Melanomacrophage Centers Research Articles

Efficacy of whole-cell-based monovalent and bivalent vaccines against Streptococcus iniae and Flavobacterium covae in fingerling Asian seabass (Lates calcarifer)

Streptococcosis and columnaris caused by Streptococcus spp. and Flavobacterium spp. have been recognized as critical problems in Asian seabass aquaculture development because they cause severe mortality. In this study, we identified various isolates of S. iniae and F. covae from diseased Asian seabass farmed in Thailand for use as candidates for vaccine development. The efficacy of the vaccines was evaluated by challenge tests and immune parameter analyses in fish that received whole-cell-based monovalent and bivalent vaccines containing S. iniae (Sin) and F. covae (Fco) delivered by top-dressed feed (TD) and intraperitoneal injection (IP). The results showed that all vaccinated groups exhibited increased antibody titers compared with control fish that peaked on day 28 after booster administration with high detection levels in the Sin-IP and Fco-IP groups. Moreover, the immune responses to the injected monovalent vaccines (Sin-IP and Fco-IP) were better than the responses in the other vaccinated groups. The hematological and innate immunological parameters were significantly increased by Sin-IP and Fco-IP, particularly lysozyme activity, nitroblue tetrazolium (NBT) activity, bactericidal activity, and white blood cell numbers, and immune-related genes, including IgM, MHC-IIα, TCRß and CD4, were significantly upregulated in the head kidney, whole blood and spleen (P < 0.05). After experimental challenge, survival in the Sin-IP and Fco-IP groups was significantly higher than that in the Sin-TD, Fco-TD, Sin + Fco-TD, and Sin + Fco-IP groups, with 80.0 % and 60.0 % survival after S. iniae and F. covae infection, respectively. In contrast, survival after bacterial challenge in the control groups was 10 % in each group. Histopathological analysis revealed that Sin-IP- and Fco-IP-vaccinated fish exhibited significantly more goblet cells in the intestines and melanomacrophage centers (MMCs) in the head kidney and spleen than those in the other groups (P < 0.05). Overall, the results of our study indicated that the monovalent vaccines Sin-IP and Fco-IP provoked better vaccine efficacy and immune responses than their orally administered counterparts, and these results are consistent with those from the immunological assays that showed significantly increased responses after immunization.

Effect of polypropylene microplastics on virus resistance in spotted sea bass (Lateolabrax maculatus)

Microplastics (MPs) pollution is a hot issue of global concern. Polypropylene microplastics (PP-MPs) age quickly in the marine environment and break down into smaller particles because of their relatively low temperature resistance, poor ultraviolet resistance, and poor antioxidant capacity, making them one of the major pollutants in the ocean. We assessed whether long-term exposure to micron-sized PP-MPs influences fish susceptibility to viral diseases. We found that exposure to PP-MPs (1–6 μm and 10–30 μm) at concentrations of 500 and 5000 μg/L resulted in uptake into spleen and kidney tissues of Lateolabrax maculatus. Increased activation of melanomacrophage centers was visible in histopathological sections of spleen from fish exposed to PP-MPs, and greater deterioration was observed in the spleen of fish infected by largemouth bass ulcerative syndrome virus after PP-MPs exposure. Additionally, exposure to PP-MPs led to significant cytotoxicity and a negative impact on the antiviral ability of cells. PP-MPs exposure had inhibitory or toxic effects on the immune system in spotted sea bass, which accelerated virus replication in vivo and decreased the expression of the innate immune- and acquired immune related genes in spleen and kidney tissues, thus increasing fish susceptibility to viral diseases. These results indicate that the long-term presence of micron-sized PP-MPs might impact fish resistance to disease, thereby posing a far-reaching problem for marine organisms.

Histopathological and Immunohistochemical Features and Expression Patterns of Cytochrome p450 1 Family Genes in Black Rockfish (Sebastes schlegelii): Exposure to 2,3,7,8-Tetrachlorodibenzo-p-dioxin and β-Naphthoflavone

The climate crisis and growing petroleum demand have put the health of aquatic animals in jeopardy. Fish are sensitive to chemical pollutants in aquatic environments, such as polycyclic aromatic hydrocarbons, dioxins, and dibenzofurans. This study investigated the effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and β-naphthoflavone (β-NF) exposure on histopathological and immunohistochemical features and expression patterns of cytochrome P450 1 (CYP1) family genes in black rockfish, Sebastes schlegelii. Histopathological alterations in the liver included congested central vein, sinusoidal dilatation, lymphocyte infiltration, and severe vacuolation within hepatocytes. The most prevalent alterations in TCDD-exposed kidneys were glomerular enlargement, narrowing of tubular lumen, melanomacrophage centers (MMCs), and necrosis. Moreover, CYP1A immunostaining was strong in renal tubules following TCDD exposure. All CYP1 family genes (CYP1A, CYP1B, CYP1C1, and CYP1C2) were significantly increased in the gills, liver, and kidney exposed to TCDD. Similarly, a significant increase of CYP1A mRNA expression in the kidney was observed upon exposure to TCDD (30.9-folds) and β-NF (25.5-folds) compared with that of the control group (p &lt; 0.05). TCDD and β-NF exposure exerted more adverse effects on the kidney than the liver, and TCDD had a greater in vivo toxic effect than β-NF. The combined histopathological, immunohistochemical, and molecular alternations may be helpful for diagnosing chemical contaminant exposure in S. schlegelii.

Hepatic melanomacrophage centers in the arctic cultured fish Cyclopterus lumpus are not indicative of its health state

The lumpfish, Cyclopterus lumpus, holds significant promise as a candidate for large-scale aquaculture production, particularly in its role as a cleaner fish used to manage sea lice infestations in Atlantic salmon Salmo salar farming. Melanomacrophage centers (MMCs) represent polymorphic structures present in the hemolymphopoietic organs of various vertebrates, serving as a widely applicable histological indicator of the fish immune and health status. This study aims to investigate the histochemical characteristics of MMCs within lumpfish livers and to compare MMC density between hatchery-produced (farmed) and wild individuals. Liver samples were collected from 34 lumpfish and subjected to a range of staining techniques, including haematoxylin-eosin, Azan-Mallory's trichrome, Masson-Fontana, Perls-Van Geison, Mallory's hemofuscin, immunohistochemical detection of cytochrome P450 monooxygenase 1 A (CYP1A), and the terminal deoxynucleotidyl transferase-mediated d'UTP nick-end labelling (TUNEL) method. Hepatocytes from hatchery-produced males exhibited notably high lipid content. Additionally, cells showing positive staining with Masson-Fontana, likely associated with the monocyte/macrophage lineage, were identified. Furthermore, small MMCs containing melanin, lipofuscin-ceroids, and ferric ions were detected. While the density of single monocytes/macrophages was markedly higher in hatchery-produced males, no significant discrepancies in MMCs density were observed between wild and hatchery-produced fish, or between males and females of the same origin. The study also revealed the presence of necrotic foci, characterized by hypertrophic hepatocytes positive for both TUNEL and CYP1A staining. These hypertrophic hepatocytes displayed large lipid droplets and pycnotic nuclei, with hatchery-produced males showing a higher numerical density of such foci. In contrast to findings in other fish species, the study found that MMCs did not appear to serve as reliable markers of health status in lumpfish. This conclusion was reached as MMCs density did not exhibit a correlation with necrotic foci or hepatocyte lipid content.

Immunotoxicological, histopathological, and ultrastructural effects of waterborne pyrogallol exposure on African catfish (Clariasgariepinus)

Pyrogallol is a naturally occurring polyphenol derived from natural plants, such as Acer rubrum and Eucalyptus sp. The current study was designed to evaluated pyrogallol-mediated toxicity at sublethal levels (1, 5, and 10 mg/L), derived from 96 h-LC50 values previously determined for African catfish (Clarias gariepinus). Immunotoxicological indices, histological, histochemical, and ultrastructural alterations in C. gariepinus were evaluated following a 15-day pyrogallol exposure. Pyrogallol decreased immune parameters [lysozyme activity (LYZ), immunoglobulin M (IgM), and phagocytic activity] and increased pro-inflammatory cytokines, interleukin-1 beta (IL-1β), interleukin-6 (IL-6) in the serum of C. gariepinus. In addition, histopathology analysis demonstrated that exposure to pyrogallol induced injury in the liver and spleen of fish. Cellular changes in the liver include hepatocyte hydropic degeneration, melanomacrophage, vacuolated hepatocytes, congested blood, severe structural deformation, and hemorrhage. In the spleen, ellipsoid structures, melanomacrophage centers, and infiltration of inflammatory cells were evident. Together, a high frequency of histopathological lesions was scored in both the liver and spleen of C. gariepinus, which showed a dose-dependent relationship between pyrogallol exposure and histopathological indices. Our data suggest that dysfunction in the immune system may be mediated by pyrogallol-induced changes in cytokines.

Cylindrospermopsin exposure promotes redox unbalance and tissue damage in the liver of Poecilia reticulata, a neotropical fish species

ABSTRACT There is a growing concern regarding the adverse risks exposure to cylindrospermopsin (CYN) might exert on animals and humans. However, data regarding the toxicity of this cyanotoxin to neotropical fish species are scarce. Using the fish species Poecilia reticulata, the influence of CYN concentrations equal to and above the tolerable for drinking water may produce on liver was determined by assessing biomarkers of antioxidant defense mechanisms and correlated to qualitative and semiquantitative histopathological observations. Adult females were exposed to 0.0 (Control); 0.5, 1 and 1.5 μg/L pure CYN for 24 or 96 hr, in triplicate. Subsequently the livers were extracted for biochemical assays and histopathological evaluation. Catalase (CAT) activity was significantly increased only by 1.5 μg/L CYN-treatment, at both exposure times. Glutathione -S-transferase (GST) activity presented a biphasic response for both exposure times. It was markedly decreased after exposure by 0.5 μg/L CYN treatment but significantly elevated by 1.5 μg/L CYN treatment. All CYN treatments produced histopathological alterations, as evidenced by hepatocyte cords degeneration, steatosis, inflammatory infiltration, melanomacrophage centers, vessel congestion, and areas with necrosis. Further, an IORG >35 was achieved for all treatments, indicative of the presence of severe histological alterations in P. reticulata hepatic parenchyma and stroma. Taken together, data demonstrated evidence that CYN-induced hepatotoxicity in P. reticulata appears to be associated with an imbalance of antioxidant defense mechanisms accompanied by histopathological liver alterations. It is worthy to note that exposure to low environmentally-relevant CYN concentrations might constitute a significant risk to health of aquatic organisms.

Cold-blooded vertebrates evolved organized germinal center-like structures.

Germinal centers (GCs) or analogous secondary lymphoid microstructures (SLMs) are thought to have evolved in endothermic species. However, living representatives of their ectothermic ancestors can mount potent secondary antibody responses upon infection or immunization, despite the apparent lack of SLMs in these cold-blooded vertebrates. How and where adaptive immune responses are induced in ectothermic species in the absence of GCs or analogous SLMs remain poorly understood. Here, we infected a teleost fish (trout) with the parasite Ichthyophthirius multifiliis (Ich) and identified the formation of large aggregates of highly proliferating IgM+ B cells and CD4+ T cells, contiguous to splenic melanomacrophage centers (MMCs). Most of these MMC-associated lymphoid aggregates (M-LAs) contained numerous antigen (Ag)-specific B cells. Analysis of the IgM heavy chain CDR3 repertoire of microdissected splenic M-LAs and non-M-LA areas revealed that the most frequent B cell clones induced after Ich infection were highly shared only within the M-LAs of infected animals. These M-LAs represented highly polyclonal SLMs in which Ag-specific B cell clonal expansion occurred. M-LA-associated B cells expressed high levels of activation-induced cytidine deaminase and underwent significant apoptosis, and somatic hypermutation of Igμ genes occurred prevalently in these cells. Our findings demonstrate that ectotherms evolved organized SLMs with GC-like roles. Moreover, our results also point to primordially conserved mechanisms by which M-LAs and mammalian polyclonal GCs develop and function.

Prevalence, molecular typing, antibiogram and histopathological changes of V. harveyi and V. parahaemolyticus isolated from Gilthead sea bream (Sparus aurata)

Vibrionaceae are a common bacterial disease that affects both wild and farmed marine fishes and causes tremendous economic losses globally. In order to investigate the prevalence, molecular typing, antibiogram and pathogenicity of vibriosis among cultured sea bream, two hundred and fifty Gilthead seabream (Sparus aurata) were collected randomly from different mariculture farms at Ismailia and Port Said Governorates. The collected fish were subjected to clinical, postmortem, bacteriological, and histopathological examinations. The majority of infected fish displayed ascites, hemorrhagic protruded anus, hemorrhages in pectoral, dorsal and tail fins, rotten gills, thinning of the head, discoloration of the skin, besides ulcer in the mouth and on the skin. The prevalence of Vibrio infection mostly was noticed at summer (35%) and spring (26%) then (22.5%) in autumn, where the kidney was the most predominant affected organ (45%). Vibrio 16s rRNA gene PCR extension generated a 663 pb amplified DNA bands that characteristic for all tested vibrio isolates. Blasting identified as V. harveyi and the other V. parahaemolyticus. The histopathological examination of naturally infected Gilthead seabream exhibited mild to moderate vacuolar degeneration of the hepatic parenchyma with tubular-nephrosis and massive renal cellular destruction. The splenic tissues showed focal activated melanomacrophage centers. The antimicrobial sensitivity was carried out, where the recovered strains were completely sensitive to novobiocin and highly resistant to ampicillin. In conclusion, the synergism of phenotypic and genotypic characterization is a valuable epidemiological tool for the diagnosis of Vibrio species. Strict veterinary hygienic regulations should be implemented to control such infections and minimize the antimicrobial use in fish farms.

Potential role of dietary Boswellia serrata resin against mancozeb fungicide-induced immune-antioxidant suppression, histopathological alterations, and genotoxicity in Nile tilapia, Oreochromis niloticus

This study was established to look into the toxicological consequences of chronic exposure to a fungicide (mancozeb; MAZ) on the immune-antioxidant response, gene expressions, hepato-renal functions, and histological pictures of Nile tilapia (Oreochromis niloticus). Additionally, the effectiveness of Indian frankincense resin extract (IFRE) to mitigate their toxicity was taken into account. Fish (n =240; average body weight: 22.45 ± 2.21 g) were randomized into four groups for eight weeks in six replicates (control, IFRE, MAZ, and IFRE + MAZ), where ten fish were kept per replicate. The control and IFRE groups received basal diets that included 0.0 and 5 g/kg of IFRE without MAZ exposure. The MAZ and IFRE+MAZ groups received the same diets and were exposed to 1/10 of the 96-h of LC50 of MAZ (1.15 mg/L). The outcomes displayed that MAZ exposure resulted in a lower survival rate (56.67 %) and significantly decreased levels of immune-antioxidant variables (antiprotease, complement3, phagocytic activity, lysozyme, glutathione peroxidase, superoxide dismutase, and total antioxidant capacity) compared to the control group. The MAZ-exposed fish showed the greatest levels of lipid peroxide (malondialdehyde), alkaline phosphatase, alanine amino-transferase, and stress indicators (cortisol and glucose). Additionally, histopathological alterations, including vacuolation, severe necrosis, degeneration, and mononuclear cell infiltrations in the hepatic, renal, and splenic tissues resulted, besides a reduction in the melanomacrophage center in the spleen. A down-regulation of immune-antioxidant-associated genes [toll-like receptors (TLR-2 and TLR-7), nuclear factor kappa beta (NF-κβ), transforming growth factor-beta (TGF-β), phosphoinositide-3-kinase regulatory subunit 3 gamma b (pik3r3b), interleukins (IL-1β and IL-8), glutathione synthetase (GSS), glutathione peroxidase (GPx), and superoxide dismutase (SOD)] were the consequences of the MAZ exposure. Remarkably, the dietary inclusion of IFRE in MAZ-exposed fish augmented the immune-antioxidant parameters, including their associated genes, decreased stress response, and increased survival rate (85 %) compared with the MAZ-exposed fish. Moreover, dietary IFRE improved hepato-renal function indices by preserving the histological architecture of the hepatic, renal, and splenic tissues. The insights of this study advocate the use of an IFRE-dietary addition to protect Nile tilapia from MAZ toxicity, which provides perspectives for future implementations in enhancing fish health for sustainable aquaculture.

Metrics Profiles of Melanomacrophages Centre on the Spleen of Carp (Cyprinus carpio) Exposed to Mercury Chloride

Increasing the number of heavy metals in the aquatic environment results in the accumulation of contaminants in fish body tissue, making fish an ideal bio-indicator of environmental pollution. The spleen included in the hematopoietic tissue contains macrophages. Macrophages form aggregates that contain pigments referred to melanomacrophage centres (MMCs). This research aims to analyse the metric profiles of MMCs on the common carp (Cyprinus carpio) spleen as a non-specific cellular immune response against mercury chloride exposure. This study used a Completely Randomized Design method with four treatments at different concentrations of mercury chloride (HgCl2) of 0 mg.l-1 , 0.01 mg.l-1 , 0.05 mg.l-1 , and 0.1 mg.l-1 . Each treatment was replicated five times. Data on the number and size of MMCs were analyzed by Analysis of Variance (ANOVA) and continued by using Duncan's Multiple Range Test (DMRT) to determine the differences between treatments. The result showed that the concentration of mercury chloride in water affects the metrics profile of spleen MMCs of carp. The number and size of MMCs of carp spleen increased at the concentration of 0.01 mg.l-1 and 0.05 mg.l-1 but decreased at the level of 0.1 mg.l-1 . The results suggest that the profile of MMCs as a non-specific cellular immune response can function as bio-indicators of environmental pollution.

Biomarcadores branquiais e hepáticos para monitoramento da sanidade de duas espécies de peixes neotropicais em área lacustre maranhense, Maranhão

The aim of the current study is to histologically assess two Neotropical fish species (Hoplerythrinus unitaeniatus and Cichlasoma bimaculatum) to monitor the health of a lake zone in Maranhão State. Forty-two (42) adult fish specimens - 21 belonged to species H. unitaeniatus and 21 to species C. Bimaculatum - were captured, euthanized and had their second right gill arch and liver fragments removed for histological analysis. Histological changes in specimens’ gill and liver were semi-quantitatively assessed by calculating the histological alterations index (HAI) based on the severity of each lesion established as stages I, II and III. Results recorded for both species were presented together; they evidenced that both assessed organs have shown changes associated with all three severity stages. Gill changes mostly observed through the analysis comprised total (85.71%) and partial (85.71%) fusion of several lamellae, epithelial lifting (80.95%), congested blood vessels (76.19%), disorganized lamellae, (76.19%) and lamellar epithelial hyperplasia (76.19%) - stage I alterations. The most common liver changes comprised vacuolization (90.48%), melanomacrophage centers (78.57%), cellular contour deformation (66.67%) and nucleus at cell periphery (52.38%), stage I alterations and hyperemia (45.24%) and cytoplasmic degeneration (40.48%) - stage II. Unlike the HAI indices recorded for gills, liver HAI values presented better distributed rates among severity classifications; therefore, it has evidenced a more severe condition. Thus, both organs analyzed in the investigated species have shown several histological changes that may have resulted from specimens interactions with stressors capable of affecting their health.

Infection and histopathological consequences in Siamese fighting fish (Betta splendens) due to exposure to a pathogenic Mycobacterium chelonae via different routes

Mycobacteriosis, a chronic progressive disease in fish caused by Mycobacterium spp., is typically characterized by granulomatous lesions that illustrate its harmful pathogenesis. This study was initiated to understand the progression of mycobacteriosis in Siamese fighting fish (Betta splendens) through various transmission modes. Experimental infections were conducted using a pathogenic isolate of Mycobacterium chelonae originally derived from a naturally infected betta fish. The bacteria were administered by five different routes at three doses (approximately 105, 106, 107 CFU/fish or CFU/mL), including intraperitoneal (IP) and intramuscular (IM) injections, oral administration, immersion, and immersion with skin trauma. Acute infections, typified by short survival, non-specific gross signs, and severe organ necrosis, were frequently observed in the IP and IM methods. The median lethal dose (LD50) for these methods was 4.16 × 105 CFU/fish and 1.66 × 106 CFU/fish, respectively, within seven days following infection. Conversely, oral administration and immersion with or without skin trauma resulted in negligible fish mortality but triggered progressive chronic disease. Chronically infected fish survived the 28-day trial but exhibited macroscopic pathology such as emaciation, aesthetic deterioration, along with an increased presence of melanomacrophage centers (MMCs) in lymphoid tissues and/or systemic granulomas, as noted in histopathology. The results indicate that the course of M. chelonae infection is strongly correlated with the exposure routes, with injuries on the fish body and/or via the digestive tract being the most probable natural pathways. This study expands the understanding of the pathogenesis of Mycobacterium species in betta fish and emphasizes the need to mitigate risk factors associated with exposure routes to effectively control the disease.

Effects of sublethal concentrations of zinc nanoparticles on bioaccumulation and cellular response in the Rabbitfish Siganus rivulatus

Zinc oxide nanoparticles (ZnONP) are increasingly utilized in many products worldwide. Eventual release into the aquatic system poses various environmental hazards and raises concerns for aquatic life and public health. In the present study, the effects of waterborne ZnONP and dissolved Zn+2 (ZnSO4 7H2O) were investigated in Siganus rivulatus through experimental exposure to environmentally relevant concentrations (40, 200, and 400 µg/L) of Zn in both forms. Fish exposed to ZnONP exhibited a higher accumulation of zinc in their muscles, whereas exposure to Zn+2 ions led to elevated zinc levels in the gills and liver. The physiological and histopathological analyses revealed distinct responses between the two experimental groups. ZnONP exposure stimulated the antioxidant system, resulting in a significant elevation (1.7–2 folds) of superoxide dismutase (SOD) and catalase (CAT) enzymes in the gills. On the other hand, Zn+2 ions increased the levels of SOD, CAT, and Glutathione-S-transferase (GST) enzymes 1.5–2.4 times in the liver, along with 1.9–2 folds higher lipid peroxidation (LPO) in both the liver and gills. Histological examination of the gills revealed lamellar fusions, degenerative alterations, and infiltration of melanomacrophagic centers, indicating the impact of both treatments. Similarly, the liver tissue showed cytoplasmic vacuolation and significant infiltration of melanomacrophagic centers engorged with brown spots. These histopathological changes were more severe in fish exposed to all concentrations of Zn+2 and in fish exposed to 400 µg/L of ZnONP. This study concludes that waterborne Zn+2 exhibited higher toxicity in fish liver and gills compared to ZnONP. However, the mechanisms of ZnONP uptake by fish tissues remain unclear and require further investigation.

Histomorphometric evaluation of melanomacrophage centers (MMCs) and CD3+ T cells of two morphs of brown trout (Salmo trutta) fed diets with immunostimulants

The brown trout (Salmo trutta) is a salmonid residing in riverine and coastal waters throughout the Northern Hemisphere, whose various populations evolved into distinct ecological morphs, differing in their migratory tendencies and preferred habitats. Unfortunately, due to progressing degradation of natural environment, the conservation of these populations is of growing importance and is undoubtedly a challenging task. Therefore, various means to refine the preparatory protocols for restocking using hatchery-reared fish are being pursued, some of which involve the administration of immunity-boosting substances. The current study assessed the effects of two dietary immunostimulants: Bioimmuno (4% inosine pranobex and 96% β-glucan) and Focus Plus (commercial preparation by Biomar, Denmark) on two morphs of the brown trout – the river trout (S. trutta morpha fario) and the sea trout (S. trutta morpha trutta). Tissue samples were obtained from ∼75 to 100g fish after 0, 2 and 4 weeks of experimental feeding. Multi-factorial analysis of conducted histological measurements of melanomacrophage centers (MMCs) revealed no changes of their parameters within spleens, but showed a decrease of the occupied tissue area and MMC counts in the livers, progressing with time regardless of the applied diet. Immunohistochemical analysis of CD3+ T cells showed their increased recruitment into mucosal folds of pyloric caeca in the 2-week sampling of trouts fed with the diet with 2% Bioimmuno addition, but this effect was not present in the 4-week sampling. When studying all groups jointly within each morph, there was a significant difference in terms of maintained CD3+ T cells levels, as sea trouts showed significantly higher tissue areas occupied by these cells than river trouts, both in the pyloric caeca and hepatic parenchyma. The study revealed that feeding with a diet enriched with Bioimmuno for 2 weeks may be a favorable enhancement of rearing protocols of brown trout stocks prior to their release, but more studies need to be conducted to test the possibility of an even shorter feeding period.

Ovarian changes and development of the branchial placenta occurring in Jenynsia lineata (Cyprinodontiformes, Anablepidae).

In viviparous teleosts, intraovarian gestation occurs intrafollicularly, as in poeciliids, or intraluminally, as in goodeids and anablepids. Furthermore, there are two different forms of embryonic nutrition: lecithotrophy and matrotrophy; depending on the species, these can be exclusive or coexist during gestation. In matrotrophic species, nutrients are transmitted from the mother to the embryo and are especially important in species with intraluminal gestation. Jenynsia lineata is a South American viviparous teleost with intraluminal gestation, characterized by eggs with scarce yolk, which is resorbed when embryos are 6 mm long, thus developing a branchial placenta. Using histological, histochemical, and immunohistochemical techniques, the present study describes the characteristics and changes of the ovarian mucosa in J. lineata during gestational and nongestational phases, and analyzes the embryonic pharyngeal epithelium in the branchial placenta. The ovaries of 30 adult female specimens were processed using histological techniques and stained with hematoxylin-eosin, Masson's trichrome, and Alcian Blue pH 2.5/periodic acid Schiff reagent. To detect cell proliferation, we used antiproliferating cell nuclear antigen antibody. In nonpregnant females, eosinophilic granular cells (EGCs) and lymphocytes were identified in the lamina propria of the tunica mucosa, and melanomacrophage centers (MMCs) and fibroblasts were identified adjacent to tissue debris in the ovarian folds'. In the cellular debris, an embryo in resorption was observed. In pregnant females, the ovarian mucosa has thin vascularization branches entering the opercular chamber of the embryos, in close contact with the forming gill processes, thereby establishing a branchial placenta. Active cell replacement was observed in these ovarian branches. The identification of fibroblasts, lymphocytes,EGCs, and MMCs adjacent to tissue debris could indicate that these cell types are involved in the embryonic resorption process. Considering the new data obtained in this study on the branchial placenta of J. lineata, we conclude that cell proliferation could be involved in the development of maternal-embryonic interaction.

Long-term crowding stress induces chronic inflammatory response and declines the immunity of grass carp (Ctenopharyngodon idella)

This study is aimed to explore the mechanism of overcrowding stress on the immunity of grass carp Ctenopharyngodon idella. Fish were reared for 120 days at 10 kg/m3 (low stocking density, LSD), 15 kg/m3 (medium stocking density, MSD) and 20 kg/m3 (high stocking density, HSD). The results showed that HSD significantly increased the serum cortisol level, implying that fish reared at higher density were subjected to physiological stress. HSD led to a remarkable decline in serum lysozyme activity. The conspicuous decrease of serum complement 3 (C3) and immunoglobulin M (IgM) contents also appeared in the HSD group. The serum antioxidant enzymes activity included total antioxidant capacity (T-AOC), total superoxide dismutase (T-SOD), glutathione peroxidase (GPx), and catalase (CAT) of fish reared at HSD group were clearly decreased. The serum glutathione (GSH) concentration of fish reared at HSD group was notably reduced. The malondialdehyde (MDA) level was obviously increased in HSD group. Long-term crowding stress gave rise to the constitution of melano-macrophage centers in spleen. The transcriptional levels of TLR4/NF-κB signaling pathway were notably up-regulated except for iκbα and mTOR pathway related genes were prominently down-regulated in fish reared at HSD group. The notably up-regulated pro-inflammatory cytokines (tnf-α, ifnγ2, il1-β, and il-8) except for il-6 and prominently down-regulated anti-inflammatory cytokines (il-10, il4/13a, il4/13b, tgf-β1 and tgf-β2) appeared in HSD group. All of the above results revealed that long-term overcrowding stress induced unbalanced inflammatory response via TLR4/NF-κB and mTOR signaling pathway and decreased the immunity in grass carp.

Using Azadirachta indica protein hydrolysate as a plant protein in Nile tilapia (Oreochromis niloticus) diet: Effects on the growth, economic efficiency, antioxidant-immune responseand resistance to Streptococcus agalactiae.

A feeding trial for 90 days was conducted on Nile tilapia (Oreochromis niloticus) (average weight: 25.50 ± 0.05 g) to evaluate the effect of dietary inclusion of Azadirachta indica seed protein hydrolysate (AIPH). The evaluation included the impact on the growth metrics, economic efficiency, antioxidant potential, hemato-biochemical indices, immune response, and histological architectures. A total of 250 fish were randomly distributed in five treatments (n = 50) and received diets included with five levels of AIPH (%): 0 (control diet, AIPH0), 2 (AIPH2), 4 (AIPH4), 6 (AIPH6)or 8 (AIPH8), where AIPH partially replace fish meal by 0, 8.7%, 17.4%, 26.1%, and 34.8%, respectively. After the feeding trial, a pathogenic bacterium (Streptococcus agalactiae, 1.5 × 108 CFU/mL) was intraperitoneally injected into the fish and the survival rate was recorded. The results elucidated that AIPH-included diets significantly (p < 0.05) enhanced the growth indices (final body weight, total feed intake, total body weight gain,and specific growth rate) and intestinal morpho-metrics (villous width, length, muscular coat thickness, and goblet cells count) in comparison to the control diet, with the AIPH8 diet recording the highest values. Dietary AIPH inclusion significantly improved (p < 0.05) the economic efficacy indicated by reduced feed cost/kg gain and increased performance index. The fish fed on the AIPH diets had noticeably significantly higher (p < 0.05) protein profile variables (total proteinsand globulin)and antioxidant capabilities (superoxide dismutase and total antioxidant capacity) than the AIPH0 group. The dietary inclusion of AIPH significantly (p < 0.05) boosted the haematological parameters (haemoglobin, packed cell volume %, and counts of red blood cells and white blood cells) and immune indices (serum bactericidal activity %, antiprotease activity, and immunoglobulin Mlevel) in a concentration-dependent manner. The blood glucose and malondialdehyde levels were significantly (p < 0.05) lowered by dietary AIPH (2%-8%). The albumin level and hepatorenal functioning parameters (aspartate aminotransferase, alanine aminotransferase, and creatinine) were not significantly (p > 0.05) altered by AIPH diets. Additionally, AIPH diets did not adversely alter the histology of the hepatic, renalor splenic tissues with moderately activated melano-macrophage centres. The mortality rate among S. agalactiae-infected fish declined as dietary AIPH levels rose, where the highest survival rate (86.67%) was found in the AIPH8 group (p < 0.05). Based on the broken line regression model, our study suggests using dietary AIPH at the optimal level of 6%. Overall, dietary AIPH inclusion enhanced the growth rate, economic efficiency, health status, and resistance of Nile tilapia to the S. agalactiae challenge. These beneficial impacts can help the aquaculture sector to be more sustainable.

Effects of exogenous elevation of corticosterone on immunity and the skin microbiome of eastern newts (Notophthalmus viridescens).

The amphibian chytrid fungus, Batrachochytrium salamandrivorans (Bsal) threatens salamander biodiversity. The factors underlying Bsal susceptibility may include glucocorticoid hormones (GCs). The effects of GCs on immunity and disease susceptibility are well studied in mammals, but less is known in other groups, including salamanders. We used Notophthalmus viridescens (eastern newts) to test the hypothesis that GCs modulate salamander immunity. We first determined the dose required to elevate corticosterone (CORT; primary GC in amphibians) to physiologically relevant levels. We then measured immunity (neutrophil lymphocyte ratios, plasma bacterial killing ability (BKA), skin microbiome, splenocytes, melanomacrophage centres (MMCs)) and overall health in newts following treatment with CORT or an oil vehicle control. Treatments were repeated for a short (two treatments over 5 days) or long (18 treatments over 26 days) time period. Contrary to our predictions, most immune and health parameters were similar for CORT and oil-treated newts. Surprisingly, differences in BKA, skin microbiome and MMCs were observed between newts subjected to short- and long-term treatments, regardless of treatment type (CORT, oil vehicle). Taken together, CORT does not appear to be a major factor contributing to immunity in eastern newts, although more studies examining additional immune factors are necessary. This article is part of the theme issue 'Amphibian immunity: stress, disease and ecoimmunology'.

Isolation, Identification, and Characterization of Aeromonas veronii from Chinese Soft-Shelled Turtle (Trionyx sinensis).

Aeromonas veronii is widespread in aquatic environments and is capable of infecting various aquatic organisms. A. veronii infection is lethal for Chinese soft-shelled turtles (Trionyx sinensis, CSST). We isolated a gram-negative bacterium from the liver of diseased CSSTs, which was named XC-1908. This isolate was identified as A. veronii based on its morphological and biochemical characteristics, and 16S rRNA gene sequence analysis. A. veronii was pathogenic for CSSTs with an LD50 of 4.17 × 105 CFU/g. The symptoms of CSSTs artificially infected with isolate XC-1908 were consistent with those of the naturally infected CSSTs. The levels of total protein, albumin, and white globule in the serum samples of the diseased turtles were decreased, whereas those of aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase were elevated. Moreover, the diseased CSSTs exhibited the following histopathological changes: the liver contained numerous melanomacrophage centers, renal glomerulus were edematous, intestinal villi were shed, and in oocytes, the number of vacuoles increased and red-rounded particles were observed. Antibiotic sensitivity tests revealed that the bacterium was sensitive to ceftriaxone, doxycycline, florfenicol, cefradine, and gentamicin, and resistant to sulfanilamide, carbenicillin, benzathine, clindamycin, erythromycin, and streptomycin. This study provides control strategies to prevent outbreaks of A. veronii infection in CSSTs.

Cytotoxic Effects of the Atrazine Herbicide on Erythrocytes and Liver Damage in Lithobates spectabilis

In Mexico, atrazine is one of the herbicides most widely authorized and used in different irrigation districts. Atrazine is a herbicide that contaminates aquatic systems. Previous studies have shown that atrazine causes damage to red blood cells and liver tissue in different aquatic species, including abnormalities in melanomacrophages. However, more information on amphibians is needed, since most of these studies have been done on fish. Furthermore, no study has determined the effect of atrazine on species native to Mexico. Therefore, in this study, we asked what the effects of atrazine are on the erythrocytes and melanomacrophages of the liver in the male frog (Lithobates spectabilis). In the present study, we analyzed (1) the cytotoxicity of atrazine using the micronucleus test, (2) the area of melanomacrophage centers and the presence of melanin, and (3) the characterization of liver damage using histological techniques. Our results show that atrazine is cytotoxic to erythrocytes, increases the area of and melanin presence in melanomacrophage centers, and causes liver damage in male L. spectabilis. Therefore, hepatotoxicity and cytotoxicity are indicators of environmental stress. We suggest monitoring Mexico’s aquatic systems and further analyzing atrazine effects and other pollutants on native species.