Analyses of the development and glycoproteins present in the ovarian follicles of Poecilia vivipara (Cyprinodontiformes, Poeciliidae)

The morphofunctional aspects of oogenesis of Poecilia vivipara were studied aiming to understand the reproductive biology and development of species with internal fertilization, particularly those belonging to the family Poeciliidae. The stages of gonadal maturation and follicular development were characterized using mesoscopic, histological, histochemical, and lectin cytochemical analyses. Through mesoscopic evaluation the ovarian development was classified in six phases of development: immature, in maturation I, in maturation II, mature I, mature II, and post-spawn. Based on microscopic examination of the ovaries, we identified the presence of oocytes types I and II during the previtellogenic phase and types III, IV, and V during the vitellogenic phase. As oogenesis proceeded the oocyte cytosol increased in volume and presented increased cytoplasmic granule accumulation, characterizing vitellogenesis. The zona radiata (ZR) increased in thickness and complexity, and the follicular epithelium, which was initially thin and consisting of pavimentous cells, in type III oocytes exhibited cubic simple cells. The histochemical and cytochemical analyses revealed alterations in the composition of the molecular structures that form the ovarian follicle throughout the gonadal development. Our study demonstrated differences in the female reproductive system among fish species with internal and external fertilization and we suggest P. vivipara can be used as experimental model to test environmental toxicity.

Effects of the 2,4-D herbicide on gills epithelia and liver of the fish Poecilia vivipara

The 2,4-dichlorophenoxyacetic acid, usually named 2,4-D is one of the most widely used herbicides in the world. Acute toxicity of 2,4-D herbicide was investigated through its effects on guppies (Poecilia vivipara Bloch et Schneider 1801). Fish were exposed to the herbicide at concentrations of 10, 20 and 40µl per liter of water for 24 hours to determine its effects on gills and liver epithelia. The estimated LC50 was 34.64µl of 2,4-D per liter of water. Histochemical analyses and Feulgen's reaction were conducted to detect glycoconjugates and DNA, respectively, in gills and liver epithelia. Histochemistry revealed qualitative variations of glycoconjugates present on mucous cells and granules. The four types of mucous cells contained neutral granules, acids, or both. Increasing amounts of syalomucins were observed from the control group to the group exposed to the highest concentration of 2,4-D, suggesting increased mucous viscosity and the formation of plaques that could inhibit gas exchange and osmoregulation. Lamellar fusion observed in the group exposed to 40µl of 2,4-D suggests a defense mechanism. Hepatocytes showed vacuolization in the 10 and 20µl/L groups. The 40 µl/L group showed normal hepatocytes as well as changed ones, many Ito cells, micronuclei, and nuclear swelling. These effects may be associated with toxicity or adaptative processes to cellular stress. The data from this study indicates the importance of assessing similar risks to aquatic species and suggests that Poecilia vivipara is an adequate biological model for analysis of environmental contamination.

Embryonic development and nourishment in the viviparous fish Poecilia vivipara (Cyprinodontiformes: Poeciliidae)

While viviparity confers protection to the embryos during gestation, it increases energetic costs for the mother, which acquires new relations to its offspring. Maternal-fetal transfer of nutrients can occur in different patterns: as lecithotrophy (nourished by yolk) or matrotrophy (nourished by the mother). The development of Poecilia vivipara embryos was described macroscopically and microscopically, and the form of nutritional provisioning was identified. Embryonic development was divided into three prefertilization and seven postfertilization stages. The first organ to appear is the notochord, followed by the nervous, digestive and cardiovascular systems, and then by muscles and eyes. Embryonic nutritional provisioning was lecithotrophic, with yolk persisting until the last developmental stages and rich in proteins and polysaccharides. This kind of embryonic nutrition confirms the pattern found in the family Poeciliidae.

Evolução do ciclo vital de Poecilia vivipara (Cyprinodontiformes: Poeciliidae) em um gradiente de predação

O presente estudo teve como objetivo testar previsões da teoria sobre a influência da predação na evolução dos ciclos vitais. Como modelo de estudo, foram estudadas populações do peixe Poecilia vivipara que habitam o Parque Nacional da Restinga de Jurubatiba, no sistema lagunar Norte-Fluminense, estado do Rio de Janeiro. Neste sistema lagunar, P. vivipara está sujeita à predação por peixes piscívoros (e.g. a traíra Hoplias malabaricus) em parte das lagoas, mas está livre destes predadores em outras lagoas. Foram estudadas três lagoas em que P. vivipara coexiste com seus predadores piscívoros e três em que estes estão ausentes. A ictiofauna das seis lagoas foi amostrada através de redes de arrasto e espinhel. Em laboratório, as fêmeas e os machos de P. vivipara foram dissecados para remoção de suas gônadas. As gônadas das fêmeas foram categorizadas em seis diferentes estágios de desenvolvimento. A partir do terceiro estágio, os embriões foram contados e medidos (maior diâmetro). Os exemplares de P. vivipara e suas gônadas foram secos em estufa a 60ºC e pesados em balança analítica (0,01 mg). Os aspectos do ciclo vital analisados foram: (1) investimento reprodutivo das fêmeas (massa gonadal/massa total da fêmea); (2) índice gonadossomático dos machos (massa gonadal/massa total do macho); (3) fecundidade (número de embriões); (4) comprimento médio dos embriões; e (5) massa seca média dos embriões (massa gonadal/fecundidade). O efeito da predação foi testado com uma ANCOVA aninhada, com regime de predação (alta intensidade, baixa intensidade), população de origem (aninhada dentro do regime de predação) e coleta como variáveis independentes, e massa somática e estágio embrionário como covariáveis. De acordo com o previsto pela teoria, as fêmeas dos ambientes de baixa predação apresentaram embriões maiores, mas, contrariando as previsões teóricas, também apresentaram maior fecundidade e...

Body size and allometric shape variation in the molly Poecilia vivipara along a gradient of salinity and predation

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Variação morfológica de Poecilia vivipara em um gradiente de predação nop litoral norte fluminense

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Caracterização transcricional dos genes CTR1 e ATP7B no peixe Poecilia vivipara: efeitos do cobre e da salinidade

Em peixes, o cobre (Cu) é absorvido a partir da água, via branquial, e pela ingestão de água e alimento, via gastrintestinal. Para evitar reações não específicas prejudiciais e suprir proteínas dependentes de Cu, existem transportadores específicos, como as proteínas de absorção de alta afinidade ao Cu (CTR1) e as Cu-ATPases (ATP7), que auxiliam na translocação intracelular do metal. No presente estudo, os genes CTR1 e ATP7B foram identificados em Poecilia vivipara e os seus transcritos foram quantificados por RT-qPCR nas brânquias, no fígado e no intestino de guarús expostos (96 h) ao Cu (0, 5, 9 e 20 µg/L) em água doce e salgada (salinidade 24). Foram identificadas novas sequências nucleotídicas dos genes CTR1 (1560 pb, completa) e ATP7B (617 pb, parcial), as quais tiveram altos valores de identidade com as descritas para Fundulus heteroclitus (CTR1=81%) e Sparus aurata (ATP7B=81%). A análise por RT-qPCR indicou níveis de transcrição para CTR1 e ATP7B em todos os tecidos analisados. Em guarús na água doce, a maior expressão da CTR1 e da ATP7B se deu no fígado. Em guarús na água salgada, a maior expressão da CTR1 ocorreu no intestino, enquanto a da ATP7B se deu no fígado e intestino. Na água doce, a exposição ao Cu aumentou o conteúdo branquial e hepático de Cu, diminuiu os transcritos de CTR1 e ATP7B nas brânquias e aumentou os transcritos destes genes no fígado, sem alterar o conteúdo corporal de Cu. Na água salgada, a exposição ao Cu aumentou o conteúdo de Cu e diminuiu o transcrito de ATP7B no intestino, sem alterar o conteúdo corporal de Cu nos P. vivipara. Estes resultados indicam que a homeostasia do Cu em P. vivipara envolve a redução da expressão do CTR1 e ATP7B nas brânquias (água doce) e intestino (água salgada) para limitar a absorção do Cu e o aumento da expressão destes genes no fígado (água doce) para facilitar o armazenamento e desintoxicação do Cu.

Identificação e expressão de genes relacionados à via esteroidogênica e resposta antioxidante após a exposição à Atrazina em Poecilia vivipara (Poeciliidae, Cyprinodontiformes)

O herbicida Atrazina (ATR) é um agrotóxico utilizado há cerca de 50 anos, responsável pelo controle seletivo de plantas daninhas em cultivo de arroz, milho e cana-de-açúcar, principalmente. Estudos recentes apontam diversos efeitos desse herbicida em invertebrados e vertebrados, através da contaminação do solo, bem como da lixiviação para os ecossistemas aquáticos. Foi demonstrado que a ATR é um desregulador endócrino, além de causar efeitos como estresse oxidativo, imunotoxicidade e distúrbios no metabolismo energético. No presente estudo, a espécie nativa Poecilia vivipara foi utilizada como modelo experimental para identificar e analisar a expressão de genes atuantes na via esteroidogênica (StAR e Cyp19a1) e genes atuantes no sistema de defesa antioxidante enzimático (SOD-1 e CAT), frente a exposição à diferentes concentrações de ATR. Sequências parciais dos genes-alvo foram obtidas e comparadas com sequências disponíveis de espécies próximas. Foram analisadas a expressão órgãoespecífica para cada um dos genes isolados, bem como a expressão dos genes frente à exposição ao herbicida atrazina. Os animais foram expostos a ATR em concentrações de 2, 10 e 100 µg/L e a expressão dos genes em gônadas e fígado desses animais foram analisadas em 24 e 96 horas de exposição. As sequências obtidas dos genes StAR, Cyp19a1, SOD-1 e CAT apresentaram 821, 80, 954, 350 pares de bases respectivamente, com identidades que variam de 86 a 100% com espécies filogeneticamente próximas a P. vivipara. Os animais apresentaram uma maior expressão dos genes StAR e Cyp19a1 nas gônadas e no fígado, enquanto a menor expressão se mostrou em órgãos como intestino e baço. Já os genes SOD e CAT apresentaram uma maior expressão no fígado, e menor expressão no intestino. Em relação à expressão gênica frente à exposição à ATR, os resultados apontaram para uma indução dos genes StAR, SOD e CAT em 24 horas, nas gônadas e no fígado, enquanto 8 que a expressão do gene Cyp19a1 foi aumentada apenas após 96 horas de exposição. Foi demonstrado que o herbicida ATR, mesmo em baixas concentrações, é capaz de desregular a expressão de genes que codificam tanto para proteínas componentes da via de síntese de hormônios esteróides, quanto para enzimas atuantes na resposta antioxidante celular de P. vivipara. 

Ichthyofauna of Rio Jurubatuba, Santos, São Paulo: a high diversity refuge in impacted lands

Ichthyofaunistic surveys in the Atlantic Rainforest have been published in relatively few works, in spite of the major biological importance of this once vast biome which is rapidly vanishing due to disordered human population growth and natural resources overexploitation. The present study aimed to access the fish fauna of a relatively well preserved basin between the cities of Santos and Cubatão (SP), an area highly modified by human activities where recent ichthyofaunistic surveys are still missing. Collections were made during three field trips in Rio Jurubatuba, a medium sized costal river, and Riacho Sabão, one of its main tributaries. A total of 2773 specimens were sampled, representing 25 species from 14 families. Six species were primary marine using the upper reaches of Rio Jurubatuba. Twelve of the 19 freshwater species are endemic of the Atlantic Rainforest and four are present in regional lists of endangered species. Only five species occurred in both Rio Jurubatuba and Riacho Sabão. The most diverse family was Characidae, followed by Poeciliidae, Rivulidae and Heptapteridae. Phalloceros caudimaculatus was the most abundant species, followed by Poecilia vivipara and Geophagus brasiliensis. The study area is considered well preserved and due to its critical location, urges for conservation policies to protect its fish diversity.

Diversidade das assembléias de peixes nas quatro unidades geográficas do rio Paraíba do Sul

Padrões na diversidade de peixes ao longo de quatro unidades geográficas (superior, médio-superior, médio-inferior e inferior) no rio Paraíba do Sul foram estudados com a finalidade de avaliar tendências de variações espaciais (unidades geográficas) e temporais (períodos de cheia e seca). Vinte e cinco locais foram amostrados entre Dezembro de 2002 e Março de 2003 (verão/cheia) e, entre Agosto e Novembro de 2003 (inverno/seca). Os peixes foram capturados com um esforço padronizado, utilizando redes de espera, tarrafas e peneiras. Um total de 81 espécies foram registradas compreendendo 9 ordens, 29 famílias e 55 gêneros. Characiformes apresentaram maior número de espécies (28) seguido de Siluriformes (23). Perciformes, principalmente Tilapia rendalli e Geophagus brasiliensis, e Cyprinodontiformes com destaque para Poecilia vivipara e Poecilia reticulata, foram os grupos numericamente mais abundantes, enquanto Siluriformes, principalmente Hypostomus luetkeni, e Perciformes com Geophagus brasiliensis apresentaram maior contribuição em biomassa. Poecilia vivipara foi encontrada apenas no período de seca. Espacialmente, Hoplosternum littorale predominou no trecho médio-superior, Pimelodus fur, Hypostomus luetkeni, Glanidium albescens no trecho médio-inferior, e Loricariichtys spixii e Prochilodus lineatus no trecho inferior. O número de espécies e a riqueza de Margalef apresentaram um aumento do trecho superior para o inferior, principalmente durante o período de cheia. Espécies que apresentaram ampla distribuição ao longo do rio (G. brasiliensis, Oligosarcus hepsetus e P. reticulata) são consideradas oportunistas por se aproveitarem dos recursos disponíveis em ambientes pobres, refletindo o estado de alterações do rio. Diferenciações na fisiografia ao longo da extensão longitudinal da bacia não coincidiram com mudanças nas assembléias de peixes, sugerindo que fatores associados a alterações de hábitats poderiam estar estruturando a comunidade de peixes a nível local.

The life history of Pygidiopsis macrostomum Travassos, 1928 (Digenea: Heterophyidae)

The life history of the trematode Pygidiopsis macrostomum Travassos, 1928 is described for the first time. Rediae and cercariae were obtained from naturally infected snails Heleobia australis (d´Orbigny), a new first intermediate host. Metacercariae were found encysted in the mesenteries of three naturally infected guppies, Phalloptychus januarius (Hensel), Jenynsia multidentata (Jenyns) (new host records) and Poecilia vivipara Bloch and Schneider. Experimental infections were successfully completed in the intermediate hosts H. australis and Poe. vivipara reared in the laboratory and hamsters Mesocricetus auratus Waterhouse were utilised as a definitive host.

Intraspecific ecomorphological variations in Poecilia reticulata (Actinopterygii, Cyprinodontiformes): comparing populations of distinct environments

ABSTRACT Morphological variations, according to the principles of ecomorphology, can be related to different aspects of the organism way of life, such as occupation of habitats and feeding behavior. The present study sought to examine the intraspecific variation in two populations of Poecilia reticulata Peters, 1859, that occur in two types of environments, a lotic (Maringá Stream) and a lentic (Jaboti Lake). Due to a marked sexual dimorphism, males and females were analyzed separately. Thus, the proposed hypotheses were that the populations that occur in distinct environments present morphological differences. The morphological variables were obtained using morphometric measurements and the ecomorphological indexes. The data were summarized in a Principal Component Analysis (PCA). A Multivariate Analysis of Variance (Manova) was made to verify significant differences in morphology between the populations. Males and females showed similar ecomorphological patterns according to the environment they occur. In general the population from Maringá Stream had fins with major areas, and the Jaboti Lake population eyes located more dorsally. Additionally, others morphological differences such as wider mouth of the males from Maringá Stream, wider heads on Jaboti Lake females and more protractible mouths on males from Jaboti Lake suggest a set of environmental variables that can possibly influence the ecomorphological patterns of the populations, as the water current, availability of food resources and predation. In summary, the initial hypotheses could be confirmed, evidencing the occurrence of distinct ecomorphotypes in the same species according to the environment type.

Monophyletic origin of multiple clonal lineages in an asexual fish (Poecilia formosa).

Despite the advantage of avoiding the costs of sexual reproduction, asexual vertebrates are very rare and often considered evolutionarily disadvantaged when compared to sexual species. Asexual species, however, may have advantages when colonizing (new) habitats or competing with sexual counterparts. They are also evolutionary older than expected, leaving the question whether asexual vertebrates are not only rare because of their 'inferior' mode of reproduction but also because of other reasons. A paradigmatic model system is the unisexual Amazon molly, Poecilia formosa, that arose by hybridization of the Atlantic molly, Poecilia mexicana, as the maternal ancestor, and the sailfin molly, Poecilia latipinna, as the paternal ancestor. Our extensive crossing experiments failed to resynthesize asexually reproducing (gynogenetic) hybrids confirming results of previous studies. However, by producing diploid eggs, female F(1) -hybrids showed apparent preadaptation to gynogenesis. In a range-wide analysis of mitochondrial sequences, we examined the origin of P. formosa. Our analyses point to very few or even a single origin(s) of its lineage, which is estimated to be approximately 120,000 years old. A monophyletic origin was supported from nuclear microsatellite data. Furthermore, a considerable degree of genetic variation, apparent by high levels of clonal microsatellite diversity, was found. Our molecular phylogenetic evidence and the failure to resynthesize the gynogenetic P. formosa together with the old age of the species indicate that some unisexual vertebrates might be rare not because they suffer the long-term consequences of clonal reproduction but because they are only very rarely formed as a result of complex genetic preconditions necessary to produce viable and fertile clonal genomes and phenotypes ('rare formation hypothesis').

Dietary lipids reduce the expression of carotenoid-based coloration in Lacerta vivipara

1. The importance of dietary lipids for carotenoid-based ornaments has rarely been investigated, although theory predicts that dietary lipids may control the development of these widespread animal signals. Dietary lipids have been suggested to enhance the expression of male carotenoid-based ornaments because they provide carotenoids with a hydrophobic domain that facilitates their absorption and transport. Dietary lipids may also enhance the uptake of tocopherols (vitamin E), which share common absorption and transport routes with carotenoids. Here, we test whether dietary lipids enhance carotenoid availability and male carotenoid-based colorations. We also explore the effects of dietary lipids on plasma tocopherol concentration, which allow disentangling between different pathways that may explain how dietary lipids affect ornamental expression. 2. Following a two-factorial design, we manipulated dietary access of naturally occurring fatty acids (oleic acid) and carotenoids (lutein and zeaxanthin) and measured its effects on the circulating concentrations of carotenoids (lutein and zeaxanthin) and vitamin E (α- and γ-(β-) tocopherols) and on the ventral, carotenoid-based coloration of male common lizards (Lacerta vivipara). 3. Lutein but not zeaxanthin plasma concentrations increased with carotenoid supplementation, which, however, did not affect coloration. Lipid intake negatively affected circulating concentrations of lutein and γ-(β-) tocopherol and led to significantly less orange colorations. The path analysis suggests that a relationship between the observed colour change and the change in plasma concentrations of γ-(β-) tocopherol may exist. 4. Our study shows for the first time that dietary lipids do not enhance but reduce the intensity of male carotenoid-based ornaments. Although dietary lipids affected plasma carotenoid concentration, its negative effect on coloration appeared to be linked to lower vitamin E plasma concentrations. These findings suggest that a conflict between dietary lipids and carotenoid and tocopherol uptake may arise if these nutrients are independently obtained from natural diets and that such conflict may reinforce signal honesty in carotenoid-based ornaments. They also suggest that, at least in the common lizard, sexual selection with respect to carotenoid-based coloration may select for males with low antioxidant capacity and thus for males of superior health.

Corticosterone regulates multiple colour traits in Lacerta [Zootoca] vivipara males.

Ornamental colours usually evolve as honest signals of quality, which is supported by the fact that they frequently depend on individual condition. It has generally been suggested that some, but not all types of ornamental colours are condition dependent, indicating that different evolutionary mechanisms underlie the evolution of multiple types of ornamental colours even when these are exhibited by the same species. Stress hormones, which negatively affect condition, have been shown to affect colour traits based on different pigments and structures, suggesting that they mediate condition dependence of multiple ornament types both among and within individuals. However, studies investigating effects of stress hormones on different ornament types within individuals are lacking, and thus, evidence for this hypothesis is scant. Here, we investigated whether corticosterone mediates condition dependence of multiple ornaments by manipulating corticosterone levels and body condition (via food availability) using a two-factorial design and by assessing their effect on multiple colour traits in male common lizards. Corticosterone negatively affected ventral melanin- and carotenoid-based coloration, whereas food availability did not affect coloration, despite its significant effect on body condition. The corticosterone effect on melanin- and carotenoid-based coloration demonstrates the condition dependence of both ornaments. Moreover, corticosterone affected ventral coloration and had no effect on the nonsexually selected dorsal coloration, showing specific effects of corticosterone on ornamental ventral colours. This suggests that corticosterone simultaneously mediates condition dependence of multiple colour traits and that it therefore accounts for covariation among them, which may influence their evolution via correlational selection.