Species-specific differences and temperature-dependence of Na+/K+-ATPase in freshwater mussels Anodonta anatina and Unio tumidus (Bivalvia: Unionidae).

The predicted global warming of surface waters can be challenging to aquatic ectotherms like freshwater mussels. Especially animals in northern temperate latitudes may face and physiologically acclimate to significant stress from seasonal temperature fluctuations. Na+/K+-ATPase enzyme is one of the key mechanisms that allow mussels to cope with changing water temperatures. This enzyme plays a major role in osmoregulation, energy control, ion balance, metabolite transport and electrical excitability. Here, we experimentally studied the effects of temperature on Na+/K+-ATPase activity of gills in two freshwater mussel species, Anodonta anatina and Unio tumidus. The study animals were acclimated to three ambient temperatures (+4, +14, +24 °C) and Na+/K+-ATPase activity was measured at those temperatures for each acclimation group. Both species had their highest gill Na+/K+-ATPase activity at the highest acclimation temperature. Na+/K+-ATPase activity of gills exhibited species-specific differences, and was higher in A. anatina than U. tumidus in all test groups at all test temperatures. Temperature dependence of Na+/K+-ATPase was confirmed in both species, being highest at temperatures between +4 and + 14 °C when Q10 values in the acclimation groups varied between 5.06 and 6.71. Our results underline the importance of Na+/K+-ATPase of gills for the freshwater mussels in warming waters. Because Na+/K+-ATPase is the driving force behind ciliary motion, our results also suggest that in warming waters A. anatina may be more tolerant at sustaining vigorous ciliary action (associated with elevated respiration rates and filter-feeding) than U. tumidus. Overall, our results indicate great flexibility of the mussel's ecophysiological characteristics as response to changing conditions.

Primary amino acid sequences of decapod (Na+, K+)-ATPase provide evolutionary insights into osmoregulatory mechanisms.

Decapod Crustacea exhibit a marine origin, but many taxa have occupied environments ranging from brackish to fresh water and terrestrial habitats, overcoming their inherent osmotic challenges. Osmotic and ionic regulation is achieved by the gill epithelia, driven by two active ATP-hydrolyzing ion transporters, the basal (Na+, K+)-ATPase and the apical V(H+)-ATPase. The kinetic characteristic of gill (Na+, K+)-ATPase and the mRNA expression of its α subunit have been widely studied in various decapod species under different salinity challenges. However, the evolution of the primary structure has not been explored, especially considering the functional modifications associated with decapod phylogeny. Here, we proposed a model for the topology of the decapod α subunit, identifying the sites and motifs involved in its function and regulation, as well as the patterns of its evolution assuming a decapod phylogeny. We also examined both the amino acid substitutions and their functional implications within the context of biochemical and physiological adaptation. The α-subunit of decapod crustaceans shows greater conservation (∼94% identity) compared to the ß-subunit (∼40%). While the binding sites for ATP and modulators are conserved in the decapod enzyme, the residues involved in the α-ß interaction are only partially conserved. In the phylogenetic context of the complete sequence of (Na+, K+)-ATPase α-subunit, most substitutions appear to be characteristic of the entire group, with specific changes for different subgroups, especially among brachyuran crabs. Interestingly, there was no consistent separation of α-subunit partial sequences related to habitat, suggesting that the convergent evolution for freshwater or terrestrial modes of life is not correlated with similar changes in the enzyme's primary amino acid sequence.

(Na+, K+)- ATPase kinetics in Macrobrachium pantanalense: highlighting intra- and interspecific variation within the Macrobrachium amazonicum complex.

The Macrobrachium amazonicum complex is composed of at least the Macrobrachium amazonicum and Macrobrachium pantanalense species, with the latter described from specimens originally identified as part of an endemic M. amazonicum population in the Brazilian Pantanal region. While there may be a reproductive barrier between these two Macrobrachium species, both are phylogenetically close, with small genetic distance. However, there is currently no available biochemical information of Macrobrachium pantanalense (Na+, K+)-ATPase. Here, we report the kinetic characteristics of the gill (Na+, K+)-ATPase in two populations of M. pantanalense from Baiazinha Lagoon (Miranda, MS, Brazil) and Araguari River (Uberlândia, MG, Brazil), and compare them with Macrobrachium amazonicum populations from the Paraná-Paraguay River Basin. (Na+, K+)-ATPase activities were 67.9 ± 3.4 and 93.3 ± 4.1 nmol Pi min-1 mg-1 protein for the Baiazinha Lagoon and Araguari River populations, respectively. Two ATP hydrolyzing sites were observed for the Araguari River population while a single ATP site was observed for the Baiazinha Lagoon shrimps. Compared to the Araguari River population, a 3-fold greater apparent affinity for Mg2+ and Na+ was estimated for the Baiazinha Lagoon population, but no difference in K+ affinity and ouabain inhibition was seen. The kinetic differences observed in the gill (Na+, K+)-ATPase between the two populations of M. pantanalense, compared with those of various M. amazonicum populations, highlight interspecific divergence within the Macrobrachium genus, now examined from a biochemical perspective.

Developing rainbow trout (Oncorhynchus mykiss) lose branchial plasma accessible carbonic anhydrase expression with hatch and the transition to pH-sensitive, adult hemoglobin polymorphs.

Salmonids possess a unique respiratory system comprised of three major components: highly pH-sensitive hemoglobins, red blood cell (RBC) intracellular pH (pHi) protection, and a heterogeneous distribution of plasma accessible carbonic anhydrase (paCA), specifically with absence of paCA at the gills. These characteristics are thought to have evolved to enhance oxygen unloading to the tissues while protecting uptake at the gills. Our knowledge of this system is detailed in adults, but little is known about it through development. Developing rainbow trout (Oncorhynchus mykiss) express embryonic RBCs containing hemoglobins that are relatively insensitive to pH; however, availability of gill paCA and RBC pHi protection is unknown. We show that pre-hatch rainbow trout express gill paCA, which is lost in correlation with the emergence of highly pH-sensitive adult hemoglobins and RBC pHi protection. Rainbow trout therefore exhibit a switch in respiratory strategy with hatch. We conclude that gill paCA likely represents an embryonic trait in rainbow trout and is constrained in adults due to their highly pH-sensitive hemoglobins.

An investigation of gill and blood carbonic anhydrase characteristics in three basal actinopterygian species: alligator gar (Atractosteus spatula), white sturgeon (Acipenser transmontanus) and Senegal bichir (Polypterus senegalus).

Many teleosts possess a unique set of respiratory characteristics allowing enhanced oxygen unloading to the tissues during stress. This system comprises three major components: highly pH sensitive haemoglobins (large Bohr and Root effects), rapid red blood cell (RBC) intracellular pH (pHi) protection, and a heterogeneous distribution of membrane-bound plasma-accessible carbonic anhydrase (paCA; absence in the gills). The first two components have received considerable research effort; however, the evolutionary loss of branchial paCA has received little attention. In the current study, we investigated the availability of branchial membrane-bound CA, along with several other CA-related characteristics in species belonging to three basal actinopterygian groups: the Lepisosteiformes, Acipenseriformes and Polypteriformes to assess the earlier hypothesis that Root effect haemoglobins constrain branchial paCA availability. We present the first evidence suggesting branchial membrane-bound CA presence in a basal actinopterygian species: the Senegal bichir (Polypterus senegalus) and show that like the teleosts, white sturgeon (Acipenser transmontanus) and alligator gar (Atractosteus spatula) do not possess branchial membrane-bound CA. We discuss the varying respiratory strategies for these species and propose that branchial paCA may have been lost much earlier than previously thought, likely in relation to the changes in haemoglobin buffer capacity associated with the increasing magnitude of the Bohr effect. The findings described here represent an important advancement in our understanding of the evolution of the unique system of enhanced oxygen unloading thought to be present in most teleosts, a group that encompasses half of all vertebrates.

Oxidative stress responses after exposure to triclosan sublethal concentrations: an integrated biomarker approach with a native (Corydoras paleatus) and a model fish species (Danio rerio).

Triclosan (TCS) is a synthetic broad-spectrum antimicrobial agent commonly used world-wide in a range of personal care and sanitizing products detected frequently in aquatic ecosystems. The aim of this study was to examine biochemical markers responses triggered by TCS in Danio rerio and in a native South American fish species (Corydoras paleatus). Further, an integrated approach comparing both test fish species was undertaken. These fish organisms were exposed to 100 or 189 µg TCS/L for 48 h. The activities of catalase (CAT), glutathione-s-transferase (GST), superoxide dismutase (SOD), and lipid peroxidation levels (LPO) and total antioxidant capacity against peroxyl radicals (ACAP) were determined in liver, gills, and brain. Acetylcholinesterase activity (AChE) was measured in the brain. Multivariate analysis showed that the most sensitive hepatic parameters were activities of GST and SOD for C. paleatus while LPO levels were for D. rerio. In gills the same parameters were responsive for C. paleatus but CAT in D. rerio. ACAP and GST activity were responsive parameters in brain of both species. Integrated biomarker responses (IBR) index demonstrated similar trends in both species suggesting this parameter might serve as a useful tool for quantification of integrated responses induced by TCS.

Short communication: Effects of acute copper exposure on ionic regulation of the freshwater crab Aegla castro.

Aeglids are unique freshwater decapods whose habitats are being impacted by metallic compounds, such as copper (Cu). Thus, we investigated the effects of acute Cu exposure on ionic regulation of Aegla castro. For this, male specimens in intermolt were collected from a reference stream and acclimated for 5 days in laboratory. After which, crabs were exposed to 11 µg L-1 Cu (Cu11) or only to water (CTR) for 24 h. Hemolymph samples were withdrawn for the determination of Na+, K+, Ca2+, and Mg2+ concentrations and the posterior gills removed for the analysis of Na+/K+-ATPase, Ca2+-ATPase, H+-ATPase, and carbonic anhydrase (CA) activities. Increased Ca2+ and Mg2+ hemolymph concentrations were observed in animals from Cu11, when compared with CTR group. In addition, decreased activity of CA was observed in animals exposed to Cu. In the current study, alterations in Ca2+ and Mg2+concentrations probably indicate that animals activated exoskeleton reabsorption mechanisms, characteristic of the premolt. Therefore, increased Ca2+ and Mg2+ concentrations in hemolymph may indicate that a biochemical signal associated with the molting cycle was triggered by Cu exposure. Despite the known harmful effects of Cu on osmoregulatory enzymes, here we observed decreased activity only in CA. However, decreased activity of CA could trigger both acid-base imbalance and ionic disruption, since CA provides H+ and HCO3- for intracellular pH maintenance, and underpins Na+ and Cl- for ionic regulation. Therefore, understanding how aeglids respond to metal contamination in laboratory conditions is crucial to assess their potential as an alternative biological model for aquatic ecotoxicology.

Effect of hypoxia and air-breathing restricted on respiratory physiology of air-breathing loach (Paramisgurnus dabryanus).

This aim of this study was to determine the respiratory physiology response in the gill and gut of Paramisgurnus dabryanus under different breathing treatment patterns. The experimental design included the following three conditions: a control group without any stress treatments, an inhibited group with intestinal respiration inhibited, and an air-exposed group with gill respiration inhibited. The results indicated that the total static metabolic rate in the air-exposed group (188.92 ± 13.67 mg h-1 kg-1) was much higher than that of the other group after 7 days, decreased significantly after the first day of recovery (81.64 ± 7.85 mg h-1 kg-1). The air metabolic rate in the air-exposed group increased significantly after 7 days (P < 0.05). There was no significant difference among the groups. Histological observation on the gill and hindgut of P. dabryanus showed that the gill filament area of inhibited group became larger, while the gill structure of air exposed group showed some damage. The number of capillariesin the hindgut mucosal epithelial in air-exposed group showed a rapidly increase (P < 0.05). Likewise, the gas diffusion distance (1.24 ± 0.36 µm) became significantly shorter (P < 0.05). Lactate dehydrogenase activity of gill in the air-exposed group (846.68 ± 88.78 U mg-1 protein) significantly increased after 7 days whereas succinate dehydrogenase (1.02 ± 0.21 U mg-1 protein) and Na+/K+ ATPase (0.57 ± 0.20 U mg-1 protein) activity decreased significantly (P < 0.05). However, there was no significant change in the hindgut. After recovery, there was no significant difference in lactate dehydrogenase, succinate dehydrogenase, and Na+/K+ ATPase activity in the gill or hindgut in groups. P. dabryanus had a high viability in air-exposed condition. When recovery occurred under normoxic conditions, the physical levels of respiration returned back to the normal level quickly.

What does the freshwater clam, Corbicula largillierti, have to tell us about chlorothalonil effects?

Chlorothalonil (CLT) is a broad spectrum, and non-systemic fungicide applied in foliar structures to prevent and treat pathogens. This compound reaches to aquatic environments and affects the biota. In this context, the main goal of this study was to assess the effects of CLT at biochemical, tissular, and individual levels of biological organization using the invasive bivalve Corbicula largillierti as a bioindicator species. Clams were exposed to different sublethal concentrations (0, 10, 20 and 50 µg. L-1 CLT) for 96 h. At biochemical level, the enzymatic activity (Glutathione-s-Transferase, Catalase, Acetyl-, Butiryl- and Carboxyl-esterases) and lipid peroxidation were measured in gills and the visceral mass. Also, the digestive gland morphometry through quantitative histological indexes was registered at the tissular level. Finally, filtering activity and burial behavior at the individual level were measured. At the highest CLT concentration, the most significant changes were observed in enzymatic activity (except for butyrylcholinesterase), lipid peroxidation and in digestive gland morphometry. It was also registered increases of the filtering activity and the latency time to burial. Most of the biomarkers assessed showed significant responses under CLT exposure. Therefore, taking into account that C. largillierti was affected by CLT, it can be expected that other species could be in a potential risk if this fungicide is present in freshwater systems.

Tissue-specific expression and activity of cytochrome P450 1A and 3A in rainbow trout (Oncorhynchus mykiss).

Piscine cytochrome P450 (CYP) enzymes play an important role in the metabolism of xenobiotics. Xenobiotics often act as inducers of CYP1A1 and CYP3A expression and activity in fish. We compared constitutive mRNA expression of CYP1A1, CYP3A27, and CYP3A45 and catalytic activity of CYP1A (7-ethoxyresorufin-O-deethylation, EROD) and CYP3A-like (benzyloxy-4-trifluoromethylcoumarin-O-debenzyloxylation, BFCOD) enzymes in the following six rainbow trout tissues: liver, gill, heart, brain, intestine, and gonad. mRNA expression and activity were present in all investigated tissues. The CYP1A1 mRNA expression was higher in the liver, gill, heart, and brain compared to gonad and intestine. The intestine was the main site of CYP3A27 and CYP3A45 expression. The highest EROD and BFCOD activity was observed in liver tissue followed in descending order by heart, brain, gill, intestine, and gonad. Such differences might be related to the role of CYP physiological functions in the specific tissue. Rainbow trout exposure to 50 mg/kg of ß-naphthoflavone for 48 h resulted in a 7.5- and 5.9-fold increase in liver EROD and BFCOD activity, respectively. In vitro EROD activity inhibition with ellipticine showed tissue-specific inhibition, while ketoconazole decreased BFCOD activity by 50-98 % in all tissues. Further studies are needed to identify all CYP isoforms that are responsible for these activities and modes of regulation.

Acetylcholine esterase and antioxidant responses in freshwater teleost, Channa punctata exposed to chlorpyrifos and urea.

We aimed to understand the toxic effects of two crop protecting agents, organophosphate pesticide, chlorpyrifos (CPF) and fertilizer, urea (U), and their binary mixtures at sublethal concentrations for 28-d in a freshwater fish Channa punctata with a battery of biochemical biomarkers in gill and liver. The study has practical value as such mixtures, so often present together in water in the agro-intensive areas, might be predicted to cause cocktail effects. Both CPF and U inhibited AChE, augmented SOD, CAT, GPx activities, and caused lipid peroxidation and depletion in tissue macromolecules in a concentration and duration-dependent manner. While U alone had less severe effects compared to CPF treatments, complex interactions were observed for three combination doses (1CPF + 1U, 2CPF + 1U, 1CPF + 2U). In their mutual effects, antagonism prevailed over other interactions when CPF and U were in equal proportion in the mixture, while synergism was observed for AchE and key antioxidant enzymes when more U was in the mixture. The present study concluded that urea in water bodies might impart adverse effects in combination with pesticides in non-target aquatic organisms such as fish, and there should be a restriction in its excessive usage.

Effects of seawater acclimation on two Na+/K+-ATPase α-subunit isoforms in the gills of the marble goby, Oxyeleotris marmorata.

The marble goby, Oxyeleotris marmorata, is a freshwater teleost, but can acclimate progressively to survive in seawater (salinity 30). As an obligatory air-breather, it can also survive long periods of emersion. Two isoforms of Na+/K+-ATPase (nka) α-subunit, nkaα1 and nkaα3, but not nkaα2, had been cloned from the gills of O. marmorata. The cDNA sequence of nkaα1 consisted of 3069 nucleotides, coding for 1023 amino acids (112.5 kDa), whereas nkaα3 consisted of 2976 nucleotides, coding for 992 amino acids (109.5 kDa). As only one form of branchial Nkaα1 was identified using molecular cloning in this study, O. marmorata lacks specific freshwater- and seawater-type Nkaα isoforms as demonstrated by some other euryhaline fish species. The nkaα1 transcript level was about 2.5-fold higher than that of nkaα3 in the gills of freshwater O. marmorata. During exposure to seawater, the branchial transcript level of nkaα1 increased significantly on day 1 (~3.3-fold) and day 6 (~2.6-fold). By contrast, the branchial transcript level of nkaα3 increased significantly on day 1 (~2.6-fold), but not on day 6, of seawater exposure. Six days of exposure to seawater also led to significant increases in protein abundances of Nkaα1 (~6.9-fold) and Nkaα3 (~2.8-fold) in the gills of O. marmorata. Hence, the mRNA and protein expressions of both nkaα1/Nkaα1 and nkaα3/Nkaα3 were up-regulated in O. marmorata during seawater acclimation. This could explain why Vmax increases but Km for Na+ and K+ remain unchanged in Nka extracted from the gills of O. marmorata acclimated to seawater as reported previously.

Acute effects of neonicotinoid insecticides on Mytilus galloprovincialis: A case study with the active compound thiacloprid and the commercial formulation calypso 480 SC.

Pesticides can enter aquatic environments potentially affecting non-target organisms. Unfortunately, the effects of such substances are still poorly understood. This study investigated the effects of the active neonicotinoid substance thiacloprid (TH) and the commercial product Calypso 480 SC (CA) (active compound 40.4% TH) on Mytilus galloprovincialis after short-term exposure to sublethal concentrations. Mussels were tested for seven days to 0, 1, 5 and 10 mg L-1 TH and 0, 10, 50 and 100 mg L-1 CA. For this purpose, several parameters, such as cell viability of haemocytes and digestive cells, biochemical haemolymph features, superoxide dismutase (SOD) and catalase (CAT) enzymatic activity of gills and digestive gland, as well as histology of such tissues were analysed. The sublethal concentrations of both substances lead to abatement or completely stopping the byssal fibres creation. Biochemical analysis of haemolymph showed significant changes (P < 0.01) in electrolytes ions (Cl-, K+, Na+, Ca2+, S-phosphor), lactate dehydrogenase (LDH) enzyme activity and glucose concentration following exposure to both substances. The TH-exposed mussels showed significant imbalance (P < 0.05) in CAT activity in digestive gland and gills. CA caused significant decrease (P < 0.05) in SOD activity in gills and in CAT activity in both tissues. Results of histological analyses showed severe damage in both digestive gland and gills in a time- and concentration-dependent manner. This study provides useful information about the acute toxicity of a neonicotinoid compound and a commercial insecticide on mussels. Nevertheless, considering that neonicotinoids are still widely used and that mussels are very important species for marine environment and human consumption, further researches are needed to better comprehend the potential risk posed by such compounds to aquatic non-target species.

Cellular oxygen consumption, ROS production and ROS defense in two different size-classes of an Amazonian obligate air-breathing fish (Arapaima gigas).

In air-breathing fish a reduction of gill surface area reduces the danger of losing oxygen taken up in the air-breathing organ (ABO) to hypoxic water, but it also reduces the surface area available for ion exchange, so that ion regulation may at least in part be transferred to other organs, like the kidney or the gut. In the air-breathing Arapaima gigas, gill lamellae regress as development proceeds, and starting as a water-breathing embryo Arapaima turns into an obligate air-breathing fish with proceeding development, suggesting that ion regulation is shifted away from the gills as the fish grows. In Arapaima the kidney projects medially into the ABO and thus, probably a unique situation among fishes, is in close contact to the gas of the ABO. We therefore hypothesized that the kidney would be predestined to adopt an increased importance for ion homeostasis, because the elevated ATP turnover connected to ion transport can easily be met by aerobic metabolism based on the excellent oxygen supply directly from the ABO. We also hypothesized that in gill tissue the reduced ion regulatory activity should result in a reduced metabolic activity. High metabolic activity and exposure to high oxygen tensions are connected to the production of reactive oxygen species (ROS), therefore the tissues exposed to these conditions should have a high ROS defense capacity. Using in vitro studies, we assessed metabolic activity and ROS production of gill, kidney and ABO tissue, and determined the activity of ROS degrading enzymes in small (~ 5g, 2-3 weeks old) and larger (~ 670 g, 3-4 months old) A. gigas. Comparing the three tissues revealed that kidney tissue oxygen uptake by far exceeded the uptake measured in gill tissue or ABO. ROS production was particularly high in gill tissue, and all three tissues had a high capacity to degrade ROS. Gill tissue was characterized by high activities of enzymes involved in the glutathione pathway to degrade ROS. By contrast, the tissues of the ABO and in particular the kidney were characterized by high catalase activities, revealing different, tissue-specific strategies in ROS defense in this species. Overall the differences in the activity of cells taken from small and larger fish were not as pronounced as expected, while at the tissue level the metabolic activity of kidney cells by far exceeded the activity of ABO and gill cells.

Chlorothalonil causes redox state change leading to oxidative stress generation in Danio rerio.

A diverse range of chemicals are used in agriculture to increase food production on a large scale, and among them is the use of pesticides such as chlorothalonil, a broad-spectrum fungicide used in the control of foliar fungal diseases. This study aimed to elucidate the effects of chlorothalonil on biochemical biomarkers of oxidative stress in tissues of the fish Danio rerio. To achieve this, animals were exposed for 4 and 7 days, to nominal concentrations of chlorothalonil at 0 µg/L (DMSO, 0.001%), 0.1 µg/L and 10 µg/L, and after the exposure period, the tissues (gills and liver) were removed for biochemical analysis. Antioxidant capacity against peroxyl radicals (ACAP) and enzyme activities, such as superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST) and glutamate cysteine ligase (GCL), were evaluated in both tissues. In addition, the concentration of reactive oxygen species (ROS), reduced glutathione (GSH) and lipid peroxidation (LPO) levels were also analysed. A significant increase in ROS concentration, ACAP levels, GST and GCL activities and a significant reduction of LPO levels in gills exposed to the highest concentration were observed after 4 days. However, there was a significant reduction of ACAP and CAT activity, as well as a significant increase of GST activity and LPO levels in gills exposed to the lower concentration after 7 days. The liver was less affected, presenting a significant reduction in CAT activity and LPO levels after 4 days. However, a significant increase in SOD activity and LPO levels occurred after 7 days. These results indicate that chlorothalonil, after 4 days, caused activation of the antioxidant defence system in gills of animals exposed to the highest concentration. However, after 7 days, the lowest concentration of this compound caused oxidative stress in this same organ. Also, the results show that gills were more affected than the liver, probably because gills can be involved in chlorothalonil metabolisation. Therefore, it is possible that the liver could be exposed to lower chlorothalonil concentrations or less toxic metabolites due to the metabolism taking place in the gills.

Effects of chronic exposure of waterborne copper on the antioxidant system and tissue accumulation in golden trout (Oncorhynchus mykiss aguabonita).

We assessed the acute and chronic effects of copper (Cu2+) on the antioxidant system in golden trout (Oncorhynchus mykiss aguabonita). The median lethal concentration after 96 h was determined as 0.24 mg L-1. We then used 0.06 (L) and 0.12 mg L-1 (H) Cu2+ to assess the responses of the antioxidant system to long-term exposure. The activities of superoxide dismutase, catalase, glutathione-S-transferase, glutathione peroxidase, glutathione reductase, reduced glutathione, and oxidized glutathione were measured in gill and liver tissue after 24 and 72 h and 7, 14, 21, and 28 days of exposure, as well as after 16 days of recovery in Cu2+-free water. Cu2+ accumulated to a greater extent in the liver than in the gill (0.61-0.75 mg kg-1 vs. 24.0-69.9 mg kg-1 in L group and 0.98-1.47 mg kg-1 vs. 33.3-66.03 mg kg-1 in H group). In the gill, we observed increases in the activities of superoxide dismutase, catalase, and glutathione peroxidase, as well as in the concentrations of reduced glutathione and oxidized glutathione. In the liver of L group, we observed increases in glutathione reductase activity and in the levels of reduced glutathione and oxidized glutathione. In L group, the activity of superoxide dismutase and reduced glutathione content increased after 24 h and then decreased over time, while catalase and glutathione reductase activity and oxidized glutathione levels increased. Data from the recovery period indicated that higher concentrations of Cu2+ may induce irreversible oxidative damage to the gill of golden trout.

Neurobehavioral, apoptotic, and DNA damaging effects of sub-chronic profenofos exposure on the brain tissue of Cyprinus carpio L.: Antagonistic role of Geranium essential oil.

Currently, the contamination of water with different insecticides like profenofos (PFF) is a critical concern in the aquatic ecosystem. There are limited studies available on the negative impacts of PFF on common carp fish (Cyprinus carpio L.). Therefore, the existing study was designed to investigate the effect of PFF exposure (1/10 of the 96 h-LC50) on the neurobehavior, growth performance, chemical composition, oxidative status, DNA damage, apoptotic status and histological indices of the brain and gill tissues. In addition, this study seeks to detect the ability of geranium essential oil (GEO) dietary supplementation to mitigate the negative impacts of PFF. Accordingly, a total of 120 healthy fish were divided into four groups: the control group, fed on basal diet only; the other groups were fed on a basal diet supplemented with 400 mg kg-1 GEO, basal diet and PFF in water (PFF group), and supplemented diet with GEO and PFF in water (GEO + PFF), respectively, for 60 days. The results showed that PFF significantly reduced fish growth performance, crude protein, and lipid contents. It caused several behavioral alterations including spiral movement, decreased activeness, and changes in feeding behavior. Moreover, PFF increased the DNA tail length, tail moment, and the level of 8-hydroxy-2'-deoxyguanosine. Histologically, PFF induced a wide array of circulatory, inflammatory, regressive and progressive alterations in the brain and gill tissues. PFF significantly downregulated Bcl-2 and upregulated caspase-3 immuno-expression in both organs. Further, it considerably depleted the antioxidant enzymes, including superoxide dismutase, catalase, and glutathione peroxidase. The GEO supplementation did not reach the respective control values but markedly improved most of the behavioral, physical, biochemical, oxidative, apoptotic, and inflammatory markers, altered by PFF exposure. It also protected the gill and brain tissues from the branchial and encephalopathic effects of PFF. These findings suggest that GEO dietary supplements could be advantageous for mitigating PFF negative impacts and presenting a promising feed additive for common carp in aquaculture.

Effects of chlorpyrifos on the crustacean Litopenaeus vannamei.

Shrimps can be used as indicators of the quality of aquatic systems exposed to a variety of pollutants. Chlorpyrifos is one of the most common pesticides found in environmental samples. In order to evaluate the effects of chlorpyrifos, adult organisms of Litopenaeus vannamei were exposed to two sublethal concentrations of the pesticide (0.7 and 1.3 µg/L) for four days. The LC50 (96-hours) value was determined and Lipid oxidation levels (LPO) and the activities of catalase (CAT), glutathion peroxidase (GPx), glutathion-S-transferase (GST) were assessed on the muscle, hepatopancreas and gills from the exposed organisms. In addition, inhibition of acetylcholinesterase (AChE) was determined in the brain. LC50 (96-hours) was 2.10 µg/L of chlorpyrifos. Catalase activity and LPO were elevated in the three tissues, whereas a decrease of AChE activities in the brain and an increase of GST activity in the hepatopancreas were observed.

Effect of hexavalent chromium exposure on the liver and kidney tissues related to the expression of CYP450 and GST genes of Oreochromis niloticus fish: Role of curcumin supplemented diet.

The present study evaluated the adverse effects of the hexavalent chromium (Cr (VI)) at sub-lethal concentrations and the ameliorative potential of curcumin (CUR) over a sub-chronic exposure period on Oreochromis niloticus. Fish were exposed to Cr (VI) (4.57 mg/L) and CUR (0.02% in diet or 200 mg/kg diet), individually or in combination for 60-days. The growth rate during the period of experiment, condition factor, body composition, hepatosomatic index (HSI), hematological parameters, oxidative stress, apoptotic and DNA damage, branchial, hepato- and nephrotoxicity were estimated in this study. Moreover, the changes in mRNA expression of Cytochromes (CYP450) and glutathione S-transferase (GST) in kidney and liver tissues were assessed by qRT-PCR. Additionally, the concentration of metallothionine in the liver, histological investigation, and lesion scoring to the branchial, hepatic, renal and gill tissues were applied. The results revealed that Cr (VI) exposure caused a significant decline in most hematological variables and growth rate with down-regulation of CYP450 and GST expression. Histologically, Cr (VI) induced diverse forms of cell injury, vascular, and inflammatory alterations with upregulation of caspase-3 and downregulation of Bcl2 expression in the examined tissues. Additionally, it elevated the levels of serum MDA and 8-hydroxy-2' -deoxyguanosine than control. CUR-supplementation resulted in a significant improvement in most indices, amelioration of histological alterations and up-regulation of CYP450 and GST expression. These results may conclude that dietary supplements with CUR could be useful for modulation of the growth with protective effects to the branchial, hepatic, and renal tissues in response to Cr (VI) exposure, thereby presenting a promising feed additive for Nile tilapia in aquaculture.

Effect of zinc on growth performance and cellular metabolic stress of fish exposed to multiple stresses.

Global warming due to increasing temperature and contamination in aquatic environment has been found to be inducing cellular metabolic stress in fish. The present study focused on temperature and contamination in aquatic ecosystems and its alleviation/mitigation. Hence, this study was conducted to evaluate the role of zinc to improve growth performance, cellular metabolic stress, and digestive enzymes of the Pangasianodon hypophthalmus reared under lead (Pb) and high temperature. Two hundred and seventy-three fishes were distributed randomly into seven treatments, each with three replicates. Three isocaloric and isonitrogenous diets with graded levels of zinc at 0 mg/kg, 10 mg/kg, and 20 mg/kg were prepared. The Pb in treated water was maintained at the level of 1/21th of LC50 (4 ppm) and maintained at a temperature of 34 °C in exposure groups. The growth performance in terms of weight gain (%), protein efficiency ratio (PER), and specific growth rate (SGR) was found to be inhibited, and the feed conversion ratio (FCR) was enhanced in the Pb and high temperature-exposed group, whereas zinc supplementation has improved weight gain (%), FCR, PER, and SGR. The liver, gill, muscle, and kidney tissues of carbohydrate metabolic enzymes (LDH and MDH), protein metabolic enzymes (ALT and AST), and liver, gill, and muscle G6PDH and ATPase as well as intestinal digestives enzymes (proteases, amylase, and lipase) and intestinal ALP were significantly affected (p < 0.01) by Pb and high temperature exposure to P. hypophthalmus. We herein report the role of zinc in mitigating cellular metabolic stress in fish exposed to Pb and high temperature.