosensors of cave-adapted tetra, Astyanax mexicanus. In addition, methods are presented for high-throughput tracking of a series of additional cavefish behaviors including hyperactivity and sleep-loss. Cavefish also show asociality, repetitive behavior and higher anxiety. Therefore, cavefish serve as an animal model for evolved behaviors. These methods use free-software and custom-made scripts that can be applied to other types of behavior. These methods provide practical and cost-effective alternatives to commercially available tracking software.

Thermal manipulation and GnRHa therapy applied to the reproduction of lambari-do-rabo-amarelo, Astyanax altiparanae females (Characiformes: Characidae) during the non-breeding season.

Lambari-do-rabo-amarelo Astyanax altiparanae in the wild reproduce during spring and summer, but females undergo vitellogenesis throughout the year, including the non-spawning winter period when water temperatures are low. The present study investigated the physiological role of temperature modulation on the hypothalamus-pituitary-gonads axis of lambari during winter, as well as the effects of gonadotropin releasing hormone agonist (GnRHa) therapy. Captive females were exposed to two different temperatures (20 °C and 27 °C) and were injected weekly with GnRHa for 21 days during winter (Control, CTR; Low dose; LD and high dose of GnRHa, HD). At the end of the 21-days period gonadosomatic index (GSI), oocyte stage of development and theoretical fecundity were evaluated, together with plasma levels of 17ß-estradiol (E2). Gene expression of the two pituitary gonadotropins follicle-stimulating hormone (fshß) and luteinizing hormone (lhß), as well as hepatic vitellogenin-A (vtgA) expression were also analyzed. At the end of the experimental period, females from the six different experimental conditions were induced to spawn using human chorionic gonadotropin (hCG). Spawning performance parameters and plasma levels of the maturation inducing steroid (MIS) were analyzed. Gene expression of fshß did not change with temperature manipulation, but females exposed to 27 °C and supplemented with a HD of GnRHa exhibited an increased fshß gene expression, associated with higher E2 levels. The higher water temperature alone was able to increase E2 levels. At both water temperatures GnRHa injections induced a decrease in E2 levels. GnRHa injected females had a lower vtgA gene expression levels at 20 °C. Even with differences in the gene expression of gonadotropins among the various temperature/GnRHa treatments, GSI and oocyte diameter did not change, but GnRHa enhanced the number of vitellogenic oocytes at 20 °C. The reproductive performance of lambari induced to spawn with hCG was better after the combined treatment with GnRHa and summer temperature.

Environmental exposure to oestrogenic endocrine disruptors mixtures reflecting on gonadal sex steroids and gametogenesis of the neotropical fishAstyanax rivularis.

Discharge of municipal wastewater promotes the entry of diverse oestrogenic compounds into the water bodies. This complex mixture of substances interferes in the steroidogenic pathway, being able to promote severe reproductive impairment in freshwater fish populations. The purpose of the present study was to evaluate the effects of oestrogenic endocrine disruptors (EDCs) mixture on gonadal sex steroids (testosterone, T; 11-ketotestosterone, 11-KT; 17ß-oestradiol, E2; 17-hydroxyprogesterone, 17-OHP) in the peak of the reproductive season of Astyanax rivularis, correlating the results obtained with the proportion of germ cells and gonadal histopathology. Three sampling sites were chosen to conduct the study, one reference site (S1), without contamination by municipal wastewater and two sites (S2 and S3) receiving discharge of municipal wastewater. Males of A. rivularis presented higher concentrations of E2, lower androgens (T and 11-KT) in gonads when compared to males from site S1. Concentrations of 17-OHP did not present significant difference among sites. In sites S2 and S3, the proportion of early spermatocytes, spermatids and Leydig cells increased while spermatozoa decreased compared to fish from S1. The following gonadal histopathologies were detected in the male fishes: intersex gonads (28% in S3) and testicular degeneration with germinal epithelium exhibiting agglutinated germ cells masses and empty cysts (57% in S2 and 71% in S3). In females, concentrations of T, E2 and 17-OHP did not present significant difference among the sites, however higher 11-KT concentrations were detected in females from sites S2 and S3. A lower proportion of perinucleolar follicles and a higher incidence of vitellogenic follicles, besides, aged oocytes and the presence of eosinophilic proteinaceous fluid in the interstitial compartment were also found in females from impacted sites. These results indicate that the urbanization and consequent release of municipal wastewater containing oestrogenic compounds in the headwater creeks are altering the levels of sex hormones and gametogenesis of A. rivularis. Further studies should be performed to determine whether oestrogenic endocrine disrupters are disrupting the reproduction of A. rivularis.

Reproductive biology of Hyphessobrycon eques (Characiformes: Characidae) in Southern Pantanal, Brazil / Biologia Reprodutiva de Hyphessobrycon eques (Characiformes: Characidae) no Pantanal Sul, Brasil

Abstract Population and reproductive aspects allow the knowledge and understanding of population dynamics and the influence of environmental factors, in addition to ensure the success of a species continuity. Thus, the aim of this study was to analyze population and reproductive traits of the species Hyphessobrycon eques in southern Pantanal, Brazil. Monthly samplings were conducted from February/2009 to January/2011, with 617 individuals analyzed, being 365 females and 262 males. A similar form of distribution in length and weight between the sexes was observed. There was no significant variation in sex ratio over time, with higher proportion of females during the entire sampled period. Both sexes presented a angular coefficient of Weight/Length (b) relationship greater than 3, with speed of increase in weight greater than in length. For females, a long reproductive period was observed, with greater reproductive intensity from January through June. No significant correlation was observed between the Gonadosomatic Index (GSI) and the average temperature, rainfall and river level, despite the fact that reproductive activity occurs in autumn/winter, when there are favorable conditions due to flooding. The size at first maturation (L50) was 20.2 mm, with confidence interval varying from 19.7 through 20.7 mm. The average fecundity was 191.9 oocytes/females and was significantly related to the standard length and total weight (g), demonstrating a relation with energy accumulation to invest in reproduction. The long reproductive period, intensified by partial spawning, higher proportion of females and low L50, show that the species has strategies necessary for survival and rapid population growth, common in small species characterized as r-strategists.

Resumo Os aspectos populacionais e reprodutivos permitem o conhecimento e a compreensão da dinâmica populacional e da influência de fatores ambientais, assegurando o sucesso da continuidade de uma espécie. Desta forma, o presente trabalho teve como objetivo analisar os traços populacionais e reprodutivos da espécie Hyphessobrycon eques no Sul do Pantanal, Brasil. Foram realizadas amostragens mensais de Fevereiro/2009 a Janeiro/2011, com um total de 617 indivíduos analisados, sendo 365 fêmeas e 262 machos. Foi observada que a distribuição foi similar em comprimento e peso entre os sexos. Não ocorreu variação significativa na proporção sexual ao longo do tempo, com maior proporção de fêmeas durante todo período amostrado. Ambos os sexos apresentaram um coeficiente angular da relação Peso/Comprimento (b) maior que 3, com velocidade do incremento em peso maior do que em comprimento. Para as fêmeas, foi observado um período reprodutivo longo, com maior intensidade reprodutiva de Janeiro e Junho. Não foi observada correlação significativa entre o Índice Gonadossomático (IGS) e a temperatura média, pluviosidade e nível do rio, apesar de ocorrer atividade reprodutiva no outono/inverno, período que há condições favoráveis decorrentes da inundação. O tamanho de primeira maturação (L50) foi de 20,2 mm, com intervalo de confiança variando entre 19,7 e 20,7 mm. A fecundidade média foi de 191,9 ovócitos/fêmea e esteve relacionada significativamente ao comprimento padrão e peso total (g), demonstrando uma relação com acúmulo de energia para investir na reprodução. O período reprodutivo longo, intensificado pela desova parcelada, maior proporção de fêmeas e baixo L50, mostram que a espécie possui estratégias necessárias para sobrevivência e rápido crescimento da população, comum em espécies de pequeno porte caracterizadas como r-estrategistas.