Modulation of miR-429 during osmotic stress in the silverside Odontesthes humensis.

Silverside fish inhabit marine coastal waters, coastal lagoons, and estuarine regions in southern South America. Although silversides are not fully adapted to freshwater, they can tolerate a wide range of salinity variations. MicroRNAs (miRNAs) are a class of ∼22 nucleotide noncoding RNAs, which are crucial regulators of gene expression at post-transcriptional level. Current data indicate that miRNAs biogenesis is altered by situations of environmental stress, thereby altering the expression of target mRNAs. Foremost, the silversides were acutely exposed to 30 g.L-1 of salt to reveal in which tissue miR-429 could be differentially expressed. Thus, fish were acclimated to freshwater (0 g.L-1) and to brackish water (10 g.L-1), and then exposed to opposite salinity treatment. Here, we reveal that miR-429, a gill-enriched miRNA, emerges as a prime osmoregulator in silversides. Taken together, our findings suggest that miR-429 is an endogenous regulator of osmotic stress, which may be developed as a biomarker to assist silverside aquaculture.

Acute exposition to Roundup Transorb® induces systemic oxidative stress and alterations in the expression of newly sequenced genes in silverside fish (Odontesthes humensis).

Roundup Transorb® (RDT) is a glyphosate-based herbicide commonly used in agricultural practices worldwide. This herbicide exerts negative effects on the aquatic ecosystem and affects bioenergetic and detoxification pathways, oxidative stress, and cell damage in marine organisms. These effects might also occur at the transcriptional level; however, the expression of genes associated with oxidative stress has not been studied well. Odontesthes humensis is a native Brazilian aquatic species naturally distributed in the habitats affected by pesticides, including Roundup Transorb® (RDT). This study evaluated the toxic effects of short-term exposure to RDT on O. humensis. Moreover, the genes related to oxidative stress were sequenced and characterized, and their expressions in the gills, hepatopancreas, kidneys, and brain of the fish were quantified by quantitative reverse transcription-polymerase chain reaction. The animals were exposed to two environmentally relevant concentrations of RDT (2.07 and 3.68 mg L-1) for 24 h. Lipid peroxidation, reactive oxygen species (ROS), DNA damage, and apoptosis in erythrocytes were quantified by flow cytometry. The expression of the target genes was modulated in most tissues in the presence of the highest tested concentration of RDT. In erythrocytes, the levels of lipid peroxidation, ROS, and DNA damage were increased in the presence of both the concentrations of RDT, whereas cell apoptosis was increased in the group exposed to 3.68 mg L-1 RDT. In conclusion, acute exposure to RDT caused oxidative stress in the fish, induced negative effects on cells, and modulated the expression of genes related to the enzymatic antioxidant system in O. humensis.

Corrigendum: Gene and Blood Analysis Reveal That Transfer from Brackish Water to Freshwater Is More Stressful to the Silverside Odontesthes humensis.

[This corrects the article DOI: 10.3389/fgene.2018.00028.].

The role of freshwater fish in the life cycle of Dioctophyme renale in Southern Brazil.

Brazil stands out by the diverse records of Dioctopphyme renale in different hosts; however, there is little information about the life cycle of the nematode in the region. This study aims to report on third-stage larvae infections in fish in southern Brazil. In this context, 324 fish of different species belonging to Characiformes, Cyprinodontiformes and Siluriformes were collected in an urban area of Rio Grande do Sul State, where domestic and wild hosts were reported with the nematode. Of the total fish examined, 25(7.7%) were found to be parasitized by third-stage larvae of D. renale which were found encysted in the stomach serous membrane and in the celoma cavity of Hoplosternum littorale (Siluriformes) with a prevalence of 53.2% (25/47) and mean intensity of infection of 4.4 larvae/host (1 to 13 larvae). The occurrence of larvae in H. littorale indicates the presence of parasitosis in the region; however, the contribution of this fish species as a source of infection for dogs in urban areas must be considered with caution given the difficulties these dogs may face in the capture and predation of the fish to the point of effectively maintaining the urban cycle of D. renale. In addition, the low level of larvae registered in the total sample of fish examined indicates that these hosts are unlikely to play an important role in the transmission of D. renale to domestic animals in the region of the study.

Roundup® Herbicide Decreases Quality Parameters of Spermatozoa of Silversides Odontesthes Humensis.

The silverside (Odontesthes humensis) is a very interesting model for toxicological studies due its high sensitivity and need for good water quality. The aim of this study was to evaluate the effects of Roundup on spermatozoa of O. humensis, after acute exposure. The fish were exposed to 0 and 7.8 mg L-1 (a.e.) of glyphosate, respectively. Through computer-assisted sperm analysis, a significant decrease in concentration, total and progressive motility, average path distance, straight line distance, path average velocity, curved line velocity, straight line velocity linearity, wobble, amplitude of lateral head displacement, cross beat frequency, and motility period of silverside spermatozoa exposed to Roundup was observed. Also, increase in membrane fluidity, ROS production and lipid peroxidation and a decrease in the mitochondrial functionality was observed in spermatozoa of Roundup exposed silversides. It was demonstrated that Roundup exposure in a concentration that can be achieve in natural water bodies soon after its application in fields is able to cause losses in several sperm quality parameters, consequently decreasing the fertilization potential of O. humensis spermatozoa.

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.

Gene and Blood Analysis Reveal That Transfer from Brackish Water to Freshwater Is More 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.

Prey species of franciscana Pontoporia blainvillei as paratenic hosts of helminths.

The distribution of franciscana Pontoporia blainvillei Gervais & d'Orbigny, 1884, is restricted to the estuarine and coastal waters of the southwestern Atlantic. The diet of this dolphin is composed of fishes, squids, and shrimps, many of which harbor helminths that may infect franciscana. Larval forms of the trematode Synthesium pontoporiae and the acanthocephalan Bolbosoma turbinella have been recorded in franciscana; however, they have not yet been identified in any of the prey species of this cetacean. We evaluated 3 components of the diet of franciscana as possible transmission sources of parasitiasis. Specimens of São Paulo squid Doryteuthis sanpaulensis (n = 50), banded croaker Paralonchurus brasiliensis (n = 43), and rough scad Trachurus lathami (n = 50) were necropsied. Organs were washed and examined under a stereomicroscope. Helminths were collected and mounted on slides. None of the species analyzed showed infection by metacercariae of S. pontoporiae. Helminths found in São Paulo squid have not been recorded in franciscana. Cistacanths of Corynosoma australe were found in the coelomic cavity and mesentery of croaker (prevalence [P] = 53.49%; mean infection intensity [MII] = 6.74) and scad (P = 4%; MII = 1.50). Cistacanths of B. turbinella were also found in the same sites in scad (P = 14%; MII = 2.14). Banded croaker and rough scad are recorded in this study as new paratenic hosts for C. australe, while scad is a new paratenic host for B. turbinella.

Neuropeptide Y gene expression around meal time in the Brazilian flounder Paralichthys orbignyanus.

Neuropeptide Y (NPY) is considered the major stimulant for food intake in mammals and fish. Previous results indicate that NPY is involved in the feeding behaviour of the Brazilian flounder, Paralichthys orbignyanus. In this study, we evaluated hypothalamic NPY expression before (-2 h), during (0 h) and after feeding (+2 h) in two independent experiments: (1) during a normal feeding schedule and (2) in fish fasted for 2 weeks. During normal feeding, changes in the levels of NPY mRNA were periprandial, with expression levels being significantly elevated at meal time (P less than 0.05) and significantly reduced 2 h later (P less than 0.05). Comparing the fasting and unfasted groups, NPY mRNA levels were significantly higher (P less than 0.05) at -2 h and +2 h in the fasting group, but there was no difference at 0 h. In addition, the higher NPY mRNA levels that were observed in the fasting group were maintained throughout the sampling period. In summary, our results show that NPY expression was associated with meal time (0 h) in food intake regulation.

Identification, tissue distribution and evaluation of brain neuropeptide Y gene expression in the Brazilian flounder Paralichthys orbignyanus.

Neuropeptide Y (NPY) is one of the most potent stimulants of food intake in vertebrates, mammals and fish. However, the present knowledge about feeding behaviour in fish is still limited and based on studies in a few species. The Brazilian flounder Paralichthys orbignyanus is being considered for aquaculture, and it is important to understand the mechanisms regulating feeding in order to improve its performance in captivity. The objectives of this study were to clone NPY cDNA, evaluate the mRNA levels in different tissues of flounder, and also evaluate brain NPY expression to associate food intake with NPY expression levels. A 597 bp NPY cDNA was cloned from Brazilian flounder brain. NPY expression was detected in all the peripheral tissues analysed. No significant differences were observed in brain NPY gene expression over 24 h after food intake at a temperature of 15 +or- 3 degrees C. No correlation was observed among plasma glucose, total protein, cholesterol, triglycerides and NPY expression levels during this 24 h period. On the other hand, mRNA levels were increased after two weeks of fasting at elevated temperatures. Our results suggest that NPY mRNA levels in Brazilian flounder are affected by temperature.

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.

Residues of persistent organochlorine contaminants in southern elephant seals (Mirounga leonina) from Elephant Island, Antarctica.

Contamination of blubber tissues by organochlorine pesticides (OC) and PCBs was assessed in female and male pups and juveniles, as well as in adult females and subdominant adult males of the Southern elephant seal, Mirounga leonina, from Elephant Island in the Antarctic Peninsula. All residues of persistent organochlorine contaminants analyzed were found in blubber samples, except for beta-HCH, endosulfan II, endrin, heptachlor, and aldrin. The relative concentrations of the analytes detected were sigmaDDT > sigmaPCB > sigmachlordane > mirex > dieldrin > HCB> sigmaendosulfan > methoxychlor > sigmaHCHs > other OC pesticides. OC and PCBs concentrations were 1 or 2 orders of magnitude lower than those found in pinnipeds from northern hemisphere. The ratio sigmaDDT/sigmaPCB was higher in southern elephant seals. The relative importance of some OC residues indicates that pesticides used either currently or in the recent past in countries in the southern hemisphere are the sources of contamination in the Antarctic region. Data showed that concentrations of contaminants generally increased from pups < juveniles < adults and suggested that pups accumulated contaminants through transfer from the mother seals via transplacental and lactational routes.

Biomarkers of exposure and effect in the Brazilian flounder Paralichthys orbignyanus (Teleostei: Paralichthyidae) from the Patos Lagoon estuary (Southern Brazil).

Biomarkers of exposure (liver metallothionein-like proteins content and catalase and glutathione S-transferase activities) and effect (liver lipoperoxidation and blood cell DNA damage) of contaminants were analyzed in the Brazilian flounder Paralichthys orbignyanus from the Patos Lagoon estuary (Southern Brazil). Flounders were collected for a year in two sites: "Coroa do Boi" (polluted site) and "Saco do Justino" (non-polluted site). Results indicated that micronucleated cells frequency was the best biomarker to distinguish flounders from the two sites. Taken together, data from DNA damage analyses (micronucleus test and comet assay) indicated that flounders from the non-polluted site efficiently repaired the DNA breaks, contrary to those from the polluted site, which probably had their DNA repair system inhibited or exhausted. Furthermore, data from enzyme activities (catalase and GST) and lipid peroxidation indicated that flounders from the polluted site were under oxidative stress in summer and autumn.

Serum immunoglobulin G concentration in Southern elephant seal, Mirounga leonina (Linnaeus, 1758), from Elephant Island (Antarctica): sexual and adrenal steroid hormones effects.

Serum immunoglobulin G (IgG; indirect enzyme-linked immunosorbent assay), as well as sexual and adrenal steroid hormones' concentrations (radioimmunoassay) were determined in 63 (male and female) Southern elephant seals (Mirounga leonine) at different developmental stages (weaned pups, juveniles and adults). In females, IgG values (mean+/-S.D.) were higher (P<0.05) in adults (15.9+/-6.5mg ml(-1)) than in juveniles (7.9+/-4.0mg ml(-1)), but similar to weaned pups (12.0+/-5.0mg ml(-1)). Estrogen concentration was higher (P<0.05) in adults than in the weaned pups. In females, a significant (P<0.05) correlation (R=0.4) between serum IgG level and progesterone concentration was observed. In males, testosterone concentration was higher (P<0.05) in adults than in the juveniles and weaned pups. Aldosterone and cortisol concentrations were higher (P<0.05) in weaned pups (1056.0+/-643.1pmol 1(-1) and 272.7+/-110.0 nmol 1(-1), respectively) than in the juveniles (638.6+/-579.7pmol1(-1) and 152.9+/-97.3nmol 1(-1), respectively) and adults (386.5+/-209.1pmol (-1) and 145.7+/-67.3nmol 1(-1), respectively). These findings indicate that weaned pups are subjected to a higher natural stressful condition in the field. Despite this, humoral immunity, measured through IgG concentration, is not impaired in weaned pups.

Movement of southern elephant seals (Mirounga leonina L.) from Elephant Is. South Shetlands, Antarctica

O presente estudo descreve os movimentos de dois exemplares de elefante-marinho do sul (Mirounga leonina) durante a fase pelágica de seu ciclo de vida. Os exemplares foram capturados no verão austral de 1999 na Ilha Elefante (61º13'S, 55º23'W), Antártica, e monitorados por aproximadamente 9 meses. Cada exemplar foi instrumentado com um medidor de tempo e profundidade de mergulho via satélite (Sattelite Time Depth Recorder, STDR mod. ST-6PPT, Telonics®, EUA) com uma antena VHF acoplada ao instrumento, montado num molde de tela e resina, fixado com cintas plásticas, e colados na pelagem dorsal do animal com resina plástica. Para a instrumentação o animal era capturado com rede de contenção e imobilizado quimicamente com anestésico dissociativo (Zoletil 50®- 1mg/kg). Um dos indivíduos (23842), deslocou-se da Ilha Elefante (61.2ºS 55.3ºW) até a Ilha Rei George (62.2ºS 58.1ºW) em fevereiro quando perdemos contato com o instrumento. Já o indivíduo 23843 permaneceu nas águas ao redor da Ilha Elefante (61.2ºS 54.4ºW a 61.6ºS 55.4ºW) por grande parte do ano (janeiro à setembro), quando começou a deslocar-se para SW. Este indivíduo foi re-avistado na temporada de 1999/2000 quando o instrumento foi recuperado. Os deslocamentos registrados podem estar relacionados com o estágio de desenvolvimento de cada indivíduo. O presente estudo demonstra a viabilidade de estudos de monitoramento de elefantes-marinhos das Ilhas Shetlands do Sul, Antártica, através de telemetria por satélite e a necessidade da continuidade destes estudos num maior número de indivíduos para uma descrição mais detalhada dos padrões de migração, distribuição e forrageio apresentados.