Ricardo Berteaux Robaldo

Lactational transfer of PCBs and chlorinated pesticides in pups of southern elephant seals (Mirounga leonina) from Antarctica

Seven pairs of southern elephant seals (Mirounga leonina) pups and their dams were sampled during the late weaning season among a breeding population of seals on Elephant Island in Antarctica. The blubber of the pups and the milk and blubber of their dams were analyzed for lipid-normalized concentrations of PCBs and organochlorines compounds in order to evaluate the lactational transfer of these contaminants. The lipid-normalized concentrations in these tissues were in the ppb range (i.e., ngg(-1) lipid). The levels of contaminants in southern elephant seals were low in comparison with residues that have been reported in pinnipeds from the northern hemisphere. The relative tissue concentrations of the analytes measured followed the pattern: SigmaDDT>mirex>SigmaPCB>Sigmachlordane>HCB>heptachlor epoxide>dieldrin>methoxychlor>SigmaHCH>other organochlorines. The very high DDE/SigmaDDT ratio (0.91) in the blubber of dams and pups was an indicative of long-term, extremely distant pollution. On the other hand, the relatively high levels of some other organochlorine pesticides (e.g. mirex, heptachlor epoxide, dieldrin, methoxychor) may reflect the continued use of these insecticides in developing countries located in the southern hemisphere. For most of the analytes measured, the lipid-normalized concentrations were lower in pup blubber and in the milk than in the maternal blubber. Lactational transfer rates were dependent on the logK(ow) (octanol/water partition coefficient) values of the analytes measured, less lipophilic compounds being more readily transferred to the pups by the lactational route.

Growth of Critically Endangered annual fish Austrolebias wolterstorffi (Cyprinodontiformes: Rivulidae) at different temperatures

Given the importance of knowledge of the biology of endangered species for the planning of conservation and management efforts, the aim of this study was to investigate the influence of temperature on the growth of Austrolebias wolterstorffi (Ahl, 1924). To clarify the thermal influence on the growth of the species, temperatures of 16, 20, 24 , and 28°C were tested in triplicate. The present study showed that a water temperature of 28°C is detrimental to the growth of the species. Among the other tested temperatures, it was found that the optimum temperature for growth decreases as an individual ages and is slightly lower for females. It was demonstrated here that males reach a higher weight and length, and grow faster than females, reaching sexual maturity earlier. To optimize the growth of this species in captivity, the ideal temperature of the water during the initial life period is 24°C, until after puberty when the temperature should decrease to about 21°C.

Probiotic actions of Bacillus cereus var. toyoi and Saccharomyces boulardii in silver catfish (Rhamdia quelen) larvae culture

The objective of this study was to evaluate the use of Bacillus cereus var. toyoi and Saccharomyces boulardii as probiotics to improve Rhamdia quelen culture. Six hundred larvaes (0.16±0.07 g) were divided in three replicate tanks (25-L recirculation, 20 oC, photoperiod of 12 h light/12 h darkness) per treatment and were randomly assigned to the following treatments: Bacillus cereus var. toyoi; Saccharomyces boulardii; B. toyoi and S. boulardii; and control (without probiotic addition) for a period of 30 days. The fish were fed five times daily (56% crude protein - Supra alevino inicial®) and the probiotics were applied in water once a day. The doses of probiotics were 5 × 108 and 2 × 109 CFU (colony forming unit)/mL for B. cereus var. toyoi and S. boulardii, respectively. Both probiotics have an inhibitory effect in vitro against Vibrio carchariae and are able to grow in media prepared with fishery water; however, no effect was observed on growth parameters when they were administered to Rhamdia quelen larvae.

Effect of temperature on growth of the threatened annual fish Austrolebias nigrofasciatus Costa & Cheffe 2001

This study evaluated the effect of temperature on growth of Austrolebias nigrofasciatus, an endemic and threatened annual killifish species of the Patos-Mirim lagoon system in Southern Brazil. In order to verify the effect of temperature on initial growth of A. nigrofasciatus, eggs stored in the laboratory were hatched and juveniles reared for eight weeks at 16 and 22 oC. The standard length of newly hatched fishes was 4.67 ± 0.25 mm and after eight weeks they reached 23.68 ± 3.73 and 22.68 ± 5.36 mm, respectively at 16 and 22 oC. However, initial growth of fish reared at 22 oC was faster and they reached sexual dimorphism at an earlier age compared to those reared at 16 oC. Final length of females reared at 22 oC was 23.00 ± 2.83 mm, they were significantly larger than those reared at 16 oC (17.91 ± 2.47 mm). Males were significantly larger than the females at 16 oC, but there was no difference for growth between sexes of fish reared at 22 oC. The sex ratios were 1:0.6 and 1:1.1 (M:F) at 16 oC and 22 oC, respectively, suggesting temperature determination of phenotypic sex. Considering the results, it appears that juveniles to be developed in captivity should be kept at 22 oC during the first six weeks of life, thus ensuring a higher growth rate until puberty.

Digenea (Trematoda) of Micropogonias furnieri (Desmarest) (Perciformes, Sciaenidae) from Rio Grande do Sul, Brazil

Micropogonias furnieri (Desmarest, 1823) from the coast of Rio Grande do Sul State were examined and six trematodes species were recovered: Aponurus laguncula Looss, 1907, A. pyriformis (Linton, 1910), Monascus filiformis (Rudolphi, 1819), Opecoeloides catarinensis Amato, 1983, O. stenosomae Amato, 1983, and Pachycreadium gastrocotylum (Manter, 1940). All species are registered for the first time in Rio Grande do Sul State and M. furnieri represents a new host record for A. laguncula, A. pyriformis and M. filiformis.

Incubation media affect the survival, pathway and time of embryo development in Neotropical annual fish Austrolebias nigrofasciatus (Rivulidae): incubation media in embryo a. nigrofasciatus

To analyse the survival, pathway and time of embryo development in the annual fish Austrolebias nigrofasciatus eggs were monitored in four liquid media and two damp media under experimental conditions for 130 days until their development was complete. Eggs kept in the same breeding water from oviposition remained in diapause I (DI) during all experiments. In constrast, up to the stage prior to entering diapause II (DII), the other media had no influence on development. Embryos at this stage (DII), however, show longer development time when treated in medium with water and powdered coconut shell so that about 80% of embryos remained in DII at 100 days. In contrast, all other treatments had a significantly lower proportion of embryos remaining in DII. When treated with Yamamotos solution in humid media, embryos showed the fastest development. The first fully developed embryos (DIII) were seen at 27 days after oviposition. It took an average of 46-58 days for 50% of eggs in each treatment to reach DIII. Compared with other studies, survival in all incubation media was high at between 70 and 98%. Taken together, it can be concluded that all incubation media were found to be viable for maintaining embryos. Altering developmental trajectories through the manipulation of diapauses in different media makes this species a potential model organism for laboratory studies.

Gene and Blood Analysis Reveal That Transfer from Brackish Water to Freshwater Is Less Stressful to the Silverside Odontesthes humensis

Silversides are fish that inhabit marine coastal waters, coastal lagoons, and estuarine regions in southern South America. The freshwater (FW) silversides have the ability to tolerate salinity variations. Odontesthes humensis have similar habitats and biological characteristics of congeneric O. bonariensis, the most studied silverside species and with great economic importance. Studies revealed that O. bonariensis is not fully adapted to FW, despite inhabiting hyposmotic environments in nature. However, there is little information about stressful environments for cultivation of silverside O. humensis. Thus, the aim of this study was to evaluate the stress and osmoregulation responses triggered by the osmotic transfers on silverside O. humensis. Silversides were acclimated to FW (0 ppt) and to brackish water (BW, 10 ppt) and then they were exposed to opposite salinity treatment. Silverside gills and blood were sampled on pre-transfer (D0) and 1, 7, and 15 days (D1, D7, and D15) after changes in environmental salinity, the expression levels of genes atp1a3a, slc12a2b, kcnh1, and hspa1a were determined by quantitative reverse transcription-PCR for evaluation of osmoregulatory and stress responses. Furthermore, glycemia, hematocrit, and osmolality were also evaluated. The expression of atp1a3a was up- and down-regulated at D1 after the FW–BW and BW–FW transfers, respectively. Slc12a2b was up-regulated after FW–BW transfer. Similarly, kcnh1 and hspa1a were up-regulated at D1 after the BW–FW transfer. O. humensis blood osmolality decreased after the exposure to FW. It remained stable after exposure to BW, indicating an efficient hyposmoregulation. The glycemia had a peak at D1 after BW–FW transfer. No changes were observed in hematocrit. The return to the pre-transfer levels at D7 after the significant increases in responses of almost all evaluated molecular and blood parameters indicated that this period is enough for acclimation to the experimental conditions. In conclusion, our results suggest that BW–FW transfer is more stressful to O. humensis than FW–BW transfer and the physiology of O. humensis is only partially adapted to FW.

Evaluation of Reference Genes to Analyze Gene Expression in Silverside Odontesthes humensis Under Different Environmental Conditions

Some mammalian reference genes, which are widely used to normalize the qRT-PCR, could not be used for this purpose due to its high expression variation. The normalization with false reference genes leads to misinterpretation of results. The silversides (Odontesthes spp.) has been used as models for evolutionary, osmoregulatory and environmental pollution studies but, up to now, there are no studies about reference genes in any Odontesthes species. Furthermore, many studies on silversides have used reference genes without previous validations. Thus, present study aimed to was to clone and sequence potential reference genes, thereby identifying the best ones in Odontesthes humensis considering different tissues, ages and conditions. For this purpose, animals belonging to three ages (adults, juveniles, and immature) were exposed to control, Roundup®, and seawater treatments for 24 h. Blood samples were subjected to flow-cytometry and other collected tissues to RNA extraction; cDNA synthesis; molecular cloning; DNA sequencing; and qRT-PCR. The candidate genes tested included 18s, actb, ef1a, eif3g, gapdh, h3a, atp1a, and tuba. Gene expression results were analyzed using five algorithms that ranked the candidate genes. The flow-cytometry data showed that the environmental challenges could trigger a systemic response in the treated fish. Even during this systemic physiological disorder, the consensus analysis of gene expression revealed h3a to be the most stable gene expression when only the treatments were considered. On the other hand, tuba was the least stable gene in the control and gapdh was the least stable in both Roundup® and seawater groups. In conclusion, the consensus analyses of different tissues, ages, and treatments groups revealed that h3a is the most stable gene whereas gapdh and tuba are the least stable genes, even being considered two constitutive genes.