The phylogeography of salmonid proliferative kidney disease in Europe and North America

Salmonid proliferative kidney disease (PKD) is caused by the myxozoan Tetracapsuloides bryosalmonae. Given the serious and apparently growing impact of PKD on farmed and wild salmonids, we undertook a phylogeographic study to gain insights into the history of genealogical lineages of T. bryosalmonae in Europe and North America, and to determine if the global expansion of rainbow trout farming has spread the disease. Phylogenetic analyses of internal transcribed spacer 1 sequences revealed a clade composed of all North American sequences plus a subset of Italian and French sequences. High genetic diversity in North America and the absence of genotypes diagnostic of the North American clade in the rest of Europe imply that southern Europe was colonized by immigration from North America; however, sequence divergence suggests that this colonization substantially pre-dated fisheries activities. Furthermore, the lack of southern European lineages in the rest of Europe, despite widespread rainbow trout farming, indicates that T. bryosalmonae is not transported through fisheries activities. This result strikingly contrasts with the commonness of fisheries-related introductions of other pathogens and parasites and indicates that fishes may be dead-end hosts. Our results also demonstrate that European strains of T. bryosalmonae infect and induce PKD in rainbow trout introduced to Europe.

The effects of stocking density in physiological parameters and growth of the endangered teleost species piabanha, Brycon insignis (Steindachner, 1877)

This study investigated the effects of stocking density on the growth and fatty acid (FA) of Brycon insignis metabolism. Fingerlings (360) were distributed into eight ponds at two stocking densities (105 and 210 g/m(3)). The analysis of growth showed that the condition factor (K) and the coefficient of variation (CV) for body mass were not affected by stocking density. However, final body mass and length, specific growth rate (SGR), and weight gain (WG) were higher in the low stocking density group, which also presented a higher feed efficiency (FE) and survival (S). By contrast, muscle protein levels were higher in the high stocking density group. The plasma and muscle lipid content were not affected by stocking density, but fish reared at lower stocking density presented higher lipid concentration in the liver, with no differences in hepatosomatic index values. Even with the differences observed in metabolic and growth parameters, plasma cortisol was not affected by stocking density. The FA profile in the muscle and liver neutral fraction were not affected by stocking density, but the FA in the polar fractions differed between the two stocking densities. In the liver, total polyunsaturated fatty acids (PUFA) and PUFA n - 3 increased in higher stocking density, mainly due to an increase in docosahexaenoic acid (DHA). In addition, PUFA n - 6 were also increased in the higher stocking density group, mainly due to an increase in arachidonic acid (AA) and docosadienoic acid (22:2n - 6). In the muscle polar fraction, the saturated fatty acids (SFA) and monounsaturated fatty acids (MUFA) decreased in the animals from the higher stocking density group, and this reduction was compensated by an increase in PUFA n - 3 and PUFA n - 6, mainly the FA with 20-22 carbons (20:4n - 6: 22:4:n - 6; 22:5n - 6, 22:5n - 3, and 22:6n - 3). A different profile was observed for the C18 PUFAs, mainly 18:2n - 6 and 18:4n - 6, which were higher in the lower density stocking group. The data suggest that when living in high stocking density, B. insignis differentially utilizes the hepatic lipids as energy source and remodels the membrane fatty acids, with higher amounts of DHA in the polar muscle fraction compensated for by a decrease in MUFA. The zootechnical and physiological indices reveal that the lower stocking density group achieve overall better performance. (C) 2010 Elsevier B.V. All rights reserved.

Aluminum as an endocrine disruptor in female Nile tilapia (Oreochromis niloticus)

The effects of aluminum on plasma ion, lipid, protein and steroid hormone concentration were evaluated in Oreochromis niloticus broodstock females. Lipid and protein concentrations from the gonads and liver were also measured Experiments were performed at neutral and acidic water pH Four groups of fish were tested for 96 h. 1) control conditions at neutral water pH, 2) control conditions at acidic water pH (CTR-Ac). 3) aluminum at neutral water pH (Al-N), and 4) aluminum at acidic water pH (Al-Ac) Aluminum and acidic water pH exposure caused no ionoregulatory disturbances Total lipid concentration increased in the mature gonads and decreased in the liver, suggesting an acceleration of lipid mobilization to the ovaries in animals exposed to aluminum However, a decreased protein concentration in ovaries was also observed Exposure of control fish to acidic water pH caused an increased concentration of plasma 17 alpha-hydroxyprogesterone However, females exposed to aluminum at acidic water pH showed a decreased of plasma 17 alpha-hydroxyprogesterone and cortisol. No differences in plasma 17 beta-estradiol were observed The physiological mechanisms underlying the disturbances observed are discussed focusing on reproduction We suggest that aluminum can be considered an endocrine disrupting compound in mature O. mloticus females (C) 2010 Elsevier Inc. All rights reserved

Gene Mapping of 18S and 5S rDNA Genes in the Karyotype of the Three-Spot Gourami Trichogaster trichopterus (Perciformes, Osphronemidae)

Ornamental fish culture is important as an economic activity and for biodiversity conservation as well. The species of the genus Trichogaster (Perciformes, Osphronemidae), popularly known as three-spot gourami, are among the several commercial species raised around the world. In the present work, eight specimens of Thrichogaster trichopterus from aquarium trade facilities were analyzed. The karyotype was composed of 23 pairs of subtelo/acrocentric chromosomes. Fluorescent in situ hybridization allowed identifying the 18S ribosomal gene at telomeric region on long arms of the largest acrocentric pair. On the other hand, the 5S rRNA gene is located at a proximal region on a pair of medium-sized chromosomes. Such information is extremely useful in face of the risks of introduction and the development of ornamental fish trade, once many fish species can be identified only by genetic studies.

(65)Zn(2+) transport by isolated gill epithelial cells of the American lobster, Homarus americanus

Gills are the first site of impact by metal ions in contaminated waters. Work on whole gill cells and metal uptake has not been reported before in crustaceans. In this study, gill filaments of the American lobster, Homarus americanus, were dissociated in physiological saline and separated into several cell types on a 30, 40, 50, and 80% sucrose gradient. Cells from each sucrose solution were separately resuspended in physiological saline and incubated in (65)Zn(2+) in order to assess the nature of metal uptake by each cell type. Characteristics of zinc accumulation by each kind of cell were investigated in the presence and absence of 10 mM calcium, variable NaCl concentrations and pH values, and 100 mu M verapamil, nifedipine, and the calcium ionophore A23187. (65)Zn(2+) influxes were hyperbolic functions of zinc concentration (1-1,000 mu M) and followed Michaelis-Menten kinetics. Calcium reduced both apparent zinc binding affinity (K (m)) and maximal transport velocity (J (max)) for 30% sucrose cells, but doubled the apparent maximal transport velocity for 80% sucrose cells. Results suggest that calcium, sodium, and protons enter gill epithelial cells by an endogenous broad-specificity cation channel and trans-stimulate metal uptake by a plasma membrane carrier system. Differences in zinc transport observed between gill epithelial cell types appear related to apparent affinity differences of the transporters in each kind of cell. Low affinity cells from 30% sucrose were inhibited by calcium, while high affinity cells from 80% sucrose were stimulated. (65)Zn(2+) transport was also studied by isolated, intact, gill filament tips. These intact gill fragments generally displayed the same transport properties as did cells from 80% sucrose and provided support for metal uptake processes being an apical phenomenon. A working model for zinc transport by lobster gill cells is presented.

Comparison of fatty acid composition in nine organs of the sympatric Antarctic teleost fish species Notothenia coriiceps and Notothenia rossii (Perciformes: Nototheniidae)

Fatty acid (FA) composition of nine organs from two closely related Antarctic fish species, Notothenia codiceps and Notothenia rossii, was determined through gas chromatography with flame ionization detection. A data set for each species was obtained using major FA profiles from specimens caught in the sea waters of Admiralty Bay during the summer season. The FA profiles for both species are overall similar, but organ peculiarities have been found, which could reflect metabolic specificities and feeding habits between species. With the exception of liver, the most abundant FA in organs was the n-3 polyunsaturated FA. The total n-6 polyunsaturated FAs were minor components in all evaluated organs. Palmitic acid was identified as the major saturated FA, whereas oleic acid was the most represented of the monounsaturated FA in almost all assessed organs of both species. The n-3/n-6 ratios of all organs were higher than 3.5. Differences in individual FA and FA metabolic profiles of some organs observed between N. coriiceps and N. rossii suggest specific requirements in the mobilization, transport, incorporation, and/or catabolism of lipids that were reinforced by differences on some FA ratios expressing the activity coefficient of enzymes implicated on the FA pathway flux. (C) 2009 Elsevier Inc. All rights reserved.

The combined effect of copper and low pH on antioxidant defenses and biochemical parameters in neotropical fish pacu, Piaractus mesopotamicus (Holmberg, 1887)

Copper sulfate is widely used in aquaculture. Exposure to this compound can be harmful to fish, resulting in oxidative metabolism alterations and gill tissue damage. Pacu, Piaractus mesopotamicus, (wt = 43.4 +/- A 3.35 g) were distributed in experimental tanks (n = 10; 180 l) and exposed for 48 h to control (without copper addition), 0.4Cu (0.4 mg l(-1)), 0CupH (without copper addition, pH = 5.0) and 0.4CupH (0.4 mg l(-1), pH = 5.0). In liver and red muscle, the superoxide dismutase (SOD) was responsive to the increases in the aquatic copper. The plasmatic intermediary metabolites and hematological variables in the fish of group 0.4Cu were similar to those of the control group. Conversely, the exposure to 0.4CupH caused an increase in the plasmatic lactate, number of red blood cells (RBC) and hemoglobin (Hb). Plasmatic copper concentration [Cu(p)] increased in group 0.4Cu and 0.4CupH, which is higher in group 0.4CupH, suggests an effect of water pH on the absorbed copper. Exposure to 0.4Cu and 0.4CupH resulted in a reduction in the Na(+)/K(+)-ATPase activity and an increase in metallothionein (MT) in the gills. Exposure to 0CupH caused a decrease in glucose and pyruvate concentrations and an increase in RBC, Hb, and the branchial Na(+)/K(+)-ATPase activity. These responses suggest that the fish triggered mechanisms to revert the blood acidosis, save energy and increase the oxygen uptake. MT was an effective biomarker, responding to copper in different pHs and dissolved oxygen. Combined-factors caused more significant disturbance in the biomarkers than single-factors.

Introdução da truta arco-íris (Oncorhynchus mykiss) e suas conseqüências para a comunidade aquática dos rios de altitude do sul do Brasil

A introdução de espécies exóticas é uma prática que acompanha a história da humanidade, sendo as espécies introduzidas a base da nutrição e economia em vários países. Porém, esse processo contínuo tem levado a uma homogeneização da flora e fauna global. Os ecossistemas aquáticos, que têm recebido menor atenção que os terrestres, têm sofrido perda de diversidade, hibridação, introdução de patógenos, degradação do habitat além da necessidade e alto custo de controle das espécies introduzidas. O desenvolvimento da aqüicultura tem acelerado a introdução de peixes exóticos, que são a base dessa produção em países como o Brasil. Além desta, entre as razões para a introdução citam-se o esporte, a manipulação ecológica com controle de organismos indesejáveis, o melhoramento dos estoques, a ornamentação ou ainda introduções acidentais. No Rio Grande do Sul, nos rios dos Campos de Cima da Serra estão ocorrendo introduções da espécie exótica truta arco- íris (Oncorhynchus mykiss) desde meados da década de 90 como forma de estímulo ao turismo rural. O objetivo dessa prática é a pesca esportiva, que atrai turistas de várias regiões. Porém, os rios da região possuem várias espécies de peixes endêmicas e as conseqüências dessa prática sobre a biota aquática são desconhecidas. O objetivo do presente trabalho é avaliar o efeito da introdução da truta arco-íris sobre o ecossistema de rios de baixa ordem no município de São José dos Ausentes – RS, Brasil. Para tanto foi descrita a biologia da truta arco- íris no novo ambiente, verificando sua alimentação, movimentação, presença e viabilidade da reprodução. A ictiofauna autóctone dos rios com ausência e presença de trutas foi comparada, bem como o efeito da predação sobre a macrofauna bentônica. A ictiofauna foi amostrada com o uso da pesca elétrica, sendo o conteúdo estomacal das trutas avaliado sazonalmente. Três trutas foram marcadas e acompanhadas por ii biotelemetria para determinação da sua área de vida. Um experimento com ninhos artificiais foi conduzido para verificação da sobrevivência de ovos de trutas nas condições dos rios de São José dos Ausentes. O experimento de exclusão de peixes foi feito a fim de avaliar o efeito da predação sobre a macrofauna bentônica, comparando a macrofauna em rios com e sem trutas. Os resultados indicaram que a truta arco- íris tem como alimento principal os invertebrados bentônicos, porém as maiores classes de tamanho incluem peixes em seus alimentos principais. Logo após sua introdução a movimentação é restrita, porém alguns exemplares foram capturados em um rio onde não foi feita introdução dessa espécie. Apesar de baixa, a sobrevivência dos adultos ocorre, assim como a reprodução. A sobrevivência dos ovos também é baixa, porém, uma pós-larva foi encontrada. A ictiofauna autóctone é diferenciada nos rios com presença de truta, apresentando menor riqueza e diversidade e uma tendência a diminuição da biomassa. A macrofauna bentônica também é diferenciada, apesar de não ser possível atribuir essa diferença somente à presença das trutas.

SEX-CHROMOSOME POLYMORPHISM AND HETEROGAMETIC MALES REVEALED BY 2 CLONED DNA PROBES IN THE ZW ZZ FISH LEPORINUS-ELONGATUS

In order to study the divergence of teleost sex chromosomes, subtractive cloning was carried out between genomic DNA of males and females of the rainbow trout (XX/XY) and of Leporinus elongatus (ZW/ZZ). Inserts cloned in a plasmid vector were individually tested on Southern blots of DNA of males and females for sex specificity. No sex-specific insert was obtained from trout, but two out of ten inserts cloned from L. elongatus showed sex-specific patterns in this species: one corresponds to a sequence present on both Z and W chromosomes, while the other is W specific. Sequences of these two inserts show neither clear homology with other known sequences, nor an open reading frame. They cross-hybridize with the genomic DNA of Leporinus friderici, but without sex-specific patterns. Twenty-four L. elongatus adults were sexed by gonadal observation, chromosomed examination and Southern hybridization with one or the other insert. Ten males and 11 females had chromosomes and hybridization patterns typical of their sex. One ZW female was recognized as a male with the W-specific probe. This was also the case for two unusual ZW males, one having a male hybridization pattern with the other probe. These three atypical individuals may result from single genetic exchanges between four regions of the Z and the W, giving rise to three atypical W chromosomes. Finding males with such atypical heterochromosomes in a female heterogametic species may indicate that a gradual transition occurs between the heterogametic systems.

Avaliação in vivo e in vitro do potencial citotóxico, genotóxico e mutagênico da água e do sedimento do rio Corumbataí - São Paulo, Brasil

Pós-graduação em Ciências Biológicas (Biologia Celular e Molecular) - IBRC

Avian Piscivores as Vectors for Myxobolus cerebralis in the Greater Yellowstone Ecosystem

Myxobolus cerebralis, the cause of whirling disease in salmonids, has dispersed to waters in 25 states within the USA, often by an unknown vector. Its incidence in Yellowstone cutthroat trout Oncorhynchus clarkii bouvieri within the highly protected environment of Yellowstone Lake, Yellowstone National Park, is a prime example. Given the local abundances of piscivorous birds, we sought to clarify their potential role in the dissemination of M. cerebralis. Six individuals from each of three bird species (American white pelican Pelecanus erythrorhynchos, double-crested cormorant Phalacrocorax auritus, and great blue heron Ardea herodias) were fed known-infected or uninfected rainbow trout O. mykiss. Fecal material produced during 10-d periods before and after feeding was collected to determine whether M. cerebralis could be detected and, if so, whether it remained viable after passage through the gastrointestinal tract of these birds. For all (100%) of the nine birds fed known-infected fish, fecal samples collected during days 1–4 after feeding tested positive for M. cerebralis by polymerase chain reaction. In addition, tubificid worms Tubifex tubifex that were fed fecal material from known-infected great blue herons produced triactinomyxons in laboratory cultures, confirming the persistent viability of the parasite. No triactinomyxons were produced from T. tubifex fed fecal material from known-infected American white pelicans or double-crested cormorants, indicating a potential loss of parasite viability in these species. Great blue herons have the ability to concentrate and release viable myxospores into shallow-water habitats that are highly suitable for T. tubifex, thereby supporting a positive feedback loop in which the proliferation of M. cerebralis is enhanced. The presence of avian piscivores as an important component of aquatic ecosystems should continue to be supported. However, given the distances traveled by great blue herons between rookeries and foraging areas in just days, any practices that unnaturally attract them may heighten the probability of M. cerebralis dispersal and proliferation within the Greater Yellowstone Ecosystem.

Lipidic and proteic absorption in digestive tract of tropical fat snook (Centropomus parallelus, POEY 1860)

The tropical fat snook Centropomus parallelus is a species of recognized ecological importance and with a high economical potential for fisheries and aquaculture. The investigations of digestive tube morphology in association with their feeding abilities are fundamental to improve techniques for aquaculture feeding procedures. Sudan black staining and Perl`s method were used to evaluate their absorption capacity of fat and protein respectively. The Sudan black stain was performed 12 h after the ingestion of lipids. The lipids are intensely absorbed in the ceca epithelium and less intensely in the intestine and rectum. The Perl`s method was performed 12 h after the ingestion of ferritin. The proteins are absorbed only in the rectum. This is the first description of fat and protein absorption ability by the digestive tube of fat snook. These data enhance the possibility of the addition of macromolecules in rations that can show a diversity of physiological effects. The histological implications of each segment of the digestive tube in association with fish biology are further discussed. (C) 2010 Elsevier B.V. All rights reserved.

Prediction of culture performance of juvenile Litopenaeus vannamei by in vitro (pH-stat) degree of feed protein hydrolysis with species-specific enzymes

Rapid in vitro methods for measuring digestibility may be useful in analysing aqua feeds if the extent and limits of their application are clearly defined. The pH-stat protein digestibility routine with shrimp hepatopancreas enzymes was previously related to apparent protein digestibility with juvenile Litopenaeus vannamei fed diets containing different protein ingredients. The potential of the method to predict culture performance of shrimp fed six commercial feeds (T3, T4, T5, T6, T7 and T8) with 350 g kg(-1) declared crude-protein content was assessed. The consistency of results obtained using hepatopancreas enzyme extracts from either pond or clear water-raised shrimp was further verified in terms of reproducibility and possible diet history effects upon in vitro outputs. Shrimps were previously acclimated and then maintained over 56 days (initial mean weight 3.28 g) on each diet in 500-L tanks at 114 ind m(-2), clear water closed system with continuous renewal and mechanical filtering (50 mu m), with four replicates per treatment. Feeds were offered four times daily (six days a week) delivered in trays at feeding rates ranging from 4.0% to 7.0% of stocked shrimp biomass. Feed was accessible to shrimp 4 h daily for 1-h feeding period after which uneaten feed was recovered. Growth and survival were determined every 14 days from a sample of 16 individuals per tank. Water quality was monitored daily (pH, temperature and salinity) and managed by water back flushing filter cleaning every 7-10 days. Feeds were analysed for crude protein, gross energy, amino acids and pepsin digestibility. In vitro pH-stat degree of protein hydrolysis (DH%) was determined for each feed using hepatopancreas enzyme extracts from experimental (clear water) or pond-raised shrimp. Feeds resulted in significant differences in shrimp performance (P < 0.05) as seen by the differences in growth rates (0.56-0.98 g week(-1)), final weight and feed conversion ratio (FCR). Shrimp performance and in vitro DH% with pond-raised shrimp enzymes showed significant correlation (P < 0.05) for yield (R-2 = 0.72), growth rates (R-2 = 0.72-0.80) and FCR (R-2 = -0.67). Other feed attributes (protein : energy ratio, amino acids, true protein, non-protein nitrogen contents and in vitro pepsin digestibility) showed none or limited correlation with shrimp culture performance. Additional correlations were found between growth rates and methionine (R-2 = 0.73), FCR and histidine (R-2 = -0.60), and DH% and methionine or methionine+cystine feed contents (R-2 = 0.67-0.92). pH-stat assays with shrimp enzymes generated reproducible DH% results with either pond (CV <= 6.5%) or clear water (CV <= 8.5%) hepatopancreas enzyme sources. Moreover, correlations between shrimp growth rates and feed DH% were significant regardless of the enzyme origin (pond or clear water-raised shrimp) and showed consistent R-2 values. Results suggest the feasibility of using standardized hepatopancreas enzyme extracts for in vitro protein digestibility.

HEMATOLOGICAL ANALYSIS OF Micropogonias furnieri, DESMAREST, 1823, SCIANIDAE, FROM TWO ESTUARIES OF BAIXADA SANTISTA, SAO PAULO, BRAZIL

Hematological alterations in fish are considered a useful tool to evaluate pathological processes resulting from the exposure to environmental pollutants. The whitemouth croaker Micropogonias furnieri is a common species in estuarine areas and potentially exposed to many contaminants. In the present study, the hematological characteristics of fish collected at two sites in Baixada Santista (Santos Estuarine System - SES, a polluted site; and the Estuary of Itanhaem River - EIR, unpolluted site) del was analysed. The following blood descriptors were analyzed: number of Erythrocytes (Er), Hematocrit (Ht), Hemoglobin (Hb), Mean Corpuscular Volume (MCV) and Mean Corpuscular Hemoglobin concentration (MCHC). Fish from SES exhibited significant lower levels of Ht and increase on MCHC and Hb. Such differences are likely related to the different contamination levels found in these estuaries.

Hematological parameters and phagocytic activity in fat snook (Centropomus parallelus) bred in captivity

The objective of this work was to determine the hematological parameters and the phagocytic capacity of peritoneal macrophages of fat snook related to sex, stage of gonadal maturation and seasonal cycle. Blood was collected from 135 animals (78 females and 57 males) and used for determinations of: erythrocyte number, hematocrit, hemoglobin, erythrocyte indices mean corpuscular volume (MCV) and mean corpuscular hemoglobin concentration (MCHC), total and differential leukocyte counts, and thrombocyte count. The phagocytic capacity and phagocytic index were determined after Saccharomyces cerevisiae inoculation in the peritoneal cavity of the animals. The hematological results according to sex showed that the erythrocyte, total leukocyte and thrombocyte counts were statistically higher in males than females, with the latter showing a higher MCV. Concerning to erythrocyte count, hematocrit and hemoglobin concentration analyzed separately by sex and stage of gonadal maturation, males were found to have significantly elevated values in the mature stage and decreased levels in the resting stage. The results of the erythrocyte and leukocyte series, thrombocytes and phagocytic activity related to seasonal cycle showed significant differences in both sexes, where hematocrit and hemoglobin concentration were lower in winter and higher in the other seasons, mean corpuscular volume was higher in the summer and lower in the winter and fall, total leukocytes and thrombocytes lower in the spring and higher in the fall, lymphocytes low in the winter and summer and high in the spring and phagocytic capacity and phagocytic index high in the summer and low in the winter and fall. The results showed that the hematological values in males are statistically higher than those in females, the erythrocyte values in males increase with the progression of gonadal maturation and that winter is the season of the year least favorable for hematological and phagocytic responses for survival of fat snook kept in captivity. The parameters studied could be utilized in the evaluation of the health status of this species in captivity. (C) 2012 Elsevier Ltd. All rights reserved.