Lamellar Epithelium Research Articles

Oxidative, biochemical and histopathological alterations in fishes from pesticide contaminated river Ganga, India

The river Ganga basin accommodates large scale of agricultural activities, where large quantities of pesticides are applied. To assess the biological impact of pesticide residues that are continuously entering in the water body, biomarkers are common approach in bio-monitoring study as early warning signals to pollutants. In the present study qualitative and quantitative analyses of gill and liver histopathological and the biochemical parameters were determined in Rita rita and Cyprinus carpio. The difference in the histopathology and oxidative stress responses emphasize the response of selected fishes to the presence of contaminants in the water. Sloughing of lamellar epithelium and their disruption, dilated vessels, lamellar fusion, smooth muscle hypertrophy in the gill and necrosis, Vacuolation in hepatocytes, inflammation and distorted arterial walls were seen in the liver. The biochemical parameters were the main contributors to discriminate the changes in the fish physiology. In conclusion, the gill and liver histopathological responses, although not reflecting specific contaminants, but can be used as biomarkers of environmental contamination.

Air sac and gill vasotocin receptor gene expression in the air-breathing catfish Heteropneustes fossilis exposed to water and air deprivation conditions.

Heteropneustes fossilis is a facultative air-breathing freshwater catfish and inhabits ponds, ditches, swamps, marshes and rivers that dry up in summers. It possesses a pair of unique tubular accessory respiratory organ (air sac), which is a modification of the gill chamber and enables it to live in water-air transition zones. In the catfish, three vasotocin (Vt) receptor gene paralogs viz., v1a1, v1a2 and v2a were identified for Vt actions. In the present study, the receptor gene transcripts were localized in the gill and air sac by in situ hybridization, and their expression levels in relation to water and air deprivation conditions were investigated by quantitative RT-PCR. The catfish were exposed to 1h and 2h in gonad inactive (resting) and gonad active (prespawning) phases. The gene paralogs showed overlapping distribution in the respiratory epithelium of primary and secondary lamellae of gills and reduced lamellae of the air sacs. In water deprivation (forced aerial mode of respiration) experiment, v2a expression showed a high fold increase in the air sac, which was unchanged or inhibited in the gill. Both v1a1 and v1a2 expression was significantly upregulated in the air sac but showed varied responses in the gill. The gill v1a1 expression was unchanged in the resting phase and modestly upregulated in the prespawning phase. The gill v1a2 expression was modestly upregulated at 1h in both phases but unchanged at 2h. In the air deprivation experiment (forced aquatic respiration), the v2a expression in the air sac was inhibited except for a mild stimulation at 1h in the prespawning phase. In the gill, the v2a expression was stimulated with a steep upregulation at 2h in the prespawning phase. Both v1a1 and v1a2 expression was significantly high in the gill but only modestly increased or unchanged in the air sac. The expression patterns point to a functional distinction; the V2 type receptor expression was higher in the air sac during forced aerial respiration, and the V1 type receptor expression was highly prominent in the gill during forced aquatic respiration. Water and air deprivation treatments caused a significant increase in plasma cortisol level, and the stimulation was higher in the water deprivation fish in the resting phase but equally prominent in the water and air deprivation groups in the prespawning phase. The results indicate that the changes in the expression patterns of Vt receptor genes may be a sequel to stress (hypoxic, metabolic and osmotic), and both Vt and cortisol may interact to counter the stress responses. This study shows that Vt has a new role in the control of air sac functions.

Behavioral and histo-pathological indices of striped catfish (Pangasionodon hypophthalmus) exposed to different salinities

Salinity intrusion due to climate changes in the coastal area is substantially jeopardized for sustainable aquaculture. This study was focused on behavioral and histo-pathological changes (gills, liver and kidney) of striped catfish (Pangasionodon hypopththalmas) exposed to four salinities (0, 4, 8 and 12 ppt), each with three replications for 56 days. Both behavioral and histological analyses were carried out for durations of 7, 14, 28 and 56 days of exposure. Mild to moderate behavioral abnormalities such as rate of swimming, food intake, gill motion and convulsions were observed in 8 ppt, but pronounced alterations were detected at higher salinity (12 ppt), increasing with longer exposures. Similarly, mild to moderate histological anomalies in gills (hypertrophy of the chloride cell, telangiectasia at the tips of secondary lamellae, epithelial necrosis, fusion of some secondary lamellae and lifting of the lamellar epithelium), liver (vacuolation, blood congestion, melanomacrophage centers, patchy degeneration, necrosis and hypertrophy nucleus) and kidney (degeneration of renal tubule, enlarged Bowman's capsule, increased space in between glomerulus center, melanomacrophage centers, vacuolation and necrotic cell in the hematopoetic tissue) were detected in 8 ppt but pronounced alterations were detected at higher salinity (12 ppt) with increasing the days of exposure. This preliminary study revealed more profound responses to higher salinity exposure in the striped catfish, and this species could serve as a model bio-indicator in costal aquaculture.

Histology, physiology, and glucose and lipid metabolism of Lateolabrax maculatus under low temperature stress.

Spotted sea bass (Lateolabrax maculatus) is a popular and important commercial fish throughout the world, but it is unknown whether introducing domesticated fish to locations that experience cold weather might alter physiological performance. In this study, we evaluated the behavior, fatty acid content, histological analysis of liver and gills, liver enzymatic activity in carbohydrate and lipid metabolism, and gene expression in liver related to carbohydrate and lipid metabolism of spotted sea bass acclimatized at 22°C (control), 16°C, 10°C, 8°C, and 4°C for 24h, and 8°C for 4 days. When L. maculatus was exposed to acute cold stress for 24h, the gill showed curling, lamellar disorganization, lamellar epithelium hyperplasia, and formed aneurysms inside of the secondary lamellae. Long term stress over four days resulted in severe lamellar epithelium hyperplasia and curling. Continued extreme cold exposure (4°C) in L. maculatus caused liver HK, PK levels and LDH activities to achieve a peak value at 0h, and decreased over time. These indicated that glucose metabolism might play critical roles in the initial time of stress. Results of carbohydrate and lipid metabolism showed that lipids appear to play roles in prolonged cold stress. The constitutive transcriptional levels of six genes related to glucose (G6Pase) and lipid metabolism (PPAR-α, PPAR-γ) and mTOR signal pathway (eif4ebp1, eif4ebp2, mlst8) genes increased significantly in most groups during cold stress.

Toxicopathological impact of sub-lethal concentrations of lead nitrate on the gill of the catfish Heteropneustes fossilis

In recent studies, fish are heavily used as biomarkers of aquatic pollution, and heavy metals are among the main contributors to water pollution. In the present study, we investigated histopathological changes along with alterations in localization and activity of enzymes alkaline phosphatase (ALP), acid phosphatase (ACP), catalase (CAT), peroxidase (PER) and Na+/K+-ATPase in the gill tissues of Indian stinging catfish Heteropneustes fossilis exposed to two different concentrations (0.4 and 4 mg/L) of lead nitrate for 15 days. Histopathological examination of gill tissues revealed hypertrophy and swelling of epithelial cells, the fusion of epithelium of gill filaments and secondary lamellae, and alteration of secondary lamellae structure. Biochemical assays and histochemical localization show a pronounced effect on enzyme alkaline phosphatase activity and acid phosphatase in the gills of both groups of treated groups. In contrast, a significant decrease was noticed in the enzymatic response including catalase and peroxidase activity. Being a vital organ gill reflects the fish's physiological condition and the severity of the contamination in the surrounding environment. Gill is also the prime organ of osmoregulation in teleosts. Decreased activity of Na+/K+-ATPase suggests lead as a potent inhibitor of Na+/K+-ATPase that causes sodium hyperregulation. Alteration in the activity of metabolic enzymes reflects the level of tissue damage and metabolic disruption. At the same time, the increased activity of antioxidant enzymes states the condition of oxidative stress. Haematological parameters also altered with the lead nitrate exposure, reflecting metal toxicity and immune response against it. Meanwhile, this study also provides a potential use of H. fossilis as a biomarker for aquatic pollution.

К вопросу о морфофункциональном состоянии органов сома обыкновенного (Silurus glanis L.)

The state of the organs of wels catfish (Silurus glanis) caught in the Volga-Caspian fishery region have been analyzed. Destructive processes in the gill apparatus manifested in the changes of the epithelium of filaments and lamellae. Signs of hemosiderosis were found in the spleen. Necrotic areas and signs of fatty degeneration were found in the liver. Pathologies were observed in the stomach and intestines, manifested by edema and desquamation of the epithelium. All examined organs were characterized by signs of impaired blood microcirculation. The pathomorphological studies indicated the response of the fish organism to the environmental conditions. The revealed pathological signs served as red flags of nonspecific adaptation taking place at the cellular and tissue levels. Such responses allowed the fish to adapt and survive in the prevailing environmental conditions.

Histopathological study of parasitic effect of Dactylogyrus vastator (Nybelin, 1924) on the Catla catla in culture ponds in Darbhanga

The current investigation was undertaken to study the gill histopathologies due to infestation of helminth parasite, monogenetic trematode, Dactylogyrus vastator (Nybelin, 1924) in the Indian major carp, Catla catla. There were only symptomatic samples of diseased fishes comprised of fingerlings and adults of Catla catla collected and examined. Behaviour respond studied- partial suffocation, lethargic swimming and clinical sign observed as their gills were very pale in colour and had haemorrhagic and inflamed areas at the sites of parasite attachments. The significant histopathological lesions and changes were seen in the gills where the parasites were found attached to the lamellar tissues causing local erosions of the epithelium and hyperplasia of the secondary lamellar epithelium and fusion of adjacent secondary lamellae, vascular congestion leading to aneurysm besides, clumbing of the tips in secondary lamellae, congested gill showed accumulation of blood leading to swelling hypertrophy in secondary lamellae, autolysis of epithelium and secondary lamellae and severe edema and necrosis were observed in gill of C. catla. No changes were noticed in other organs of the affected fishes. In severe infestations the fishes showed growth retardation. The histopathological analysis for parasites in fish will be useful tool for fish health management.

Behavioral and histological features of zebrafish following sedation with eugenol or propofol

The use of anesthesia during short or lengthy procedures is scientifically and ethically required when dealing with fish. In this setting, the present study investigated whether a 3 h exposure to eugenol (4 and 8 mg/L) or propofol (0.2 and 0.4 mg/L) at sedative levels, followed by a 1 h wash-out time, would interfere with behavioral and histological characteristics of zebrafish. Anxiety-like phenotypes were assessed in the novel tank test using an automated tracking software, and histological features of the gills were determined through light microscopy. In the eugenol trial, zebrafish showed behavioral signs of complete recovery from sedation, but the novel environment diving response was amplified at both concentrations tested, thus pointing to the production of an anxiogenic phenotype. Furthermore, transitory alterations were implemented to increase the functional surface area of the gill and decrease lamellar epithelium thickness in order to improve gas exchange in the individuals bathed in 8 mg/L eugenol. Differently, the zebrafish submitted to 0.2 and 0.4 mg/L propofol presented an anti-anxiety response, since they dwelled much longer in the upper half of the tank; together with other behavioral endpoints which indicated a certain degree of inactivity, it seems that these fish were still under the effect of the drug, despite the 1 h wash-out, and thus neurological function had not been fully restored. Moreover, both concentrations of propofol induced shrinkage of the gill structure, which may have contributed towards an incomplete recovery; hence the behavioral pattern displayed in the novelty-based tank paradigm. This work reports contrasting behavioral and histological effects produced by eugenol and propofol upon zebrafish, thus demonstrating that standardization is a challenge with psychoactive drugs. Therefore, it is paramount to refine anesthetic protocols for this highly qualified test organism in order to maximize welfare and reduce experimental variability.

Effects of benzo[a]pyrene exposure on black rockfish (Sebastes schlegelii): EROD activity, CYP1A protein, and immunohistochemical and histopathological alterations.

Cytochrome P450 1A (CYP1A) is the major phase I of metabolic enzyme that plays essential roles in the detoxification of drugs and biotransformation of environmental pollutants. This study investigated CYP1A enzyme induction using EROD activity, CYP1A protein levels, and immunohistochemistry, along with histopathology of the liver, gills, kidneys, and intestine from the black rockfish, Sebastes schlegelii, exposed to benzo[a]pyrene (B[a]P). S. schlegelii has high risks of ingestion of sediment and absorption of heavy crude oil after accidental oil spills in Korea. This study thus exposed fish to B[a]P at 2, 20, and 200 μg/g body weight. EROD activity and CYP1A protein levels in hepatic microsomes had a positive correlation with the concentration of B[a]P (2-200 μg/g); in particular, exposure to 200 μg/g of B[a]P resulted in a 4- and 6-fold increase in hepatic EROD activity and CYP1A protein level, respectively. Hyperplasia of primary lamellar epithelium and atrophy of renal tubules were observed in the gills and kidney, respectively, following exposure to B[a]P at 200 μg/g. In contrast, severe histological alteration was not seen in intestinal tissues. Immunohistochemical analysis of the distribution of cellular CYP1A in four tissues showed strong immunostaining in the cytoplasm and nuclear membranes of the liver against B[a]P at 200 μg/g. Polycyclic aromatic hydrocarbons (PAHs), such as B[a]P, cause adverse histological changes in tissues of fish and provide evidence that PAH metabolism is inducible in fish liver, leading to increased CYP1A induction. Furthermore, the CYP1A induction in specific tissues might assist in monitoring and field assessment of marine ecosystems.

Effects of melatonin feed on histology and antioxidant ability of the gills and oxygen consumption of Chinese mitten crab (Eriocheir sinensis), exposed to acute hypoxia stress

The Chinese mitten crab (Eriocheir sinensis), as a benthic animal, adequate oxygen is very important for their life activities. This study was designed to evaluate the damage of the gill structure and function of E. sinensis under acute hypoxia (DO = 0.5 mgO2L−1,4 h) and the anti-injury effects of melatonin (MT). Histological examination showed predominantly regressive changes in the gills of crab exposed to hypoxia. The histoarchitecture of gills in the hypoxia group showed disrupted pillar cells (PC), the accumulation of hemocytes (HeM) in the hemocoelic space, lifting of lamellar epithelium and the appearance of bigger size of distal tips. Whereas, no such changes were found in MT group. Compared with the Control+hypoxia (C_hyp) group, the MDA of MT + hypoxia (MT_hyp) was significantly decreased (p < 0.05), while the Na+-K+ATPase content obviously increased in MT_hyp (p < 0.05). Hypoxia promoted the CAT and GSH-PX activities in gills, meanwhile, SOD, CAT and GSH-PX activities significantly increased in MT_hyp group after 4 h of hypoxia compared to Control+normoxia (C_nor) group (p < 0.05). Next, we observed the changes of oxygen consumption rate (OC) and the number times of opening-and-closing water inlet (OCWI) in each group within 1 h after reoxygenation. We found that both MT + normoxia (MT_nor) and MT_hyp groups in oxygen consumption rate after reoxygenation obviously higher than that of C_hyp and C_nor group at 2 mins. The number times of OCWI in C_hyp and MT_hyp groups were significantly higher than that of C_nor and MT_nor groups (p < 0.05) after reoxygenation for 2 mins. In summary, the number times of OCWI be used as an index to evaluate hypoxia for E. sinensis. MT is demonstrated as an antioxidant not only related to the antioxidant ability, but also to the protective gill structure and regulation respiratory for E. sinensis.

Quality of water from fish farms and histopathological analysis of tilapia ( Oreochromis sp.) in São José de Ribamar and Paço do Lumiar, state of Maranhão, Brazil

The present work aimed to use histopathological gill biomarkers in tilapia (Oreochromis sp.) and physicochemical and microbiological analyses of water in the evaluation of impacts to fish farms located in São José de Ribamar and Paço do Lumiar, state of Maranhão, northeastern Brazil. Histopathological analyses performed on gills indicated morphological alterations in all tissues analysed. The lesions found were as follows: epithelial lifting, lamellar epithelium hyperplasia, blood vessel congestion, lamellar fusion, blood sinus dilation and lamellar aneurysm. Temperature, D.O., salinity and nitrite values obtained were considered within recommended standards. The pH verified in a pisciculture located in Paço do Lumiar is considered potentially stressful to fish and, therefore, predisposing to pathologies. Ammonia in a fish farm in São José de Ribamar is considered sublethal. In all fish farms evaluated, the presence of high counts of total coliforms for fish farms of São José de Ribamar was verified. Escherichia coli was verified in a cultivation in São José de Ribamar indicating faecal contamination of the nursery water by the flow of some source of domestic effluent. The integration of studies with physical, chemical and biological variables generates important data to evaluate the quality of fish production.

Hydrogen peroxide treatment of Atlantic salmon temporarily decreases oxygen consumption but has negligible effects on hypoxia tolerance and aerobic performance

Hydrogen peroxide is widely used as a treatment of ectoparasite (e.g. salmon lice, amoebic gill disease) affected Atlantic salmon (Salmo salar) in aquaculture but has frequently caused unacceptable levels of mortality. Hydrogen peroxide toxicity increases with dose, exposure duration and temperature, and can cause an acute stress response, oxidative stress, gill damage, respiratory acidosis and altered gas and ion exchange. The combined stress effects have the potential to cause sub-lethal impacts on aerobic capacity that affects hypoxia tolerance and swimming performance. Here, we tested the sub-lethal effects of hydrogen peroxide treatment on the aerobic capacity, hypoxia tolerance and gill histopathology of healthy Atlantic salmon. Hydrogen peroxide treatment (1250 ppm at 12 °C for 20 min) caused a significant but negligible reduction in acute hypoxia tolerance (lost equilibrium at slightly higher levels of dissolved oxygen) immediately following treatment, but not in the 14 days afterwards. Immediately following hydrogen peroxide treatment (1250 ppm at 16 °C for 20 min) oxygen consumption was reduced for up to 20 min in normoxia and 40 min in hypoxia. However, following transient reductions in oxygen consumption the maximum metabolic rate and Ucrit measured in a swim flume were unaffected by hydrogen peroxide exposure (1250 ppm at 16 °C). Resting metabolic rate was unaffected by hydrogen peroxide treatment in all experiments. Hydrogen peroxide exposure caused non-homogenous lamellar epithelium lifting with subepithelial oedema and multi-focal lamellar fusion. The reduction in oxygen consumption following hydrogen peroxide treatment was likely caused by an increase in blood oxygen due to the breakdown of hydrogen peroxide by catalase and other oxidative enzymes in the fish tissues. Ultimately, hydrogen peroxide did not affect aerobic performance and had negligible impacts on hypoxia tolerance, suggesting that hydrogen peroxide has minimal sub-lethal impacts on aerobic performance of healthy Atlantic salmon at the doses used here.

Microplastics induced histopathological lesions in some tissues of tilapia (Oreochromis niloticus) early juveniles

Although microplastics (MPs) have received increasing focus and currently have become an emerging area of research, there is limited knowledge about their effect on whole body histology of fish. In this study, tilapia (Oreochromis niloticus) early juveniles were exposed to 1, 10, or 100 mg/L of MPs for 15 days and 15 days post-exposure, after which whole body histological examinations were performed. Histological analysis of kidney revealed congestion of blood capillaries, inflammatory cells, loss of basophilic cytoplasm in several tubules, vacuolated tubules, shrinking of convoluted tubules, widening of intertubular space, complete deformation, glomerular atrophy, vacuolated glomerular cells, and signs of fatty tubules. The liver tissue exhibited vacuoles, hydropic degeneration, necrotic area, severe deformation of hepatocytes, pyknotic nuclei, and dilation and congestion of blood sinusoids. The pancreatic tissue revealed shrunken and degenerated acini with pyknotic nuclei, hemorrhage, necrotic area, inflammatory cells, fatty cells, and congested blood capillaries. In the muscle tissue, fiber core dissociation, edema, necrosis, segmented fibers, and inflammatory cells were detected. The gill tissue demonstrated dilation and congestion of blood vessels, complete lamellar fusions, lifting of epithelium, shortening and degeneration of secondary lamellae, hyperplasia, and deposition of MPs between primary lamellae. In the spinal cord and notochord, the effects were degeneration and protrusion of meninges, deformation and deviation of notochord from its central axis, edema, degeneration of notochord (disappearance of vacuolar cells), deviation of spinal cord from the central axis, and loss of vacuolar cells in notochord. The intestinal tissue exhibited degeneration of basement membrane, inflammatory cells, goblet cells, atrophy of submucosa, pyknotic nuclei, hemorrhage, and vacuolization of mucosal cells. The histopathological changes in different organs were noticed even post-exposure in fish exposed to MPs compared to those in control fish and these changes were concentration dependent. In conclusion, these data together with our previous data suggest that MPs can cause different changes, ranging from biochemical alterations in single cells to lesions in the entire tissue, which can affect the vitality and life of fish.

Alterations in gills and intestine of Danio rerio after exposure to acaricide yoksorrun-5EC (hexythiazox): histopathologic and morphometric evaluation

In this study, histopathological and morphological changes in gill and intestinal tissue of zebra fish exposed to acaricide yoksorrun were aimed to determine. Yoksorrun was applied to the experimental groups as 0.01 mL/L, 0.02 mL/L, and 0.03 mL/L. Histopathological findings which showed a parallel increase with the amount of exposure in the gill were determined. In the gills, disruption of lamellae shape, shortening and breakage of primary and secondary lamellae, edema, fusion, and separation in the secondary lamellae epithelium, hyperplasia were observed. In the intestine tissue of some groups, advanced necrosis at the tip of the villi and deterioration of the overall integrity of the villi in these regions, epithelial hyperplasia, increasing in eosinophilic cells in the submucosa, and dissolution in muscle fibers of tunica muscularis were observed. In the morphometric analysis of the gills, a significant decrease in gas exchange (PAGE) percentages (p < 0.0001) was observed in the experiment group compared to the control group. Compared to the control group, a significant decrease in the interlamellar distance and secondary lamellar length measurements of the gills and a significant increase in the secondary lamellar width measurement were observed. In basal epithelial thickness measurement, the result was insignificant between the groups. According to the results of the morphometric analysis of the intestine, a significant decrease in musculus externa was observed only in the group 2. In total wall thickness, there was a significant thinning (p < 0.0001) in all experimental groups. There was a significant shortening of the villi length (p < 0.0001) and a significant increase (p < 0.0001) in the villus width only in the group 2 and group 3. A significant increase in epithelial thickness after application was observed (p < 0.0001) in all groups compared to the control group. Based on the findings, it was decided that the living in the aquatic ecosystem would be adversely affected by this acaricide if exposed.

Anesthetic potential of the essential oils of Aloysia triphylla, Lippia sidoides and Mentha piperita for Colossoma macropomum

The aim of this study was to evaluate the anesthetic potential of the essential oils of Aloysia triphylla (EOAT), Lippia sidoides (EOLS) and Mentha piperita (EOMP) in relation to juvenile tambaqui (Colossoma macropomum), based the swimming behavior of these fish and possible histopathological damage to the gills. To induce anesthesia, juvenile tambaqui (130.55 ± 22.4 g; 16.8 ± 0.95 cm) were individually exposed to different concentrations of the essential oils (EOAT: 5, 10, 20, 40, 60, 80 and 150 mg L−1; EOLS: 2, 5, 10, 20, 30, 40 and 50 mg L−1; EOMP: 5, 10, 20, 40, 60, 70 and 90 mg L−1), for exposure periods of between four minutes and six hours. The three essential oils evaluated presented anesthetic properties. The concentration intervals over which the fish reached all the stages of anesthesia (1, 2, 3a, 3b and 4) were between 40 and 150 mg L−1 for EOAT; 20 and 50 mg L−1 for EOLS; and 40 and 90 mg L−1 for EOMP. The times taken to reach the anesthesia stages were inversely proportional to the increases in the concentration of the essential oils. The histopathological damage to the gills of the tambaqui that resulted from using essential oil concentrations that were defined as anesthetic (150 mg L−1 EOAT, 30 mg L−1 EOLS and 90 mg L−1 EOMP) were also evaluated. The main damage recorded was hypertrophy and hyperplasia of the lamellar epithelium, lamellar fusion and proliferation of chloride cells. According to the histopathological alteration index (HAI), there were no irreversible damage (such as fibrosis and necrosis) to the gills of the tambaqui. However, while EOLS and EOMP caused mild damages in the gills, the EOAT generated more severe damages. Overall, for the tambaqui, the essential oils of A. triphylla, L. sidoides and M. piperita are efficient anesthetic in the doses of 150, 30 and 90 mg L−1, respectively.

A practical guide to unbiased quantitative morphological analyses of the gills of rainbow trout (Oncorhynchus mykiss) in ecotoxicological studies.

Rainbow trout (Oncorhynchus mykiss) are frequently used as experimental animals in ecotoxicological studies, in which they are experimentally exposed to defined concentrations of test substances, such as heavy metals, pesticides, or pharmaceuticals. Following exposure to a broad variety of aquatic pollutants, early morphologically detectable toxic effects often manifest in alterations of the gills. Suitable methods for an accurate and unbiased quantitative characterization of the type and the extent of morphological gill alterations are therefore essential prerequisites for recognition, objective evaluation and comparison of the severity of gill lesions. The aim of the present guidelines is to provide practicable, standardized and detailed protocols for the application of unbiased quantitative stereological analyses of relevant morphological parameters of the gills of rainbow trout. These gill parameters inter alia include the total volume of the primary and secondary gill lamellae, the surface area of the secondary gill lamellae epithelium (i.e., the respiratory surface) and the thickness of the diffusion barrier. The featured protocols are adapted to fish of frequently used body size classes (300–2000 g). They include well-established, conventional sampling methods, probes and test systems for unbiased quantitative stereological analyses of light- and electron microscopic 2-D gill sections, as well as the application of modern 3-D light sheet fluorescence microscopy (LSFM) of optically cleared gill samples as an innovative, fast and efficient quantitative morphological analysis approach. The methods shown here provide a basis for standardized and representative state-of-the-art quantitative morphological analyses of trout gills, ensuring the unbiasedness and reproducibility, as well as the intra- and inter-study comparability of analyses results. Their broad implementation will therefore significantly contribute to the reliable identification of no observed effect concentration (NOEC) limits in ecotoxicological studies and, moreover, to limit the number of experimental animals by reduction of unnecessary repetition of experiments.

Ultrastructural alteration in Gill and Hepatopancrease of freshwater prawn Macrobrachium rosenbergii exposed to 60Co gamma radiation.

The present study was designed to evaluate the impact of gamma radiation (60Co) on freshwater prawn Macrobrachium rosenbergii by using electron microscopic (SEM, TEM) studies. One set of prawns (experimental group) was irradiated (3, 30, 300, and 3000 mGy) by Theratron Phoenix TeleCobalt Unit [P-33], while other set of prawns (control group) was maintained (non-irradiated) separately. Scanning electron microscopic observations of gills and hepatopancreas showed fused and swollen lamella, abnormal gill tips, wrinkled lamellar epithelium, and necrotic epithelium surface in irradiated groups, while no such abnormalities were obvious in the control group. Transmission electron microscopic studies showed damaged nucleus, granulated mitochondria, vacuoles with crystalline granular inclusions, destructed membrane, vacuoles filled with granules, rough endoplasmic reticulum with residual bodies, shrunken mitochondria, dilated rough endoplasmic reticulum, and dilated cisternae of the Golgi body in irradiated groups. The structural abnormalities of vital organs could affect physiological functions such as respiration, osmo-ionic regulation and storage, secretion of the gills, and hepatopancreas, which in turn could adversely affect the growth and survivability of M. rosenbergii.

Biochemical and morphological biomarker responses in the gills of a Neotropical fish exposed to a new flavonoid metal-insecticide

The flavonoid metal-insecticide [Mg(hesp)2(phen)], denominated MgHP, has high potential for controlling agricultural pests. If applied in large scale, it may reach aquatic ecosystems and be harmful to the biota. This study evaluated the effects of MgHP in the gills of the Neotropical fish, Prochilodus lineatus by determining the activity of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione s-transferase (GST), and the levels of glutathione (GSH) and lipid peroxidation (LPO) after 24 and 96 h exposure to 0, 1, 10, 100 and 1000 μg L−1. The histopathological changes with emphases to mitochondria-rich cells (MRC) were evaluated as well. After 24 h exposure the enzyme activities and the GSH and LPO levels were unchanged however, after 96 h exposure to high MgHP concentration (1000 μg L−1), the GST activity and GSH levels increased. Oxidative stress measured as LPO levels did not occur after MgHP exposure in both periods. Gill tissue alterations increased after MgHP exposure to 10, 100 and 1000 μg L−1. Cellular atrophy, pillar cells changes, filament epithelium hyperplasia and hypertrophy, lamellar epithelium hyperplasia were the most frequent histopathology. MRC in the filament epithelium decreased after exposure to 24 h and increased after 96 h indicating possible transitory osmo-ionic disruption. P. lineatus exhibited high tolerance to MgHP. The increased GST activity and GSH levels after 96 h exposure suggested possible MgHP accumulation and concentration- and time-dependent response. Histopathology in the gills of exposed fish occurred at high MgHP concentrations. These results suggested that the MgHP into water, at high concentrations, affect the gills by changing GST activity, GSH levels and histology being useful biomarkers for MgHP water contamination.

Assessment of cytotoxicity in gills of fish Labeo rohita reared in untreated and treated sewage water.

The unregulated discharge of untreated municipal sewage water to the natural water bodies is a major threat to the aquatic ecosystems. In the present study, the fingerlings of Labeo rohita were exposed to treated sewage water and 1/10th of LC50 and 1/20th of LC50 of untreated sewage water (UT) obtained from sewage water treatment plant, Ludhiana, India. After determining 96-h LC50 value of UT, fingerlings were divided into four groups: control, treated, 1/10th of LC50 UT and 1/20th of LC50 UT and exposed for a period of 2months. Our study revealed that 1/10th LC50 UT and 1/20th LC50 UT groups had significant reduction in body weight, total body length, standard body length and gills somatic index in comparison with the control and treated groups. Histopathological alterations in cellular structure of gills such as ruptured primary lamellae, broken secondary lamellae, distorted chloride cells and goblet cells, missing lamellae and disintegrated lamellar epithelium were also observed in 1/10th LC50 UT and 1/20th LC50 UT groups. A significantly high frequency of micronucleated (MN) cells was observed in 1/10th LC50 UT and 1/20th LC50 UT groups along with the presence of binucleated cells (BN), elongated nuclei (EN) and nuclear buds in gill cells. Genotoxic nature of UT was further confirmed from significantly high values of genetic damage index (GDI) and percentage (%) DNA damage in gill cells of fingerlings exposed to sub-lethal concentrations of UT. The study concluded that untreated sewage water has potential to induce anatomical and physiological defects in gills cells and the severity of toxicity increases with the increase in duration of exposure.

Functional re-organization of the gills of metamorphosing sea lamprey (Petromyzon marinus): preparation for a blood diet and the freshwater to seawater transition.

Sea lamprey (Petromyzon marinus) begin life as filter-feeding larvae (ammocoetes) before undergoing a complex metamorphosis into parasitic juveniles, which migrate to the sea where they feed on the blood of large-bodied fishes. The greater protein intake during this phase results in marked increases in the production of nitrogenous wastes (N-waste), which are excreted primarily via the gills. However, it is unknown how gill structure and function change during metamorphosis and how it is related to modes of ammonia excretion, nor do we have a good understanding of how the sea lamprey's transition from fresh water (FW) to sea water (SW) affects patterns and mechanisms of N-waste excretion in relation to ionoregulation. Using immunohistochemistry, we related changes in the gill structure of larval, metamorphosing, and juvenile sea lampreys to their patterns of ammonia excretion (Jamm) and urea excretion (Jurea) in FW, and following FW to artificial seawater (ASW) transfer. Rates of Jamm and Jurea were low in larval sea lamprey and increased in feeding juvenile, parasitic sea lamprey. In freshwater-dwelling ammocoetes, immunohistochemical analysis revealed that Rhesus glycoprotein C-like protein (Rhcg-like) was diffusely distributed on the lamellar epithelium, but following metamorphosis, Rhcg-like protein was restricted to SW mitochondrion-rich cells (MRCs; ionocytes) between the gill lamellae. Notably, these interlamellar Rhcg-like proteins co-localized with Na+/K+-ATPase (NKA), which increased in expression and activity by almost tenfold during metamorphosis. The distribution of V-type H+-ATPase (V-ATPase) on the lamellae decreased following metamorphosis, indicating it may have a more important role in acid-base regulation and Na+ uptake in FW, compared to SW. We conclude that the re-organization of the sea lamprey gill during metamorphosis not only plays a critical role in allowing them to cope with greater salinity following the FW-SW transition, but that it simultaneously reflects fundamental changes in methods used to excrete ammonia.