Mucous Cell Density Research Articles

Mucosal epithelial homeostasis: Reference intervals for skin, gill lamellae and filament for Atlantic salmon and other fish species.

Mucosal barriers are gatekeepers of health and exhibit homeostatic variation in relation to habitat and disease. Mucosal Mapping technology provides an in-depth examination of the dynamic mucous cells (MCs) in fish mucosal barriers on tangential sections, about 90° from the view of traditional histology. The method was originally developed and standardized in academia prior to the establishment of QuantiDoc AS to apply mucosal mapping, now trademarked as Veribarr™ for the analysis of skin, gills and gastrointestinal tracts. Veribarr™ uses design-based stereology for the selection and measurement of cell area (size) (μm2), the volumetric density of MCs in the epithelium (MCD, amount of the epithelia occupied by MCs, in %) and the calculated abundance of the MCs (barrier status or defence activity). MC production was mapped across the skin and gill epithelia in 12 species, discovering that gills consistently have two distinct groups of MCs, one on the lamellae where MCs are few and small and one on the filament where MCs are larger and more abundant. MCs were usually much larger in the skin than in the gills, with the latter requiring fewer and smaller cells for adequate respiration. The difference observed between MCs in gill lamella and gill filament is likely a result of functional demands. In addition, our findings also highlight a variation in the mucosal parameters between the species skin, which cannot be explained by the weight differences, and a potential link between MC distribution and species-specific lifestyles in the gill lamella. This diversity necessitates the development of species and tissue site-specific reference intervals for mucosal health evaluation. Mucosal bivariate reference intervals were developed for MC production, including size (trophy) and calculated defence activity (plasia) in the skin and gills of Atlantic salmon, to contrast new measurements against historical data patterns. The application of mucosal reference intervals demonstrates that stress from parasites and treatments can manifest as changes in mucosal architecture, as evidenced by MC hypertrophy and hyperplasia within the gill lamellae. These reference intervals also facilitate comparisons with wild Atlantic salmon, revealing a somewhat higher MC level in farmed salmon gill lamellae. These findings suggest that MC hyperplasia and hypertrophy in the gills are stress/environmental responses in aquaculture. They also advocate for developing specific mucosal bivariate homeostatic reference intervals in aquaculture to improve fish health and welfare across all farmed species.

Fermentation and fortification of Sargassum linearifolium with multi-strain probiotics improves mucosal barrier status, inflammatory response and resistance to Vibrio harveyi infection in barramundi Lates calcarifer

While aquaculture intensification increases productivity and profitability, it can lead to increased susceptibility of fish to disease outbreaks. As such, the production of aquafeeds with immunomodulatory properties is currently of significant interest to the aquaculture sector with a view of improving on-farm welfare. This study aimed to assess the effectiveness of the brown seaweed Sargassum linearifolium fortified with the probiotics Lactobacillus casei and Saccharomyces cerevisiae through submerged fermentation in improving growth, overall health and the ability to resist Vibrio harveyi infection in barramundi, Lates calcarifer. Two seaweed-based diets, probiotic-fortified S. linearifolium (referred to as PF-SL) and S. linearifolium without probiotic fortification (referred to as SL), were compared with a standard barramundi diet (control). Barramundi juveniles weighing 4.25 ± 0.09 g were fed the test diets three times daily to satiety for 56 days. The results demonstrated a significantly higher growth performance in fish fed the PF-SL diet when compared to the control. However, there were no notable differences in the apparent digestibility coefficient (ADC) of dry matter, protein, and lipid between PF-SL and control. Fish fed the PF-SL diet showed improved mucosal barrier function in both the skin and intestine, as indicated by the increased density of mucous cells. Similarly, fish fed the PF-SL diet exhibited higher glutathione peroxidase and total antioxidant activity than the control. The qRT-PCR revealed significant upregulation in the expression of anti-inflammatory genes (occludin, claudin1 and nrf2) and downregulation in the expression of proinflammatory genes (hsp70 and tnf-α) in fish fed the PF-SL diet. Furthermore, the PF-SL diet resulted in a 27% mortality in a 14-day challenge trial with V. harveyi, which was lower than the mortality rates of the SL (46%) and control (67%). Overall, this study demonstrates that feeding juvenile barramundi with probiotic-fortified brown seaweed S. linearifolium was able to improve growth, immunity, and resistance against Vibrio harveyi infection, suggesting significant potential for application across the aquaculture industry for improved fish welfare.

The Effects of Stocking Density and Food Deprivation on Mucous Cells and Lysozyme Activity in the Skin and Gills of Silver Catfish.

This study aimed to identify the regions of the body surface of silver catfish (Rhamdia quelen) with a higher population of mucous cells in the skin. Additionally, the effects of stressful conditions, such as different stocking densities and food deprivation, on the proliferative response of mucous cells in the skin and gill epithelium and their impact on cutaneous mucous lysozyme activity were investigated. Silver catfish were divided into four experimental groups: high stocking density (32 kg/m3) and fed (HSD-F), high stocking density and fasted (HSD-FS), low stocking density (2.5 kg/m3) and fed (LSD-F), and low stocking density and fasted (LSD-FS). Fish in the fed groups received commercial feed twice a day, amounting to 1% of the tank biomass. After a 14-day experimental period, the fish were anesthetized and euthanized. Samples of cutaneous mucous and skin fragments from seven different points and the second left branchial arch were collected. Histological slides of the skin and gills were stained with PAS + Alcian Blue at pH 2.5, and the epidermal mucous lysozyme activity was assessed using the turbidimetric method. The ventral point in front of the ventral fin was found to be the optimal location for collecting cutaneous epithelia due to its higher density of mucous cells. The population of mucous cells in both the skin and gills varied based on the collection point and treatment applied. The highest lysozyme activity in the epidermal mucous was observed in fish from the HSD-F group. Overall, these findings suggest that stocking density and food deprivation create stressful conditions for silver catfish, which modulate their mucosal response to each situation.

Integrating chemical and biological data by chemometrics to evaluate detoxification responses of a neotropical bivalve to metal and metalloid contamination

Mangroves represent a challenge in monitoring studies due to their physical and chemical conditions under constant marine and anthropogenic influences. This study investigated metals/metalloids whole-body bioaccumulation (soft tissues) and the risk associated with their uptake, biochemical and morphological detoxification processes in gills and metals/metalloids immobilisation in shells of the neotropical sentinel oyster Crassostrea rhizophorae from two Brazilian estuarine sites. Biochemical and morphological responses indicated three main mechanisms: (1) catalase, superoxide dismutase and glutathione played important roles as the first defence against reactive oxygen species; (2) antioxidant capacity against peroxyl radicals, glutathione S-transferase, metallothionein prevent protein damage and (3) metals/metalloids sequestration into oyster shells as a mechanism of oyster detoxification. However, the estimated daily intake, target hazard quotient, and hazard index showed that the human consumption of oysters would not represent a human health risk. Among 14 analysed metals/metalloids, chemometrics indicate that Mn, As, Pb, Zn and Fe overload the antioxidant system leading to morphological alterations in gills. Overall, results indicated cellular vacuolization and increases in mucous cell density as defence mechanisms to prevent metals/metalloids accumulation and the reduction in gill cilia; these have long-term implications in respiration and feeding and, consequently, for growth and development. The integration of data from different sites and environmental conditions using chemometrics highlights the main biological patterns of detoxification from a neotropical estuarine bivalve, indicating the way in which species can cope with metals/metalloids contamination and its ecological consequences.

Tocopherol Enhances the Antioxidant Defense System and Histomorphometric Parameters in The Gastrointestinal Tract of Rats Treated with Sodium Arsenite.

Arsenic compromises the gastrointestinal integrity and function via the body's anti-oxidative system breakdown. Hence, this study aimed to investigate the effects of tocopherol on redox imbalance and histoarchitectural alterations in rats' gastrointestinal tract exposed to sodium arsenite. Sodium arsenite and graded doses of tocopherol were administered orally into experimental rats assigned to different groups for four weeks concurrently. Redox status assay was done in homogenized samples by spectrophotometry. Parietal cell mass and mucous cell density (stomach), villus height and crypt depth (ileum), goblet cells count, and crypt depth (colon) were evaluated by histomorphometry. Inflammatory cells infiltration was also assessed using a semi-quantitative procedure.Sodium arsenite caused a significant increase in Malondialdehyde and Myeloperoxidase but, decreased Superoxide dismutase, Catalase, Nitric oxide, Glutathione peroxidase, Glutathione, and Glutathione-S-Transferase. Tocopherol treatment reversed the changes (p<0.05) though not largely dose-dependent. Furthermore, tocopherol annulled sodium arsenite-induced increase in parietal cell mass and decrease in mucous cell density in the stomach, decrease in villus height and villus height/crypt depth ratio in the ileum, and decrease in goblets cells and increase in crypt depth in the colon. Moreover, activated inflammatory cell infiltration by sodium arsenite was mitigated by tocopherol. Sodium arsenite provokes not only marked inflammatory cellular infiltration but a focal loss of glands, hyperplasia of crypts, atrophic villi, and hypertrophy of Peyer's patches in the intestines, which are all lessened with tocopherol treatment. These findings underscore the anti-oxidative properties of tocopherol as a potent dietary factor against sodium arsenite toxicity in the gastrointestinal tract. Keywords: Tocopherol, arsenic, stomach, ileum, colon.

Nutrient Digestibility, Growth, Mucosal Barrier Status, and Activity of Leucocytes From Head Kidney of Atlantic Salmon Fed Marine- or Plant-Derived Protein and Lipid Sources.

Nutrient digestibility, growth, and mucosal barrier status of fish skin, gills, and distal intestine were studied in Atlantic salmon fed feeds based on marine or plant-derived ingredients. The barrier status was assessed by considering the expression of four mucin genes, five genes that encode antimicrobial proteins, distal intestine micromorphology, and design-based stereology of the midgut epithelium. In addition, the head kidney leukocytes were examined using flow cytometry; to understand the differences in their counts and function. Five experimental feeds containing the main components i) fishmeal and fish oil (BG1), ii) soybean meal (BG2; to induce enteritis), iii) fishmeal as the main protein source and rapeseed oil as the main lipid source (BG3), iv) a mix of plant protein concentrates as the protein sources and fish oil as the lipid source (BG4), and v) plant and marine ingredients in the ratio 70:30 (BG5) were produced for the study. Atlantic salmon with initial weight 72.7 ± 1.2 g was offered the experimental feeds for 65 days. The results revealed that the weights of all fish groups doubled, except for fish fed BG2. Fish fed the BG2 diet had lower blood cholesterol concentration, developed enteritis, had lower expression of muc2 in the distal intestine, and had a compromised barrier status in the intestine. Expression of both the mucin genes and genes that encode antimicrobial peptides were tissue-specific and some were significantly affected by diet. The fish fed BG1 and BG3 had more head kidney lymphocyte-like cells compared to BG5-fed fish, and the phagocytic activity of macrophage-like cells from the head kidney was the highest in fish fed BG1. The intestinal micromorphology and the mucosal mapping suggest two different ways by which plant-based diets can alter the gut barrier status; by either reducing the mucous cell sizes, volumetric densities and barrier status (as noted for BG2) or increasing volumetric density of mucous cells (as observed for BG4 and BG5). The results of the compromised intestinal barrier in fish fed plant ingredients should be further confirmed through transcriptomic and immunohistochemical studies to refine ingredient composition for sustainable and acceptable healthy diets.

Inflammatory reactions in rainbow trout fins and gills exposed to biocides.

Several biocides are widely used in rainbow trout aquaculture against various ectoparasites and ectobionts, but the inflammation induced in treated fish is less well described. Dose-response studies were conducted to elucidate the effects on rainbow trout (gills and fins) induced by a series of biocides including formalin, hydrogen peroxide (H2O2), peracetic acid (PAA) and the surfactant SPH6, which was isolated from the bacterium Pseudomonas H6. The compounds have documented antiparasitic effects, but the specific effects on fish needs further documentation. This study was performed over 24 h, and inflammatory reactions were evaluated in gills and fins. A dose-dependent effect was noted for expression of immune genes encoding for IL-1β, TNFα, IFNγ, IL-10, IL-8, lysozyme, serum amyloid A (SAA), hepcidin, precerebellin and complement factor C3. PAA induced the strongest upregulation of cytokine and acute phase reactant genes followed by H2O2 and formalin. SPH6 showed a lower effect, and in several cases the compound induced downregulation of several genes. Gills showed a stronger response compared to fins. The mucous cell density in fins showed a range of changes which varied by compound. PAA, and to a lesser degree H2O2 and formalin, initially induced mucous cell hyperplasia, whereas SPH6 immediately decreased the number of cells containing mucus.

Dynamic morphometrics of mucous cells reveal the minimal impact of therapeutic doses of peracetic acid on Atlantic salmon gill health

Mucous cells, the microscopic structural hallmark of mucosal surfaces, are highly responsive to environmental changes. Here we report how the gills of Atlantic salmon (Salmo salar) smolts responded to peracetic acid (PAA), a potent oxidative disinfectant and a candidate chemotherapeutant in aquaculture, through the Mucosal Mapping showing mucous cell size, volumetric density and defence activity, coupled with two-way histopathological scoring strategies. Two hundred and forty smolts were exposed to therapeutic doses of PAA on two occasions. The initial exposure included a 5-min bathing at concentrations of 0, 0.6, 1.2, and 2.4 ppm PAA. After a two-week recovery, the treatment groups were re-exposed to the same PAA concentrations for 30 min. Gill samples were collected at 2 h, 2 days, and 2 weeks after each exposure. The dynamic changes (i.e., size, volumetric density and defence activity) of the mucous cells were analysed on the distinct mucous cell populations in the gill filament and the lamella, as well as the lamellar thickness. Lamellar mucous cells were always significantly smaller (<70 μm2) and less dense (<2% volume) than those in the filament (70–100 μm2 and 8–11% volume) giving defence activities (combination of mean mucous cell area and volumetric density in a given tissue) of 0.1–0.4 for lamellae and about 0.6–1.4 for filaments, consistent with the functions of these branchial areas. A transient sub-acute mucous cell hypertrophy was a striking response in the gill lamella to PAA during the initial and re-exposure, particularly demonstrated by the groups exposed to 0.6 and 1.2 ppm where this size change was significant. Nonetheless, the recovery was quick, suggesting more an effect of general stress than dose of PAA. Similarly, a transient reduction in hyperplasia was noted as mucous cell density decreased on the filament, but generally significant hyperplasia was not detected, and volumetric density remained unaltered regardless of treatment doses and duration of exposure. The defence activity of the lamella and the filament demonstrated a transient tendency to decrease after the initial exposure but was minimally affected by re-exposure. Lamellar thickness was not markedly affected by the highest PAA dose and overall was positively correlated with mucous cell size. The results from two independent histopathological scorings revealed that at least 93% of the evaluated gill filaments per fish were categorised as healthy. Taken together, mucous cells in the gills of salmon smolts responded with transient hypertrophy of mucous cells to therapeutic doses of PAA however the impact was minimal, and the mucosal morphometrics were in agreement with the marginal alterations in tissue structure and integrity. The data suggest that the PAA doses used in the study are safe for salmon and do not pose substantial impact on gill mucosal health.

Sedative and anesthetic potential of the essential oil and hydrolate from the fruit of Protium heptaphyllum and their isolated compounds in Colossoma macropomum juveniles

This study aimed to evaluate the sedative and anesthetic potential of the essential oil (PHEO) and hydrolate (PHH) from the fruit of Protium heptaphyllum and the isolated compounds (IC) of PHEO in tambaqui (Colossoma macropomum) juveniles. Fish were placed in aquaria with different concentrations of PHEO, PHH or IC (sabinene, terpinen-04-ol, γ-terpinene and α-pinene) to evaluate sedative and anesthetic efficacy. In another experiment, fish were transported for 22 h with 10% anesthetic concentrations added to the water of transport. No similarities regarding the major compounds of PHEO and PHH were observed. No mortality was observed during the experiments, and PHH did not show any sedative or anesthetic effect. PHEO induced sedation at 250 mg L−1 and deep anesthesia at 500, 750 and 1000 mg L−1. The recovery time increased with increasing concentration of PHEO. There was a significant difference in the time to induce deep anesthesia between PHEO and IC. Water conductivity and mucous cell density were lower in the transport with 50 mg L−1 PHEO. Tambaqui transported with 50 mg L−1 PHEO and 11.6, 17.4, and 23.2 mg L−1 terpinen-04-ol presented lower net whole body K+ and Cl− effluxes. PHEO is an alternative as sedative and anesthetic for tambaqui juveniles, and the concentration of 500 mg L−1 was the best for deep anesthesia. The use of 50 mg L−1 PHEO was effective for the transport of this species because it reduced net K+ and Cl− effluxes and did not stimulate proliferation mucous cells.

Ablation of IL-33 Suppresses Th2 Responses but Is Accompanied by Sustained Mucus Obstruction in the Scnn1b Transgenic Mouse Model.

Cystic fibrosis is characterized by dehydration of the airway surface liquid layer with persistent mucus obstruction. Th2 immune responses are often manifested as increased mucous cell density (mucous cell metaplasia) associated with mucus obstruction. IL-33 is a known inducer of Th2 immune responses, but its roles in mucus obstruction and related phenotypes in a cystic fibrosis-like lung disease model (i.e., Scnn1b-Tg-positive [Tg+]) mouse, remain unclear. Accordingly, IL-33 knockout (IL-33KO) Tg+ mice were examined and compared with IL-33 heterozygous (IL-33HET) Tg+ mice. As compared with IL-33HET/Tg+ mice, IL-33KO/Tg+ mice had complete absence of bronchoalveolar lavage fluid eosinophilia, accompanied with significant reduction in bronchoalveolar lavage fluid concentration of IL-5, a cytokine associated with eosinophil differentiation and recruitment, and IL-4, a major Th2 cytokine. As compared with IL-33HET/Tg+ mice, IL-33KO/Tg+ mice had significantly reduced levels of Th2-associated gene signatures (Slc26a4, Clca1, Retnla, and Chi3l4), along with complete loss of intracellular mucopolysaccharide staining in the airway epithelium. As compared with IL-33HET/Tg+ mice, although the IL-33KO/Tg+ mice had significantly reduced levels of MUC5AC protein expression, they showed no reduction in the degree of mucus obstruction, MUC5B protein expression, bacterial burden, and neonatal mortality. Interestingly, the histological features, including subepithelial airway inflammation and alveolar space enlargement, were somewhat exaggerated in IL-33KO/Tg+ mice compared with IL-33HET/Tg+ mice. Taken together, our data indicate that although IL-33 modulates Th2 inflammatory responses and MUC5AC protein production, mucus obstruction is not dependent on IL-33.

Antioxidative, histological and immunological responses of rainbow trout after periodic and continuous exposures to a peracetic acid-based disinfectant

Peracetic acid (PAA)-based disinfectants are considered as sustainable alternatives in aquaculture due to their harmless residues from spontaneous decay. The key components of PAA-based disinfectants are PAA and hydrogen peroxide (H2O2). Little is known if the exposure to exogenous PAA and H2O2 from PAA-based disinfectants interferes with endogenous reactive oxygen species (ROS) regulation and innate immunity in fish. In the present study, rainbow trout (Oncorhynchus mykiss) were exposed to a PAA-based disinfectant for 6 weeks in flow-through systems by either periodic (pulse) or continuous modes. For the periodic exposure, a single dose of 1 mg L−1 PAA (and 1.4 mg L−1 H2O2) was applied biweekly and spontaneously degraded within hours. For the continuous exposure, a constant drip was applied which aimed to maintain 0.2 mg L−1 PAA (and 0.28 mg L−1 H2O2) in the inflow water but was counteracted by escalating degradation by the second week. The exogenous PAA/H2O2 elevated the level of endogenous total free radicals (TFR), probably through diffusion at the gill, as well as stress-activated endogenous generation (by periodic exposure only at the higher PAA/H2O2 dose). In response, the total antioxidant capacity (TAC) in gill and serum was significantly elevated. Liver showed no significant changes in the levels of TFR and TAC. Epidermal mucous cell density was significantly lower in response to persistent scarce exogenous PAA/H2O2 of the continuous exposure compared to the control. This sensitive response was absent in the periodic exposure probably due to rapid epidermal recovery during the exposure intervals. In contrast, the branchial structural adjustment of trout in response to exogenous ROS was less sensitive. Minimal hyperplasia of lamellae was present only in the periodic exposure. Enzymes responsible for innate cutaneous and humoral immunity including lysozyme, alkaline phosphatase, myeloperoxidase and esterase were not influenced by either exposure. However, ceruloplasmin was highly sensitive to exogenous PAA/H2O2; it was significantly elevated in fish skin, independent of the exposure modes, suggesting potential antioxidant protection of ceruloplasmin in fish skin. In serum, ceruloplasmin reduction was positively correlated to the reduction of antiprotease activity which indicates that ceruloplasmin may have anti-proteolytic function in fish blood. In conclusion, both exposure modes of the PAA-based disinfectant triggered mild antioxidant defenses in rainbow trout. To minimize the risk of oxidative damage while applying PAA-based disinfectants, sufficient intervals between periodic applications might be a better option than the persistent low PAA/H2O2 concentration of the continuous application.

Hydrolate toxicity of Lippia alba (Mill.) N. E. Brown (Verbenaceae) in juvenile tambaqui (Colossoma macropomum) and its potential anaesthetic properties

The study aimed to establish the lethal concentration at 96 h (LC50-96h) of hydrolate of Lippia alba (citral chemotype) (LAH) in juvenile tambaqui (Colossoma macropomum), as well as to identify the anaesthetic induction and recovery times. The acute toxicity test LC50-96h was performed in five animals per replicate (three replicates per treatment). The concentrations tested were 5, 6, 7 and 8% hydrolate diluted in water, and a control treatment containing only water. During the LC50-96h test, the physical and chemical parameters of the water were evaluated (temperature, dissolved oxygen, pH, and electrical conductivity). Water was also collected at the beginning and end of the experiment for the analysis of alkalinity, hardness, total ammonia, Na+, K+ and Cl− levels. Gills were collected from the survival fish to determine the density of gill mucous cells (DGMC). The concentrations of 10, 15, 20 and 25% of the hydrolate diluted in water were used for anaesthesia tests, which lasted up to 30 min (15 animals/treatment). After anaesthesia, the recovery time was evaluated. The toxicity test indicated LC50-96h 7.43% (confidence interval: 6.9–8.2%). During LC50-96h, ion loss and increased mucous gill cell density was observed in treatments with LAH. However, a long exposure time (up to 96 h) at lower concentrations (6, 7 and 8%) of the by-product caused osmoregulatory (ion loss) and cellular (increased DGMC) disturbances. The data also indicated that increasing the concentration of hydrolate proportionally decreases the time for anaesthesia induction. Anaesthesia recovery time was not significantly affected by LAH concentration. The concentrations of 20 and 25% of LAH showed the best times for anaesthesia induction in juvenile tambaqui (1.76 min and 2.01 min, respectively).

Effects of salinity on the growth and mucous cells of the abalone Haliotis diversicolor Reeve, 1846

This study was conducted to determine the influence of salinity on the growth of abalone Haliotis diversicolor Reeve, including the density and size of mucous cells. Abalone individuals were reared in the laboratory at salinities of 20, 25, 31, 35 and 40 ppt. The mucous cells of the lips, gills and digestive gut of H. diversicolor, which react to some forms of stress such as suboptimal salinity, were characterized following staining with Alcian Blue–Periodic Acid–Schiff`s Reagent (AB–PAS). The specific growth rate in wet weight and shell length of H. diversicolor were highest at 31 ppt and lowest at 20 ppt (0.52 vs 0.15% d−1, and 0.058 vs 0.021 mm d−1, respectively). The abalone H. diversicolor tolerated salinity fluctuations within the range of 20–40 ppt, but growth was optimum at 25–35 ppt. Mucous cells of the lips and gills showed significant differences (ANOVA, df = 4, P = <0.001) in cell density and cell size, being less dense and larger at 31 ppt than at 40 ppt, which could be an effect of osmotic and ionic regulation. Consistent with reports in literature, salinity ranges of 25–35 ppt are suitable for growth of H. diversicolor. Results of this study indicated that areas with such salinity are favorable for stock enhancement and mariculture of the abalone H. diversicolor.

The use and effects of hydrogen peroxide on salmon lice and post-smolt Atlantic salmon

When salmon lice, Lepeophtheirus salmonis, infect Atlantic salmon, they are typically removed by chemical or mechanical treatments. Delousing side effects can lead to poor welfare outcomes for fish, however reducing lice abundance in farms is crucial to reduce their ecological impacts. To determine which delousing treatments causes the highest salmon mortality, we used national-level databases to compare mortality for all delousing treatments (i.e. in-feed, bath, well-boat based, cage-based, thermal and mechanical treatments) used from 2012 to 2015 in Norway. As hydrogen peroxide (H2O2) is one of the riskiest delousing treatments with high mortality levels, we determined how hydrogen peroxide concentration (0, 1, 1.25, 1.5, 1.75, 2 and 2.25gL−1) for 20min at 10°C influenced post-smolt salmon mortality and mucous cells, pre-adult lice attachment, and the re-infection success of lice copepodids. H2O2 concentration strongly affected salmon mortality, but did not alter mucous cell area or density, pre-adult lice removal efficiency, or the re-infection success of lice copepodids. We recorded salmon mortality of 0% at 1gL−1, 18% at 1.5gL−1 and 100% at 2.25gL−1. Further, previous exposure to lice had no effect on salmon welfare or the likelihood of re-infection, as salmon mortality and copepodid infection success did not differ for salmon infected with lice and treated, or salmon not infected with lice and treated. We found discrepancies in salmon mortality between our experiment and industry observed mortality, with higher mortalities observed in our experiment compared to the industry. This is most likely due to the overestimation of water volume during in-cage treatments at farms, and also less organic matter present during our experiment. As we found no difference in lice removal efficacies between 1 and 2gL−1 and salmon mortality was high at concentrations of 1.5gL−1 upwards, we do not recommend increasing H2O2 dosage during treatment as a form of combatting H2O2 resistance in lice.

Lippia alba(Verbenaceae) hydrolate as sedative of tambaqui (Colossoma macropomum) juveniles in simulated transport conditions

Lippia alba hydrolate (LAH) is produced as a secondary product in essential oil extraction. The objective of this study was to evaluate the use of LAH in a simulation of the transport of tambaqui (Colossoma macropomum) juveniles. A series of 17 hr simulations were performed using load densities of 30, 60 and 90 juveniles per L and four LAH concentrations of 0.4%, 1%, 2% and 5%, with a control of just water. Fish survival, temperature, dissolved oxygen, conductivity, pH, ammonia excretion, alkalinity water hardness and net Na+, K+, Cl- fluxes, were all evaluated before and after the transport simulations. Mucous cell densities in the gills were also determined microscopically, post simulation. The results showed that addition of 5% LAH achieved better survival during the simulation than the water control. Dissolved oxygen was significantly lower with an increased load density. Also, conductivity, NH4 excretion and net ionic fluxes (K+ and Cl−) were lower with 5% LAH, although mainly where higher load densities were tested. Mucous cell density was lower in LAH concentrations of 2% and 5%, again with higher load densities. In view of these findings, it is suggested that 5% LAH is an effective sedative for use in the transport of tambaqui, mainly with high load densities. This work is the first study of LAH and highlights its potential applications in aquaculture management.

Preclinical mouse model to monitor live Muc5b-producing conjunctival goblet cell density under pharmacological treatments.

PurposeModification of mucous cell density and gel-forming mucin production are established hallmarks of mucosal diseases. Our aim was to develop and validate a mouse model to study live goblet cell density in pathological situations and under pharmacological treatments.MethodsWe created a reporter mouse for the gel-forming mucin gene Muc5b. Muc5b-positive goblet cells were studied in the eye conjunctiva by immunohistochemistry and probe-based confocal laser endomicroscopy (pCLE) in living mice. Dry eye syndrome (DES) model was induced by topical application of benzalkonium chloride (BAK) and recombinant interleukine (rIL) 13 was administered to reverse the goblet cell loss in the DES model.ResultsAlmost 50% of the total of conjunctival goblet cells are Muc5b+ in unchallenged mice. The decrease density of Muc5b+ conjunctival goblet cell population in the DES model reflects the whole conjunctival goblet cell loss. Ten days of BAK in one eye followed by 4 days without any treatment induced a −18.3% decrease in conjunctival goblet cell density. A four days of rIL13 application in the DES model restored the normal goblet cell density.ConclusionMuc5b is a biological marker of DES mouse models. We bring the proof of concept that our model is unique and allows a better understanding of the mechanisms that regulate gel-forming mucin production/secretion and mucous cell differentiation in the conjunctiva of living mice and can be used to test treatment compounds in mucosal disease models.

Parasites and a host's sense of smell: reduced chemosensory performance of fathead minnows (Pimephales promelas) infected with a monogenean parasite.

Parasites residing within the central nervous system of their hosts have the potential to reduce various components of host performance, but such effects are rarely evaluated. We assessed the olfactory acuity of fathead minnows (Pimephales promelas) infected experimentally with the monogenean Dactylogyrus olfactorius, the adults of which live within the host's olfactory chambers. Olfactory acuity was compared between infected and uninfected hosts by assessing electro-olfactography (EOG) neural responses to chemical stimuli that indicate the presence of food (L-alanine) or the presence of conspecifics (taurocholic acid). We also compared differences in gross morphology of the olfactory epithelium in infected and uninfected minnows. Differences in EOG responses between infected and uninfected minnows to both cue types were non-significant at 30days post-exposure. By days 60 and 90, coincident with a two times increase in parasite intensity in the olfactory chambers, the EOG responses of infected minnows were 70-90% lower than controls. When infected fish were treated with a parasiticide (Prazipro), olfactory acuity returned to control levels by day 7 post-treatment. The observed reduction in olfactory acuity is best explained by the reduced density of cilia covering the olfactory chambers of infected fish, or by the concomitant increase in the density of mucous cells that cover the olfactory chambers. These morphological changes are likely due to the direct effects of attachment and feeding by individual worms or by indirect effects associated with host responses. Our results show that infection of a commonly occurring monogenean in fathead minnows reduces olfactory acuity. Parasite-induced interference with olfactory performance may reduce a fish's ability to detect, or respond to, chemical cues originating from food, predators, competitors or mates.

Alterations in the skin of Labeo rohita exposed to an azo dye, Eriochrome black T: a histopathological and enzyme biochemical investigation.

Histopathological changes and alterations in the activity of certain metabolic and antioxidant enzymes were analyzed in the head skin of Labeo rohita, exposed to sublethal test concentrations of the azo dye, Eriochrome black T for 4days, using 24h renewal bioassay method. Hypertrophied epithelial cells, increased density of mucous goblet cells, and profuse mucous secretion at the surface were considered to protect the skin from toxic impact of the azo dye. Degenerative changes including vacuolization, shrinkage, decrease in dimension, and density of club cells with simultaneous release of their contents in the intercellular spaces were associated to plug them, preventing indiscriminate entry of foreign matter. On exposure of fish to the dye, significant decline in the activity of enzymes-alkaline phosphatase, acid phosphatase, carboxylesterase, succinate dehydrogenase, catalase, and peroxidase-was associated with the binding of dye to the enzymes. Gradual increase in the activity of lactate dehydrogenase was considered to reflect a shift from aerobic to anaerobic metabolism. On transfer of azo dye exposed fish to freshwater, skin gradually recovers and, by 8days, density and area of mucous goblet cells, club cells, and activity of the enzymes appear similar to that of controls. Alteration in histopathology and enzyme activity could be considered beneficial tool in monitoring environmental toxicity, valuable in the sustenance of fish populations.

Salinity influence on growth, osmoregulation and energy turnover in juvenile pompano Trachinotus marginatus Cuvier 1832

The effects of salinity on growth and parameters related to osmoregulation and energy turnover were evaluated in juvenile pompano Trachinotus marginatus. In a first experiment, fish were reared at different salinities (4, 8, 12, 16 and 20ppt) for 15days. No fish mortality was observed. Also, no significant difference in fish size (length and weight), hematocrit and plasma glucose concentration was observed among treatments. Although a significant increase in plasma Na+, Cl− and K+ concentrations was noted with increasing water salinities, while no significant changes in plasma osmolality were observed. The isosmotic point was calculated as 357.5mOsm/kg H2O, corresponding to the osmolality of the ambient water at salinity 13.1ppt. These findings indicate that juvenile pompanos T. marginatus acclimated to 20ppt are fully acclimated to a wide range of salinities (4–20ppt) after 15days of transfer to the new salinity condition. Also, they show that the plasma isosmotic point of juvenile T. marginatus (13ppt) is within the range of values reported for some euryhaline teleost fish from different habitats (10–14ppt). In a second experiment, fish were reared at different salinities (3, 6, 12 and 32ppt) for 28days. No significant difference in survival, feed conversion, Fulton's condition factor, body weight coefficient of variation, feed intake and intestinal trypsin activity was observed among salinity treatments. However, fish reared at 32ppt showed lower body weight, specific growth rate, oxygen consumption, and gill Na+,K+-ATPase activity than those reared at 3 and 6ppt. Also, their gills showed higher chloride cell area, epithelium thickness, chloride cell density and mucous cell density than those of fish reared at 3, 6 and 12ppt. These findings indicate that juvenile pompano T. marginatus is euryhaline in the range of salinities tested (3–32ppt) and grows better when reared at low salinities (3–6ppt) for 28days. This performance is associated with a good osmoregulatory ability resulting from structural and biochemical adaptive responses of the gill epithelium to salinity changes paralleled by increased aerobic metabolism and elevated use of liver carbohydrate store. Although it would be cost effective to maintain higher salinities in coastal aquaculture systems given the proximity to the marine environment, our findings support the idea of rearing juvenile pompano T. marginatus in low salinities (3–6ppt) to improve growth. Statement of relevancePompano rearing performance is improved in low salinities.

Dietary Mannan Oligosaccharides: Counteracting the Side Effects of Soybean Meal Oil Inclusion on European Sea Bass (Dicentrarchus labrax) Gut Health and Skin Mucosa Mucus Production?

The main objective of this study was to assess the effects of 4 g kg−1 dietary mannan oligosaccharides (MOS) inclusion in soybean oil (SBO)- and fish oil (FO)-based diets on the gut health and skin mucosa mucus production of European sea bass juveniles after 8 weeks of feeding. Dietary MOS, regardless of the oil source, promoted growth. The intestinal somatic index was not affected, however dietary SBO reduced the intestinal fold length, while dietary MOS increased it. The dietary oil source fed produced changes on the posterior intestine fatty acid profiles irrespective of MOS dietary supplementation. SBO down-regulated the gene expression of TCRβ, COX2, IL-1β, TNFα, IL-8, IL-6, IL-10, TGFβ, and Ig and up-regulated MHCII. MOS supplementation up-regulated the expression of MHCI, CD4, COX2, TNFα, and Ig when included in FO-based diets. However, there was a minor up-regulating effect on these genes when MOS was supplemented in the SBO-based diet. Both dietary oil sources and MOS affected mean mucous cell areas within the posterior gut, however the addition of MOS to a SBO diet increased the mucous cell size over the values shown in FO fed fish. Dietary SBO also trends to reduce mucous cell density in the anterior gut relative to FO, suggesting a lower overall mucosal secretion. There are no effects of dietary oil or MOS in the skin mucosal patterns. Complete replacement of FO by SBO, modified the gut fatty acid profile, altered posterior gut-associated immune system (GALT)-related gene expression and gut mucous cells patterns, induced shorter intestinal folds and tended to reduce European sea bass growth. However, when combined with MOS, the harmful effects of SBO appear to be partially balanced by moderating the down-regulation of certain GALT-related genes involved in the functioning of gut mucous barrier and increasing posterior gut mucous cell diffusion rates, thus helping to preserve immune homeostasis. This denotes the importance of a balanced dietary n–3/n–6 ratio for an appropriate GALT-immune response against MOS in European sea bass juveniles.