Salinity tolerance in Clarias gariepinus (Burchell, 1822): insight on blood parameter variations and gill histological changes.

This study was designed to evaluate the tolerance of Clarias gariepinus juveniles to a gradual and abrupt increase in salinity over time. To this effect, C. gariepinus juveniles were exposed to three salinity incremental protocols namely 1 g L-1 day-1, 5 g L-1 day-1, and 10 g L-1 day-1. Changes in the hematological parameters and the gill histology of fish were analyzed to determine the impact of osmotic stress on the health status of the fish and its osmoregulatory ability. The result obtained showed that juveniles of C. gariepinus can tolerate salinity stress up to 14 g L-1. At 15 g L-1 and beyond, all samples died regardless of gradual (i.e., 1 g L-1 day-1 administered for 15 days) or abrupt salinity exposure (i.e., 5 g L-1 day-1 administered for three days and 10 g L-1 day-1 administered for two days). Interestingly, more than 90% of the fish survived a direct 10 g L-1 exposure for 24 h without prior acclimation. The hematological parameters accessed in the fish exposed to 10 g L-1 (either gradually or abruptly) showed a significant increase in the white blood cells and a decrease in the red blood cells, packed cell volume, hemoglobin concentration, and all derived blood parameters. The results of the serum biochemistry show a lower total protein and albumin in the salinity-treated fish compared to the control group. However, the serum glucose and the plasma electrolytes (i.e., K+, Na+, and Cl-) were higher in the former group than in the latter. Aside from the stress response expressed in the blood parameters, severe gill degenerations were seen in the histological micrograph obtained for the salinity-treated fish, while the control had a near-normal gill architecture. It was concluded that C. gariepinus could tolerate salinity exposure of 10 g L-1 day-1 (administered gradually or abruptly) and below without killing the fish within 24 h.

Long-term hypoxia-induced physiological response in turbot Scophthalmus maximus L.

Hypoxia affects fish's survival, growth, and physiological metabolism processes. In this study, turbot plasma glucose and cortisol contents, hepatic glycolysis (hexokinase [HK], phosphofructokinase [PFK], pyruvate kinase [PK]) and lipolysis (fatty acid synthetase [FAS], lipoprotein lipase [LPL]) enzyme activities, anti-oxidant enzyme (superoxide dismutase [SOD], catalase [CAT], glutathione peroxidase [GSH-Px]) activities, malondialdehyde (MDA), lactate and glycogen contents, gill histological parameters (lamellar length [SLL], width [SLW], interlamellar distance [ID]), respiratory frequency (RF), the proportion of the secondary lamellae available for gas exchange (PAGE), and hifs (hif-1α, hif-2α, hif-3α) expression were determined during long-term hypoxia and reoxygenation. Results showed that long-term hypoxia (3.34 ± 0.17 mg L-1) significantly elevated plasma cortisol and glucose contents; increased hepatic HK, PK, PFK, FAS, and LPL activity; decreased hepatic glycogen, lactate contents, and lipid drop numbers; and caused changes of hepatocyte (vacuolation, pyknotic, and lytic nucleus) after treatment for 4 weeks. Hepatic SOD, CAT, GSH-Px activity, and MDA contents; lamellar perimeter, SLL, ID, RF, and PAGE; and hepatic hif-1α, hif-2α, and hif-3α manifested similar results. Meanwhile, hif-1α is significantly higher than hif-2α, and hif-3α. Interestingly, females and males demonstrated no sex dimorphism significantly different from the above parameters (except hepatic FAS, LPL activity, and lipid drop number) under hypoxia. The above parameters recovered to normal levels after reoxygenation treatment for 4 weeks. Thus, long-term hypoxia promotes turbot hepatic glycogenolysis and lipolysis, induces oxidative damage and stimulates hepatic antioxidant capacity, and alters gill morphology to satisfy insufficient energy demand and alleviate potential damage, while hif-1α plays critical roles in the above physiological process.

Essential oils from Cymbopogon citratus and Lippia sidoides in the anesthetic induction and transport of ornamental fish Pterophyllum scalare.

This study verified the effects of essential oils from Lippia sidoides (EOLS) and Cymbopogon citratus (EOCC) on the anesthesia of freshwater angelfish (Pterophyllum scalare) of two different sizes (juveniles I (0.82 g) and II (2.40 g)) and the transport (8 h) of juveniles II. Fish were exposed to different concentrations of EOLS and EOCC: 0, 10, 25, 50, 75, 100, 150, 200, and 250 mg L-1. Ventilatory rate (VR) and transport for 8 h with 0, 10, and 15 mg L-1 of each essential oil were evaluated in juveniles II. The major components found in EOLS and EOCC were carvacrol (44.50%) and α-citral (73.56%), respectively. The best sedation and anesthesia times for both essential oils were obtained with 10 and 25 mg L-1 and 200 and 250 mg L-1 for juveniles I and II, respectively. Fish sedated with EOLS had lower VR values than the other treatments. Blood glucose levels were higher in ornamental fish transported with 10 and 15 mg EOLS L-1 and 15 mg EOCC L-1. Hepatic glycogen values were higher in the control group. In general, fish transported with 10 mg EOLS L-1 showed fewer gill histological alterations than other transported fish. When the type of lesion was evaluated, the highest gill alterations occurred in fish transported with EOCC. In conclusion, 10 mg EOLS L-1 could be used to transport of juveniles II because although this concentration increased blood glucose levels, it decreased the VR and muscle glycogen levels and caused only mild alterations to the gills.

Eugenol and Lippia alba essential oils as effective anesthetics for the Amazonian freshwater stingray Potamotrygon wallacei (Chondrichthyes, Potamotrygonidae).

This study assessed the potential of eugenol and the essential oil of Lippia alba (EOLA) in providing suitable anesthetic induction and recovery times, and their consequent effects on the blood and respiratory physiology, as well as the gill architecture of an Amazonian freshwater stingray, Potamotrygon wallacei, shortly after reaching the recovery and 48 h later. Juveniles of P. wallacei were exposed to increasing concentrations of eugenol (75, 100, 125, and 150 µL L-1) and EOLA (150, 175, 200, and 225 µL L-1) in an immersion bath. Anesthetic induction was found to be faster with the use of eugenol compared to EOLA. On the other hand, the stingrays anesthetized with eugenol displayed a longer recovery time than those exposed to EOLA. The highest concentrations of eugenol caused moderate to severe histological changes in the gills. No significant changes were found for hematocrit and plasma energy metabolites in the stingrays anesthetized with all concentrations of both eugenol and EOLA shortly after reaching the recovery from the ansthesia, when compared to those recovered after 48 h. Investigations regarding the potential use of these natural anesthetics are unprecedented for freshwater stingray species, and 200 µL L-1 EOLA is recommended as the most suitable anesthetic for use in juveniles of P. wallacei.

Altered physiological response and gill histology in black rockfish, Sebastes schlegelii, during progressive hypoxia and reoxygenation.

Hypoxia has gradually become common in aquatic ecosystems and imposes a significant challenge for fish farming. The loss of equilibrium (LOE), 50% lethal time (LT50), plasma cortisol, glucose, red blood cells (RBC), hemoglobin (Hb), gill histological alteration, and related parameters (lamellar length [SLL] and width [SLW], interlamellar distance [ID], basal epithelial thickness [BET], lamellar surface area [LA], and gill surface area [GSA]); respiratory rate; the proportion of the secondary lamellae available for gas exchange (PAGE); and hypoxia-inducible factor (hif-1α, hif-2α) mRNA expression were determined during progressive hypoxia and reoxygenation (R-0, R-12, R-24 h) to illustrate the underlying physiological response mechanisms in black rockfish Sebastes schlegelii. Results showed that the DO concentration significantly decreased during progressive hypoxia, while DO at LOE and LT50 were 2.42 ± 0.10 mg L-1 and 1.67 ± 0.38 mg L-1, respectively. Cortisol and glucose were significantly increased at LOE and LT50, with the highest levels observed at LT50, and then gradually recovered to normal within reoxygenation 24 h. RBC number and Hb results were like those of glucose. Hypoxia stress resulted in lamellar clubbing, hypertrophy, and hyperplasia. Respiratory frequency significantly increased at LOE and decreased at LT50. Lamellar perimeters, SLL, ID, LA, GSA, and PAGE, significantly increased at LOE and LT50, with the highest values observed at LT50. However, SLW and BET significantly decreased at LOE, LT50, and R-0. These parameters recovered to nearly normal levels at R-24 h. hif-1α mRNAs in gill and liver were significantly upregulated at LOE and LT50, and recovery to normal after reoxygenation 24 h. hif-2α mRNAs in gill was similar to that of hif-1α, whereas hepatic hif-2α mRNAs remained unchanged during hypoxia-reoxygenation. These results indicated that progressive hypoxia stress elevated RBC number, Hb, cortisol, and glucose levels, induced the alteration of gill morphology, increased LA and GSA, stimulated respiratory frequency and PAGE, and upregulated the transcription of hif-1α and hif-2α in gill and liver. Reoxygenation treatment for 24 h alleviated the stress mentioned above effects. These findings expand current knowledge on hypoxia tolerance in black rockfish Sebastes schlegelii.

Effect of copper exposure and recovery period in reared Diplodus sargus.

The aquaculture growth can be followed by the occurrence of more and new pathogenic agents, since the production leads to higher fish densities in confined areas more appropriate to the appearance and propagation of pathologies. Copper sulfate has been widely used in preventing and controlling fish parasites. The objective of this study is to investigate the effects of copper treatments in the fish tissues (bioaccumulation and histological changes in different organs), mortality and evaluate what happens during the recovery period. White sea bream (Diplodus sargus) were exposed to copper sulfate (0.25 and 0.5 mg L-1) during 60 days followed with a 75-day recovery period. The results showed that the concentration of copper in fish liver was significantly higher in the 0.5 mg L-1 treatment than in the 0.25 mg L-1 treatment. Conversely, copper load in the muscle did not differ significantly between treatments and control. Copper levels in muscle, and especially in liver, increased during copper exposure (up to 60 days). In summary, at higher concentrations copper sulfate treatment (0.5 mg L-1) might be toxic to fish, which showed histological alterations and copper accumulation in their tissues, mainly in the liver. Nevertheless, individuals returned to their original state after a 75-day recovery period and the tested copper concentrations does not represents risk for food safety.

Environmental health in the upper-middle Luján River basin from a multi-biomarker approach.

The objectives of this study were to evaluate the ecophysiological state of the biota using a set of biomarkers in the upper-middle Luján River. To this aim, we collected adult Cnesterodon decemmaculatus fish, biofilm and water at three sampling sites in the upper-middle Luján River (S1: rural area, S2: Luján City and S3: urban area after passing Lujan City). For each site we determined physicochemical variables, heavy metal concentration in water, 19 biomarkers in fish (morphometric, histological, genotoxic, oxidative stress, metabolic and neurotoxic) and six biomarkers in biofilm (oxidative stress and extracellular enzyme). Additionally, we compared the responses of fish and biofilm with those of laboratory controls obtained from outdoor cultures. Our results indicated increased heavy metal concentration at all sites, mainly As and Cd, and decreased dissolved oxygen at S1 and S3. In fish, genotoxic biomarkers showed significant differences with respect to the control. The comet assay indicated damage in fish at the urbanized sites (S2 and S3) and an increased frequency of erythrocytes with nuclear aberrations at all sites. The CEA index (cellular energy allocation), calculated from the metabolic biomarkers and lipid concentration were significantly increased at S1. The gill damage evaluated histologically and with three indices indicated severe damage at all sites. Gills showed thickened primary and secondary lamellae and fusion of filaments at all sites, but a significant increase in mucous cells was only found at S1 and S3. Biofilm showed increased values of extracellular enzymes (ß-glucosidase and alkaline phosphatase, lipid peroxidation and oxidative stress enzymes (i.e., catalase) at S3. These results are novel in that they incorporated laboratory controls allowing for comparisons with fish and biofilm from the field. They provided information on the status of a fish population and biofilm community, indicating the negative effect of river water deterioration on the tested organisms. Moreover, results showed what biomarkers were most sensitive for each biological sample.

Effects of low salinity stress on osmoregulation and gill transcriptome in different populations of mud crab Scylla paramamosain.

Animals living in estuaries suffer from rapid and continuous salinity fluctuations, while the global warming and extreme precipitation aggravate this situation. Osmoregulation is important for estuarine animals adapt to salinity fluctuations. The present study investigated the effects of low salinity stress on osmoregulation and gill transcriptome in two populations of mud crab from Hangzhou Bay and Zhangzhou Bay of China, respectively. Crabs were transferred from salinity 25 ppt to 5 ppt for 96 h. Edematous swelling in gill filaments was caused by low salinity stress and was more serious in Zhangzhou Bay population. Gill Na+/K+-ATPase activity increased (p < 0.01) in both populations under the low salinity stress and was significantly higher (p < 0.01) in Hangzhou Bay population than in Zhangzhou Bay population. According to transcriptome analysis, there were 191 genes differentially expressed under the low salinity stress in gill tissue of both populations. Several ion transport and energy metabolism related pathways, as well as the arginine and proline metabolism pathway, were enriched by these genes. On the other hand, 272 genes were identified to differentially express between two populations under the low salinity stress, but not under the control salinity. The enrichment analysis showed that these genes were mainly related to ion transport, energy metabolism, osmolytes metabolism and methyltransferase activity. In conclusion, the present study suggested that mud crab exploited a combination of extracellular anisosmotic regulation and intracellular isosmotic regulation for osmoregulation under the low salinity stress. Hangzhou Bay population showed a greater osmoregulatory capacity, which is probably due to the enhanced ion transport, energy supply, and osmolytes regulation. Meanwhile, epigenetic modification might also contribute to an inherent osmoregulation ability for Hangzhou Bay population to response to salinity fluctuation rapidly.

Comparison of cellular mechanisms induced by pharmaceutical exposure to caffeine and its combination with salicylic acid in mussel Mytilus galloprovincialis.

Urban and hospital-sourced pharmaceuticals are continuously discharged into aquatic environments, threatening biota. To date, their impact as single compounds has been widely investigated, whereas few information exists on their effects as mixtures. We assessed the time-dependent biological impact induced by environmental concentrations of caffeine alone (CAF; 5 ng/L to 10 µg/L) and its combination with salicylic acid (CAF+SA; 5 ng/L+0.05 µg/L to 10 µg/L+100 µg/L) on gills of mussel Mytilus galloprovincialis during a 12-day exposure. Although no histological alteration was observed in mussel gills, haemocyte infiltration was noticed at T12 following CAF+SA exposure, as confirmed by flow cytometry with increased hyalinocytes. Both the treatments induced lipid peroxidation and cholinergic neurotoxicity, which the antioxidant system was unable to counteract. We have highlighted the biological risks posed by pharmaceuticals on biota under environmental scenarios, contributing to the enhancement of ecopharmacovigilance programmes and amelioration of the efficacy of wastewater treatment plants.

Sublethal Effects of Arsenic on Oxygen Consumption, Hematological and Gill Histopathological Indices in Chanos chanos.

BACKGROUND: The current study was performed aiming to evaluate possible changes in the effect on oxygen consumption, hematology and gill histopathological parameters in fish (Chanos chanos) upon exposure to sublethal concentration of the metalloid arsenic. METHODS: Bioassay tests were conducted for determining the LC50 values of arsenic for 96 h. Oxygen consumption in control and arsenic-exposed fish was estimated using Winkler's method. Red blood corpuscular (RBC) count was examined with a Neubauer counting chamber under a phase contrast microscope. Hemoglobin (Hb) was estimated following the acid hematin method. Histopathological studies were carried by processing and staining the gill tissues with hematoxylin and eosin in accordance with standard histological techniques. They were then subjected to examination under a scanning electron microscope. RESULTS: Chanos chanos exposed to 1/10th of LC50 (24.61%) for a period of 30 days exhibited a maximum decline in the rate of respiration, followed by a decline in RBC and Hb above 45.59% and 51.60%, respectively. Significant toxic lesions encompassing fused gill lamellae, detached gill epithelium, hyperplasia and hypertrophy of respiratory epithelium became heavy handed on the 30th day. CONCLUSION: Information synthesized from our study serves to be useful in monitoring and managing (As) contamination in the aquatic environment.

Physiological performance of common carp (Cyprinus carpio, L., 1758) exposed to a sublethal copper/zinc/cadmium mixture.

In a natural ecosystem, fish are subjected to a multitude of variable environmental factors. It is important to analyze the impact of combined factors to obtain a realistic understanding of the mixed stress occurring in nature. In this study, the physiological performance of juvenile common carp (Cyprinus carpio) exposed for one week to an environmentally relevant metal mixture (4.8 µg/L of copper; 2.9 µg/L of cadmium and 206.8 µg/L of zinc) and to two temperatures (10 °C and 20 °C), were evaluated. After 1, 3 and 7 days, standard (SMR) and maximum metabolic rate (MMR) were measured and aerobic scope (AS) was calculated. In addition, hematocrit, muscle lactate, histology of the gills and metal accumulation in gills were measured. While SMR, MMR and AS were elevated at the higher temperature, the metal mixture did not have a strong effect on these parameters. At 20 °C, SMR transiently increased, but no significant changes were observed for MMR and AS. During metal exposure, hematocrit levels were elevated in the 20 °C group. The bioaccumulation of Cd in the gills reflected the increased metabolic rate at the higher temperature, with more accumulation at 20 °C than at 10 °C. Anaerobic metabolism was not increased, which corresponds with the lack of significant histopathological damage in the gill tissue. These results show that common carp handled these metal exposures well, although increased temperature led to higher Cd accumulation and necessitated increased hematocrit levels to maintain aerobic performance.

Potash mining effluents and ion imbalances cause transient osmoregulatory stress, affect gill integrity and elevate chronically plasma sulfate levels in adult common roach, Rutilus rutilus.

Secondary salinization is a growing global ecological issue. One cause is the discharge of effluents by the potash mining industry into surface waters such as the River Werra in Germany. Increases of major ions require various physiological responses of freshwater organisms to maintain the hydromineral balance of body fluids. However, only little is known about the acute and chronic effects of high concentrations and imbalances of ions on osmoregulation in freshwater teleosts. The present study aimed to elucidate the effects of potash mining effluents and different cation ratios on the osmoregulatory capacity and gill histopathology of a native fish species. Individuals of Rutilus rutilus were exposed to the currently allowed (HT) and intended future (LT) thresholds as well as to high concentrations of Mg2+ (Mg), K+ (K), and Mg2+ and K+ (Mg + K) for a period of 24 h, 7 d, 21 d and 8 wk. Plasma osmolarity, [Na+], [Mg2+], [K+], [Ca2+], [Cl-] and [SO42-] and branchial Na+/K+-ATPase activity were determined. Moreover, histological gill alterations after 21 d and muscle water content after 8 wk were examined. HT transiently (24 h) elevated plasma osmolarity, plasma [Na+] and [Ca2+], whereas [SO42-] was chronically increased even after 8 wk. Exposure to LT, Mg and Mg + K led to increased [SO42-] levels for at least 21 d. It seems that [SO42-] is mainly disturbed by multiple ions at high concentrations and long-term effects are unknown. Hydromineral homeostasis was maintained as indicated by unchanged Na+/K+-ATPase activity and muscle water content. However, mild structural alterations of the gills were observed in all exposure groups suggesting adaptational responses but with the potential to affect gas exchange capacity. Hence, the current thresholds for potash mining effluents affect osmomineral regulation in roach and further investigations should address potential impacts on reproduction in native fish species and physiological effects of SO42-.

Plasma Stress Responses in Juvenile Red-Spotted Grouper (Epinephelus akaara) exposed to Abrupt Salinity Decrease.

The objective of the current study was to determine acute plasma stress responses in two size groups of juvenile Epinephelus akaara (average body weight: 8.4±2.1 and 3.3±0.6 g; 150 and 120 days after hatch, respectively) exposed to abrupt salinity drops (from 34 practical salinity unit, PSU seawater to 18, 10 PSU (experiment 1) or 26, 18, 10 PSU (experiment 2), respectively). Plasma glucose, glutamic oxalate transaminase, glutamic pyruvate transaminase, red blood cell counts, and gill histology were determined during 72 h exposure. Significantly increased plasma glucose, glutamic oxalate transaminase levels, and red blood cell counts were observed in fish exposed to 18 or 10 PSU. Histological changes, such as hyperplasia and lifting of epithelium in the gill secondary lamellae, were also observed in fish exposed to 18 or 10 PSU at 72 h post-drop. E. akaara exposed to sudden salinity drops to 18 or 10 PSU still seems to undergo the primary adjustment phase before fish reaches a new homeostasis, whereas fish exposed to 26 PSU seems to mount osmotic changes. Therefore, the no observed adverse effect levels for 72 h acute salinity challenge was 26 PSU in our study, and salinity drop to 18 PSU and below can possibly cause acute adverse effect, in which fish could be vulnerable to additional stresses such as a temperature changes or handling stress.