Use of chromosome microdissection in fish molecular cytogenetics

Chromosome microdissection is a technique in which whole chromosomes or chromosomal segments are dissected under an inverted microscope yielding chromosome-specific sequences. Several protocol modifications introduced during the past 15 years reduced the number of chromosomes required for most applications. This is of particular interest to fish molecular cytogenetics, since most species present highly uniform karyotypes which make impossible the collection of multiple copies of the same chromosome. Probes developed in this manner can be used to investigate chromosome homologies in closely related species. Here we describe a protocol recently used in the gymnotiform species group Eigenmannia and review the major steps involved in the generation of these markers focusing on protocol modifications aiming to reduce the number of required chromosomes.

Spatial and temporal distribution of fish in Palmas bay, Ubatuba, Brazil

The aim of this study was to analyze the distribution and abundance of the fish fauna of Palmas bay on Anchieta Island in southeastern Brazil. Specimens were caught in the summer and winter of 1992, using an otter trawl at three locations in the bay. The specimens were caught in both the nighttime and daytime. Data on the water temperature and salinity were recorded for the characterization of the predominant water mass in the region, and sediment samples were taken for granulometric analysis. A total of 7 656 specimens (79 species), with a total weight of approximately 300 kg, were recorded. The most abundant species were Eucinostomus argenteus, Ctenosciaena gracilicirrhus, Haemulon steindachneri, Eucinostomus gula and Diapterus rhombeus, which together accounted for more than 73% of the sample. In general, the ecological indices showed no differences in the composition of species for the abiotic variables analyzed. The multivariate analysis showed that the variations in the distribution of the fish fauna were mainly associated with intra-annual differences in temperature and salinity, resulting from the presence of South Atlantic Central Water (SACW) in the area during the summer. The analysis also showed an association with the type of bottom and a lesser association with respect to the night/day periods.

Histopathological, immunohistochemical and ultraestructural evaluation of inflammatory response in Arius genus fish under experimental inoculation of BCG

The aim of this study was to evaluate the inflammatory response kinetics after experimental inoculation with BCG in the primitive Arius sp. fish. The BCG was applied through the intramuscular injection in the caudal peduncular region, and the samples were collected for the analyses at days 1, 3, 7, 14, 21, and 33 post-injection. Acute phase inflammatory infiltrate was characterized by the predominant mononuclear cells, intersticial edema, and muscular tissue necrosis. As the inflammatory response evolved, a large number of multinuclear giant cells were formed containing the BCG. These giant cells were positive for the S100 protein at the histochemical analysis, which demonstrate the macrofage activity, confirmed by the ultra-structural analysis showing the lack of the cytoplasmic membrane enveloping the many nuclei within the giant cell. These results led to the conclusion that Arius sp. fish injected with the BCG showed a difuse inflammatory response characterized by a large number of mononuclear cells, absence of granuloma formation, and predominant giant cells.

Effect of n-3 polyunsaturated fatty acids on oxidative stress in rats supplemented with different doses of fish oil

The diet and plasma lipid patterns associated with lipid oxidation susceptibility in rats fed different doses of polyunsaturated fatty acids (n-3 PUFA) from fish oil were evaluated. Wistar rats were assigned into three groups and received diets containing 8% soybean oil (SOY), 4% soybean oil + 4% fish oil (SOY-FISH) and 8% fish oil (FISH) for 21 days. Linoleic, oleic and ?-linolenic acids in SOY diets were substituted by myristic, palmitic, palmitoleic, eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids in SOY-FISH and FISH diets reducing the n-6/n-3 ratio and increasing the peroxidability index (PI). Increased dietary EPA and DHA were observed in SOY-FISH and FISH plasma at the expense of linoleic and arachidonic acid levels. Saturated fatty acids, which were significantly different between the three diets (P < 0.01), were found at the same concentration in the plasma (P = 0.23). No changes were observed in oxidative stress as measured by the concentration of thiobarbituric acid reactive substances (TBARS) expressed in brain homogenates. However, TBARS concentration in the plasma of the SOY-FISH group was higher than the other two groups (P = 0.02). The major differences between these three groups were the n-3 PUFA content (0.4, 1.8 and 3.2 g/100 g diet) and the saturates/polyunsaturates ratio (0.3, 0.5 and 0.8) for SOY, SOY-FISH, and FISH groups, respectively. Thus, n-3 PUFA intake from fish oil only when followed by a decrease in saturated/polyunsaturated fatty acids ratio increased oxidative susceptibility in rats measured by plasma TBARS concentration

Total and inorganic mercury determination in fish tissue by flow injection cold vapour atomic fluorescence spectrometry

A simple and reliable method for Hg determination in fish samples has been developed. Lyophilised fish tissue samples were extracted in a 25% (w/v) tetramethylammonium hydroxide (TMAH) solution; the extracts were then analysed by FI-CVAFS. This method can be used to determine total and inorganic Hg, using the same FI manifold. For total Hg determination, a 0.1% (w/v) KMnO(4) solution was added to the FI manifold at the sample zone, followed by the addition of a 0.5% (w/v) SnCl(2) solution, whereas inorganic Hg was determined by adding a 0.1% (w/v) L-cysteine solution followed by a 1.0% (w/v) SnCl(2) solution to the FI system. The organic fraction was determined as the difference between total and inorganic Hg. Sample preparation, reagent consumption and parameters that can influence the FI-CVAFS performance were also evaluated. The limit of detection for this method is 3.7 ng g(-1) for total Hg and 4.3 ng g(-1) for inorganic Hg. The relative standard deviation for a 1.0 mu gL(-1) CH(3)Hg standard solution (n = 20) was 1.1%, and 1.3% for a 1.0 mu gL(-1) Hg(2+) standard solution (n = 20). Accuracy was assessed by the analysis of Certified Reference Material (dogfish: DORM-2, NRCC). Recoveries of 99.1% for total Hg and 93.9% inorganic Hg were obtained. Mercury losses were not observed when sample solutions were re-analysed after a seven day period of storage at 4 degrees C.

Frozen Chicken for Wild Fish: Nutritional Transition in the Brazilian Amazon Region Determined by Carbon and Nitrogen Stable Isotope Ratios in Fingernails

Objectives: Amazonian populations are experiencing dietary changes characteristic of the nutrition transition. However, the degree of change appears to vary between urban and rural settings. To investigate this process, we determined carbon and nitrogen stable isotope ratios in fingernails and dietary intake of Amazonian populations living along a rural to urban continuum along the Solimoes River in Brazil. Methods: Carbon and nitrogen stable isotope ratios were analyzed from the fingernails of 431 volunteer subjects living in different settings ranging from rural villages, small towns to urban centers along the Solimoes River. Data from 200 dietary intake surveys were also collected using food frequency questionnaires and 24-h recall interviews in an effort to determine qualitative aspects of diet composition. Results: Fingernail delta(13)C values (mean standard deviation) were -23.2 +/- 1.3, 20.2 +/- 1.5, and 17.4 +/- 1.3 parts per thousand and delta(15)N values were 11.8 +/- 0.6, 10.4 +/- 0.8, and 10.8 +/- 0.7 parts per thousand for those living in rural villages, small towns, and major cities, respectively. We found a gradual increase in the number of food items derived from C(4) plant types (meat and sugar) and the replacement of food items derived from C(3) plant types (fish and manioc flour) with increasing size of urban centers. Conclusion: Increasing urbanization in the Brazilian Amazon is associated with a significant change in food habits with processed and industrialized products playing an increasingly important role in the diet and contributing to the nutrition transition in the region. Am. J. Hum. Biol. 23:642-650, 2011. (C) 2011 Wiley-Liss, Inc.

The recent occurrence, establishment and potential impact of Geophagus proximus (Cichlidae : Perciformes) in the Tiete River reservoirs: an Amazonian fish species introduced in the Parana Basin (Brazil)

The introduction of allochthonous fish species happens constantly in large bodies of freshwater, like as the reservoirs of Parana Basin, located in Brazilian southeast, representing a threat for local biodiversity. The fish species Plagioscion squamosissimus and Cichla ocellaris were introduced from the 1970s in several water bodies of this basin and had successfully established themselves in all six reservoirs located in the middle and lower Tiete River (SP, Brazil), particularly. After six decades from the first recorded species introduction, this hydrographic system remains open to the invasion of further fish species, owing to widespread fish-farming activity and by the channels opened between this system and other reservoirs and river basin. This study was an effort to confirm the Geophagus proximus occurrence in the six Tiete River reservoirs, verifying the actual introduction status and analyzing its potential environmental impacts on local species by the analysis of the population structure (abundance, body dimensions and feeding habits). By the results, this species was confirmed in the Ibitinga, Nova Avanhandava and Tres Irmaos reservoirs. The abundance and feeding analysis shows, respectively, it is successfully established in the Tres Irmaos reservoir with the same feeding habitats of local species, such as Geophagus brasiliensis. It was further shown to be very likely that G. proximus would spread throughout the reservoir system of the middle and lower Tiete River, in the manner of P. squamosissimus and C. ocellaris, and the competition pressure for food resources between G. proximus and the local species which represents a potential environmental impact system. These scientific evidences fortifies the knowledge basin for the implantation of a fish management system, to control and reduce the abundance of the invader and to prevent its becoming established in all the Tiete River Basin, avoiding the disastrous consequences for the native species of Parana River Basin.

Determination of heavy metals by inductively coupled plasma-optical emission spectrometry in fish from the Piracicaba River in Southern Brazil

A total of 202 fish, representing 16 species, were collected during 2008 (March-October) in the Tanquan region of the Piracicaba River using nets. Flesh samples were collected and analyzed, using inductively coupled plasma-optical emission spectroscopy for Al, As, Cd, Co Cr, Cu, Mn, Mo, Ni, Ph, Se, Sn, Sr, and Zn. The results showed that the flesh of these fish all contained extremely high levels of Al and Sr, and moderately high levels of Cr, As, Zn, Ni. Mn and Pb. The metals were higher in these fish during rainy season, with fish collected during the months of March and October being the highest. In addition, the accumulation of metals was species-dependent. Cascudos (Hypostomus punctatus) and piranhas (Serrasalmus spilopleura) exhibited high levels of almost all of the metals, while curimbata (Prochilodus lineatus) had moderate levels. A few species, including pacu (Piaractus mesopotamicus) and dourado (Salminus maxillosus), had very low levels of most metals. The results show that the Piracicaba River Basin is widely contaminated with high levels of many toxic heavy metals, and that human consumption of some fish species is a human health concern. (C) 2009 Elsevier B.V. All rights reserved.

EFFECT OF n-3 POLYUNSATURATED FATTY ACIDS ON OXIDATIVE STRESS IN RATS SUPPLEMENTED WITH DIFFERENT DOSES OF FISH OIL

The diet and plasma lipid patterns associated with lipid oxidation susceptibility in rats fed different doses of polyunsaturated fatty acids (n-3 PUFA) from fish oil were evaluated. Wistar rats were assigned into three groups and received diets containing 8% soybean oil (SOY), 4% soybean oil + 4% fish oil (SOY-FISH) and 8% fish oil (FISH) for 21 days. Linoleic, oleic and alpha-linolenic acids in SOY diets were substituted by myristic, palmitic, palmitoleic, eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids in SOY-FISH and FISH diets reducing the n-6/n-3 ratio and increasing the peroxidability index (PI). Increased dietary EPA and DHA were observed in SOY-FISH and FISH plasma at the expense of linoleic and arachidonic acid levels. Saturated fatty acids, which were significantly different between the three diets (P < 0.01), were found at the same concentration in the plasma (P = 0.23). No changes were observed in oxidative stress as measured by the concentration of thiobarbituric acid reactive substances (TBARS) expressed in brain homogenates. However, TBARS concentration in the plasma of the SOY-FISH group was higher than the other two groups (P = 0.02). The major differences between these three groups were the n-3 PUFA content (0.4, 1.8 and 3.2 g/100 g diet) and the saturates/polyunsaturates ratio (0.3, 0.5 and 0.8) for SOY, SOY-FISH, and FISH groups, respectively. Thus, n-3 PUFA intake from fish oil only when followed by a decrease in saturated/polyunsaturated fatty acids ratio increased oxidative susceptibility in rats measured by plasma TBARS concentration. PRACTICAL APPLICATIONS Because fish oil intake is associated with risk reduction for cardiovascular disease, individuals are taking supplements containing a high dose of fish oil. However, there is no scientific consensus if the intake of a high dose of fish oil could increase the oxidative stress. Thus, more studies are necessary to assure the safety of this kind of supplementation.

Evaluation of protective effects of fish oil against oxidative damage in rats exposed to methylmercury

The present study evaluates a possible protective effect of fish oil against oxidative damage promoted by methylmercury (MeHg) in sub-chronically exposed rats. Reduced glutathione peroxidase and catalase enzyme activity and reduced glutathione levels were observed in MeHg-exposed animals compared to controls. Methylmercury exposure was also associated with DNA damage. Administration of fish oil to the methylmercury-exposed animals did not ameliorate enzyme activity or glutathione levels. On the other hand, a significant DNA protective effect (about 30%) was observed with fish oil treatment. There were no differences in the total mercury concentration in rat liver, kidney, heart or brain after MeHg administration with or without fish oil co-administration. Histopathological analyses showed a significant leukocyte infiltration in rat tissues after MeHg exposure, but this effect was significantly reduced after co-administration of fish oil. Taken together, our findings demonstrate oxidative damage even after low-level MeHg exposure and the protective effect of fish oil. This protection seems not to be related to antioxidant defenses or mercury re-distribution in rat tissues. It is probably due to the anti-inflammatory effects of fish oil. (C) 2010 Elsevier Inc. All rights reserved.

Use of Carnobacterium maltaromaticum cultures and hydroalcoholic extract of Lippia sidoides Cham. against Listeria monocytogenes in fish model systems

Minimally processed refrigerated ready-to-eat fishes may offer health risk of severe infection to susceptible individuals due to contamination by the psychrotolerant bacterium L monocytogenes. In this work, inhibition of L monocytogenes by a plant extract and lactic acid bacteria (IAB) was studied in model fish systems kept at 5 degrees C for 35 days. For that, fillets of tropical fish ""surubim"" (Pseudoplatystoma sp.) and hydroalcoholic extract of the plant Lippia sidoides Cham. (""alecrim pimenta"") were used. Fish peptone broth (FPB), ""surubim"" broth and ""surubim"" homogenate were inoculated with combinations of L monocytogenes and bacteriocin-producing Carnobacterium maltaromaticum (C2 and A9b(+)) and non bacteriocin-producing C. maltaromaticum (A9b(-)), in the presence or absence of extract of ""alecrim pimenta"" (EAP). In all model systems, monocultures of L monocytogenes and carnobacteria reached final populations >= 10(8) CFU/ml after 35 days, except for L monocytogenes in ""surubim"" homogenate (10(4) CFU/ml). In FPB, EAP alone and combined with cultures of LAB inhibited L monocytogenes but carnobacteria without EAP were only weakly antilisterial. In ""surubim"" broth, EAP alone did not prevent L monocytogenes growth but cultures of carnobacteria combined or not with EAP inhibited L monocytogenes, with more pronounced effect being observed for C maltaromaticum C2, which produced bacteriocin. In ""surubim"" homogenate, EAP alone and combined with cultures of C. maltaromaticum A9b(-) and A9b(+) were strongly inhibitory to L monocytogenes, while C maltaromaticum C2 with EAP caused transient inhibition of L monocytogenes. No significant inhibition of L monocytogenes was observed for carnobacteria in ""surubim"" homogenate without EAP. In conclusion, it was observed that the use of EAP and cultures of carnobacteria have potential to inhibit L monocytogenes in fish systems and the applications should be carefully studied, considering the influence of food matrix. (c) 2011 Elsevier B.V. All rights reserved.

Patterns of species diversity in the gastrointestinal helminths of a coral reef fish, Epinephelus merra (Serranidae), from French Polynesia and the south Pacific Ocean

Large-scale patterns of species diversity in the gastrointestinal helminth faunas of the coral reef fish Epinephelus merra (Serranidae) were investigated in French Polynesia and the South Pacific Ocean. The richer barrier reef community in French Polynesia supported richer parasite communities in E. merra than that on the fringing reef. While parasite communities among fish from the same archipelago were similar, differences in potential host species and the distance between archipelagos may have contributed to a qualitative difference in parasite communities between archipelagos. Digenean community diversity in coral reef fishes was greater in the western South Pacific, following similar patterns in free-living species. However, overall species diversity of camallanid nematodes of coral reef fishes does not appear to have been similarly affected.

Catecholamine release in heat-stressed Antarctic fish causes proton extrusion by the red cells

Two species of Antarctic fish were stressed by moving them from seawater at -1 degrees C to seawater at 10 degrees C and holding them for a period of 10 min. The active cryopelagic species Pagothenia borchgrevinki maintained heart rate while in the benthic species Trematomus bernacchii there was an increase in heart rate. Blood pressure did not change in either species. Both species released catecholamines into the circulation as a consequence of the stress. P. borchgrevinki released the greater amounts, having mean plasma concentrations of 177 +/- 54 nmol.l(-1) noradrenaline and 263 +/- 131 nmol.l(-1) adrenaline at 10 min. Pla.sma noradrenaline concentrations rose to 47 +/- 14 nmol.l(-1) and adrenaline to 73 +/- 28 nmol.l(-1) in T. bernacchii. Blood from P. borchgrevinki was tonometered in the presence of isoprenaline. A fall in extracellular pH suggests the presence of a Na+/H+ antiporter on the red cell membrane, the first demonstration of this in an Antarctic fish. Treatment with the beta-adrenergic antagonist drug sotalol inhibited swelling of red blood cells taken from temperature-stressed P. borchgrevinki, suggesting that the antiporter responds to endogenous catecholamines.

Studies of evolutionary temperature adaptation: Muscle function and locomotor performance in Antarctic fish

1, Studies of evolutionary temperature adaptation of muscle and locomotor performance in fish are reviewed with a focus on the Antarctic fauna living at subzero temperatures. 2. Only limited data are available to compare the sustained and burst swimming kinematics and performance of Antarctic, temperate and tropical species. Available data indicate that low temperatures limit maximum swimming performance and this is especially evident in fish larvae. 3, In a recent study, muscle performance in the Antarctic rock cod Notothenia coriiceps at 0 degrees C was found to be sufficient to produce maximum velocities during burst swimming that were similar to those seen in the sculpin Myoxocephalus scorpius at 10 degrees C, indicating temperature compensation of muscle and locomotor performance in the Antarctic fish. However, at 15 degrees C, sculpin produce maximum swimming velocities greater than N, coriiceps at 0 degrees C, 4, It is recommended that strict hypothesis-driven investigations using ecologically relevant measures of performance are undertaken to study temperature adaptation in Antarctic fish, Recent detailed phylogenetic analyses of the Antarctic fish fauna and their temperate relatives will allow a stronger experimental approach by helping to separate what is due to adaptation to the cold and what is due to phylogeny alone.

A systemic secondary vessel system is present in the teleost fish Tandanus tandanus and absent in the elasmobranchs Carcharhinus melanopterus and Rhinobutos typus and in the dipnoan Neoceratodus forsteri

A system of secondary vessels emerging from the primary vessels as numerous coiled capillaries has been described in numerous teleost and holost fishes. The systemic secondary vessels of the teleost Tandanus tandanus are typical of this system and are described in this study. The existence of a secondary vessel system has been postulated in the elasmobranch group. No secondary vessel origins, as seen in the teleosts, are present in the elasmobranchs Rhinobatos typus and Carcharhinus melanopterus. Vessels with a similar distribution to secondary arteries are observed but these are venous rather than arterial in nature and do not connect with the primary arteries. Like the secondary veins in teleosts, the cutaneous veins in R. typus contain blood with a low haematocrit. There is no morphological evidence for a secondary vessel system in the dipnoan Neoceratodus forsteri.