First Record of Clinostomum sp. (Digenea: Clinostomidae) in Danio rerio (Actinopterygii: Cyprinidae) and the Implication of Using Zebrafish from Pet Stores on Research.

Many scientific studies still use zebrafish from pet stores as animal models, even cutting-edge researches. However, these animals differ genotypically and phenotypically between them. The importance of the use of standardized models is widely recognized. Besides that, another consequence of using zebrafish from unknown origins is the acquisition of parasitized animals. This study aimed to relate the infection by Clinostomum sp. in zebrafish. Animals sold as "high standard" were acquired from a commercial company. Swimming alterations and superficial yellow dots were observed in five zebrafish with clinical signs, which were isolated, euthanized, and necropsied. Muscular yellow cysts with metacercaria associated with lesions were observed. The muscular cysts were responsible for the superficial yellow dots as well as the swimming alterations. The prevalence was 2.5%, and the mean infection intensity was 7 digeneans/host. The cysts measured a mean of 1251.43 µm long × 784.28 µm wide. Metacercariae measured a mean of 4847 µm long × 1353 µm wide. This first report about infection by Clinostomum sp. in zebrafish is globally relevant since the host and the parasite genus currently overlap worldwide. Furthermore, this study sheds light on the importance of the specific pathogen-free commercial creations or laboratory-reared zebrafish for research.

The Effects of Acute Copper and Ammonia Challenges on Ammonia and Urea Excretion by the Blue Crab Callinectes sapidus.

Copper (Cu) is a persistent environmental contaminant that elicits several physiological disturbances in aquatic organisms, including a disruption in ammonia regulation. We hypothesized that exposure to Cu in a model crustacean (blue crab, Callinectes sapidus) acclimated to brackish water (2 ppt) would lead to hyperammonemia by stimulating an increase in ammonia production and/or by inhibiting ammonia excretion. We further hypothesized that urea production would represent an ammonia detoxification strategy in response to Cu. In a pilot experiment, exposure to 0, 100, and 200 µg/L Cu for 6 h caused significant concentration-dependent increases in ammonia excretion (J amm). Based on these results, an acute 24-h 100 µg/L Cu exposure was conducted and this similarly caused an overall stimulation of J amm during the 24-h period, indicative of an increase in ammonia production. Terminal haemolymph total ammonia content (T amm) was unchanged, suggesting that while ammonia production was increased, there was no inhibition of the excretion mechanism. In support of our second hypothesis, urea excretion (J urea) increased in response to Cu exposure; haemolymph [urea] was unaffected. This suggested that urea production also was increased. To further test the hypothesis that J urea increased to prevent hyperammonemia during Cu exposure, crabs were exposed to high environmental ammonia (HEA; 2.5 mmol/L NH4HCO3) for 12 h in a separate experiment. This led to a fourfold increase in haemolymph T amm, whereas J urea increased only transiently and haemolymph [urea] was unchanged, indicating that urea production likely does not contribute to the attenuation of hyperammonemia in blue crabs. Overall, Cu exposure in blue crabs led to increased ammonia and urea production, which were both eliminated by excretion. These results may have important implications in aquaculture systems where crabs may be exposed to elevated Cu and/or ammonia.

mRNA Expression and activity of ion-transporting proteins in gills of the blue crab Callinectes sapidus: effects of waterborne copper.

Waterborne Cu effects on the transcription of genes encoding ion-transporting proteins and the activities of these proteins were evaluated in gills of the blue crab Callinectes sapidus acclimated to diluted (2‰) and full (30‰) seawater. Crabs were exposed (96 h) to an environmentally relevant concentration of dissolved Cu (0.78 µM) and had their posterior (osmoregulating) gills dissected for enzymatic and molecular analysis. Endpoints analyzed were the activity of key enzymes involved in crab osmoregulation (sodium-potassium adenosine triphosphatase [Na(+)/K(+)-ATPase], hydrogen adenosine triphosphatase [H(+)-ATPase], and carbonic anhydrase [CA]) and the mRNA expression of genes encoding these enzymes and the sodium-potassium-chloride (Na(+)/K(+)/2Cl⁻) cotransporter. Copper effects were observed only in crabs acclimated to diluted seawater (hyperosmoregulating crabs) and were associated with an inhibition of the expression of mRNA of genes encoding the Na(+)/K(+)-ATPase and the Na(+)/K(+)/2Cl⁻ cotransporter. However, Cu did not affect Na(+)/K(+)-ATPase activity, indicating that the gene transcription is downregulated before a significant inhibition of the enzyme activity can be observed. This also suggests the existence of a compensatory response of this enzyme to prevent osmoregulatory disturbances after short-term exposure to environmentally relevant Cu concentrations. These findings suggest that Cu is a potential ionoregulatory toxicant in blue crabs C. sapidus acclimated to low salinity. The lack of Cu effect on blue crabs acclimated to full seawater would be due to the reduced ion uptake needed for the regulation of the hemolymph osmotic concentration in full seawater (30‰). Also, this could be explained considering the lower bioavailability of toxic Cu (free ion) associated with the higher ionic content and dissolved organic matter concentration in high salinity (30‰) than in diluted seawater (2‰).