Periderm fate and independence of tooth formation are conserved across osteichthyans.

BACKGROUND: Previous studies have reported that periderm (the outer ectodermal layer) in zebrafish partially expands into the mouth and pharyngeal pouches, but does not reach the medial endoderm, where the pharyngeal teeth develop. Instead, periderm-like cells, arising independently from the outer periderm, cover prospective tooth-forming epithelia and are crucial for tooth germ initiation. Here we test the hypothesis that restricted expansion of periderm is a teleost-specific character possibly related to the derived way of early embryonic development. To this end, we performed lineage tracing of the periderm in a non-teleost actinopterygian species possessing pharyngeal teeth, the sterlet sturgeon (Acipenser ruthenus), and a sarcopterygian species lacking pharyngeal teeth, the axolotl (Ambystoma mexicanum). RESULTS: In sturgeon, a stratified ectoderm is firmly established at the end of gastrulation, with minimally a basal ectodermal layer and a surface layer that can be homologized to a periderm. Periderm expands to a limited extent into the mouth and remains restricted to the distal parts of the pouches. It does not reach the medial pharyngeal endoderm, where pharyngeal teeth are located. Thus, periderm in sturgeon covers prospective odontogenic epithelium in the jaw region (oral teeth) but not in the pharyngeal region. In axolotl, like in sturgeon, periderm expansion in the oropharynx is restricted to the distal parts of the opening pouches. Oral teeth in axolotl develop long before mouth opening and possible expansion of the periderm into the mouth cavity. CONCLUSIONS: Restricted periderm expansion into the oropharynx appears to be an ancestral feature for osteichthyans, as it is found in sturgeon, zebrafish and axolotl. Periderm behavior does not correlate with presence or absence of oral or pharyngeal teeth, whose induction may depend on 'ectodermalized' endoderm. It is proposed that periderm assists in lumenization of the pouches to create an open gill slit. Comparison of basal and advanced actinopterygians with sarcopterygians (axolotl) shows that different trajectories of embryonic development converge on similar dynamics of the periderm: a restricted expansion into the mouth and prospective gill slits.

L3-L4/L4-L5 Type II-A spondylolisthesis: A case report.

INTRODUCTION AND IMPORTANCE: Double level isthmic spondylolisthesis at L3-L4/L4-L5 is exceedingly rare with only a few documented cases in the literature, but to our knowledge no detailed case reports have been written. CASE PRESENTATION: 49 year old male with L3-4, L4-5 isthmic spondylolisthesis with neurologic symptoms and failed conservative management treated with L3-4, L4-5 Gill laminectomy, transforaminal interbody fusion with bone grafting and L3-5 posterior instrumented fusion. CLINICAL DISCUSSION: While rare, this condition can be successfully treated with posterior decompression and instrumented interbody fusion similar to single level spondylolisthesis. Surgeons should feel confident that they can achieve a good outcome for patients and feel comfortable offering this procedure. CONCLUSION: This case report may offer guidance for surgeons in the future as it explores the successful treatment of double level isthmic spondylolisthesis at L3-L4/L4-5 from initial presentation to final post-operative follow-up where the patient had complete resolution of symptoms.

Establishment and identification of the gill cell line from the blunt snout bream (Megalobrama amblycephala) and its application in studying gill remodeling under hypoxia.

To probe the mechanisms of gill remodeling in blunt snout bream under hypoxic conditions, we selected gill tissue for primary cell culture to establish and characterize the first blunt snout bream gill cell line, named MAG. The gill cells were efficiently passaged in M199 medium supplemented with 8% antibiotics and 15% fetal bovine serum at 28 °C, exhibiting primarily an epithelial-fibroblast mixed type. Additionally, the MAG cells (17th generation) were subjected to four experimental conditions-normoxia, hypoxia 12 h, hypoxia 24 h, and reoxygenation 24 h (R24h)-to evaluate the effects of hypoxia and reoxygenation on MAG cells during gill remodeling. We found that the MAG cell morphology underwent shrinkage and mitochondrial potential gradually lost, even leading to gradual apoptosis with increasing hypoxia duration and increased reactive oxygen species (ROS) activity. Upon reoxygenation, MAG cells gradually regain cellular homeostasis, accompanied by a decrease in ROS activity. Analysis of superoxide dismutase (SOD), glutathione (GSH), lactate dehydrogenase (LDH), catalase (CAT), anti-superoxide anion, and other enzyme activities revealed enhanced antioxidant enzyme activity in MAG cells during hypoxia, aiding in adapting to hypoxic stress and preserving cell morphology. After reoxygenation, the cells gradually returned to normoxic levels. Our findings underscore the MAG cells can be used to study hypoxic cell apoptosis during gill remodeling. Therefore, the MAG cell line will serve as a vital in vitro model for exploring gill remodeling in blunt snout bream under hypoxia.

Microbiome and network analysis reveal potential mechanisms underlying Carassius auratus diseases: The interactions between critical environmental and microbial factors.

Despite the rapid development of research on aquatic environment microbiota, limited attention has been paid to exploring the complex interactions between microbial communities and aquatic environments. Particularly, the mechanisms underlying fish diseases based on such dynamic interactions remain unknown. This study aimed to address the gap by conducting microbiome and co-occurrence network analyses on the typical freshwater aquaculture systems. High-throughput 16S rRNA gene sequencing results revealed significant differences in the microbiota between the disease and healthy groups. Notably, disease mortality varied consistently with the gradient of relative abundance of Proteobacteria (intestine, R2 = 0.46, p < 0.05) and Cyanobacteria (gill, R2 = 0.67, p < 0.01), indicating their potential use as diagnostic criteria. Furthermore, the elevated hepatosomatic index, NO3-N, COD and TC (sediment) were directly correlated with diseases (r > 0.54, p < 0.01). Mean concentrations of NO3-N, COD and TC were elevated by 78.87%, 25.63% and 44.2%, respectively, in ponds where diseases occurred. Quantitative analysis (qPCR) revealed that Aeromonas sobria infected hosts through a potential pathway of "sediment (4.4 × 105 copy number/g)-water (1.1 × 103 copy number/mL)-intestine (1.2 × 106 copy number/g)". Similarly, the potential route for Aeromonas veronii was sediment (4.9 × 106 copy number/g) to gill (5.1 × 105 copy number/g). Additionally, the complexity of microbial networks in the intestine, water, and sediment was significantly lower in the disease group, although no similar phenomenon was observed in the gill microbial network. In summary, these findings reveal that elevated concentrations of crucial environmental factors disrupt the linkages within microbiota, fostering the growth of opportunistic bacteria capable of colonizing fish gut or gills. This offers new insights into potential mechanisms by which environmental factors cause disease in fish.

Myxobolus dabryi n. sp. (Myxozoa: Myxobolidae) Infecting the Gills of Chanodichthys dabryi, Bleeker, 1871 (Cypriniformes: Cyprinidae) in Hunan Province, China.

Culters are a popular and economically important carnivorous freshwater fish, widely distributed in rivers, lakes, and reservoirs in China. An investigation of Myxozoa was conducted to enhance the understanding of Myxozoan diversity in Culters in China, as only 15 Myxosporean species have been previously reported in 6 Culters species. A new species with typical Myxobolus characteristics was discovered exclusively in the gills of Chanodichthys dabryi, Bleeker, 1871, and no other species were found in other Culters fish or organs. The new species elicited whitish plasmodia in the serosa layer of the gill arch, with no distinct inflammatory reaction observed. This species is morphologically different from all reported Myxobolus spp. from Culters, differing in plasmodium and spore size, as well as the coils of polar filaments. Molecular analysis further supports that it does not match any sequences available in GenBank. Therefore, we identified it as a new species and named it Myxobolus dabryi n. sp.

Impact of the novel chlorinated polyfluorinated ether sulfonate, F-53B, on gill structure and reproductive toxicity in zebrafish.

6:2 Chlorinated polyfluorinated ether sulfonate, commonly known as F-53B, is widely used as a mist suppressant in various industries and is frequently detected in the environment. Despite its prevalent presence, the adverse effects of F-53B are not well understood and require future investigation. This study utilized zebrafish embryos and adults to examine the toxic effects of F-53B. Our findings revealed that F-53B impaired gill structure and increased erythrocyte numbers in adult zebrafish. Notably, F-53B demonstrated a higher sensitivity for inducing mortality (LC50 at 96 h) in adult zebrafish compared to embryos. Additionally, F-53B disrupted the expression of critical steroidogenic genes and hindered sex hormone production, which negatively affecting egg production. In conclusion, this study underscores the detrimental impact of F-53B on gill structure and reproductive toxicity in zebrafish, providing valuable insights into its overall toxicity.

Gill-associated ammonia oxidizers are widespread in teleost fish.

Recent advances in sequencing methods have greatly expanded the knowledge of teleost-associated microorganisms. While fish-gut microbiomes are comparatively well studied, less attention has gone toward other, external organ-microbiome associations. Gills are particularly interesting to investigate due to their functions in gas exchange, osmoregulation, and nitrogen excretion. We recently discovered a branchial symbiosis between nitrogen-cycling bacteria and teleosts (zebrafish and carp), in which ammonia-oxidizing Nitrosomonas and denitrifying bacteria together convert toxic ammonia excreted by the fish into harmless dinitrogen (N2) gas. This symbiosis can function as a "natural biofilter" in fish gills and can potentially occur in all ammonotelic fish species, but it remains unknown how widespread this symbiosis is. In this study, we analyzed all publicly available gill microbiome data sets and checked for the presence of Nitrosomonas. We discovered that more than half of the described fish gill microbiomes contain 16S rRNA gene sequences of ammonia-oxidizing bacteria (AOB). The presence of gill-specific AOB was shown in both wild and aquacultured fish, as well as in marine and freshwater fish species. Based on these findings, we propose that ammonia oxidizers are widespread in teleost fish gills. These gill-associated AOB can significantly affect fish nitrogen excretion, and the widespread nature of this association suggests that the gill-associated AOB can have similar impacts on more fish species. Future research should address the contribution of these microorganisms to fish nitrogen metabolism and the fundamental characteristics of this novel symbiosis.IMPORTANCERecent advances in sequencing have increased our knowledge of teleost-associated microbiota, but the gill microbiome has received comparatively little attention. We recently discovered a consortium of nitrogen-cycling bacteria in the gills of common carp and zebrafish, which are able to convert (toxic) ammonia into harmless dinitrogen gas. These microorganisms thus function as a natural nitrogen biofilter. We analyzed all available gill microbiome data sets to determine how widespread gill-associated ammonia-oxidizing bacteria (AOB) are. More than half of the data sets contained AOB, representing both aquacultured and wild fish from freshwater and marine habitats. In total, 182 amplicon sequencing variants were obtained, of which 115 were found specifically in the gills and not the environmental microbiomes. As gill-associated AOB are apparently widespread in teleost fish, it is important to study their impact on host nitrogen excretion and the potential to reduce ammonia accumulation in (recirculating) aquaculture of relevant fish species.

Dead reckoning of protist viability with propidium monoazide (PMA)-quantitative PCR; a case study using Neoparamoeba perurans.

The ability to distinguish between viable and non-viable protozoan parasites is central to improved human and animal health management. While conceptually simple, methods to differentiate cell viability in situ remain challenging. Amoebic gill disease, caused by Neoparamoeba perurans is a parasitic disease impacting Atlantic salmon aquaculture globally. Although commercial freshwater treatments alleviate AGD, viable amoebae remain on gills or in used treatment water. Existing PCR-based assays are able to quantify N. perurans abundance but cannot discriminate amoeba viability. We investigated the use of propidium monoazide (PMA) application, prior to real-time PCR, to distinguish between alive and dead cells. We demonstrate that 200 µM PMA can significantly reduce amplification from non-viable (isopropanol treated) cultured amoebae across at least three logs of cell concentrations. Using a serial dilution of viable and non-viable cells, we show that non-PMA PCR amplifies both viable and non-viable amoebae, while PMA exposure suppresses (but does not completely inhibit) amplification from non-viable amoebae. The effect of freshwater treatment on N. perurans viability was assessed using the PMA-PCR. Following PMA exposure, amplification from freshwater treated amoebae was reduced by approximately 94-97 %. Taken together this study demonstrates that PMA combined with traditional real-time PCR can estimate amoeba viability.

Hypoxia induces reversible gill remodeling in largemouth bass (Micropterus salmoides) through integrins-mediated cell adhesion.

Gill remodeling is an important strategy for fish to cope with hypoxia, and many of the teleost possess this ability, but the underlying mechanism is not well understood. To investigate the mechanism of hypoxia-induced gill remodeling, largemouth bass (Micropterus salmoides) exposed to hypoxia (dissolved oxygen level: 2.0 ± 0.2 mg L-1) for 7 days, followed by 7 days of reoxygenation. Hypoxia tests were also performed on primary gill cells from largemouth bass. We found that hypoxia-induced gill remodeling increased the respiratory surface area of the gills. This change in gill morphology was reversible and recovered after reoxygenation. A reduction in the number of mucous cells and rearrangement of mitochondria-rich cells (MRCs) were observed during gill remodeling. After 7 days of reoxygenation, the number of mucous cells and the position of the MRCs were restored. Hypoxia resulted in a 2.92-fold increase in the number of primary gill cells that underwent migration over a 12-h period. The mRNA levels of nine integrin subunits (α1, α2, α5, α7, α8, α10, αL, ß1 and ß2) were significantly up-regulated after 12 h of hypoxia in vivo, and the changes in the expression of these subunits were consistent with the HIF-1α trend. Immunohistochemistry showed that integrin ß1 protein levels were significantly increased and were abundantly expressed in the interlamellar cell mass after exposure to hypoxia. Taken together, the results of the present study demonstrated that changes in mucosal cells and MRCs play an important role in hypoxia-induced gill remodeling in largemouth bass and that these changes are regulated by integrins.

Salinity and prolactin regulate anoctamin 1 in the model teleost, Fundulus heteroclitus.

To maintain internal ion balance in marine environments, teleost fishes leverage seawater (SW)-type ionocytes to actively secrete Na+ and Cl- into the environment. It is well established that SW-type ionocytes use apically expressed cystic fibrosis transmembrane conductance regulator 1 (Cftr1) as a conduit for Cl- to exit the gill. Here, we investigated whether the Ca2+-activated Cl- channel, anoctamin 1 (Ano1), provides an additional path for Cl--secretion in euryhaline mummichogs (Fundulus heteroclitus). Two ano1 gene isoforms, denoted ano1.1a and -1.1b, exhibited higher expression in the gill and opercular epithelium of mummichogs long-term acclimated to SW versus fresh water (FW). Branchial ano1.1b and cftr1 expression was increased in mummichogs sampled 24 h after transfer from FW to SW; ano1.1a and -1.1b were upregulated in the gill and opercular epithelium following transfer from SW to hypersaline SW. Alternatively, the expression of ano1.1a, -1.1b, and cftr1 in the gill and opercular epithelium was markedly decreased after transfer from SW to FW. Given its role in attenuating ion secretion, we probed whether prolactin downregulates ano1 isoforms. In addition to attenuating cftr1 expression, a prolactin injection reduced branchial ano1.1a and -1.1b levels. Given how Ano1 mediates Cl- secretion by mammalian epithelial cells, the salinity- and prolactin-sensitive nature of ano1 expression reported here indicates that Ano1 may constitute a novel Cl--secretion pathway in ionocytes. This study encourages a wider evaluation of this putative Cl--secretion pathway and its regulation by hormones in teleost fishes.NEW & NOTEWORTHY In this study, we provide evidence in a teleost fish that the Ca2+-activated Cl- channel, anoctamin 1 may provide an additional path for Cl- secretion by seawater-type ionocytes. Not only is this the first report of a Cftr-independent Cl--secreting pathway conferring survival in seawater but also the first description of its regulation by the pituitary hormone prolactin.

Assessing the vulnerability of Elasmobranch species in the Bay of Bengal: Insights from Lakkha gill net fishery of Bangladesh.

The elasmobranch population is declining in the Bay of Bengal of Bangladesh due to large-mesh gill net fishing, locally known as the Lakkha net, which primarily targets Indian threadfin (Leptomelanosoma indicum). This study was the first attempt to identify megafaunal bycatch in Lakkha fishing and assess its vulnerability using Productivity Susceptibility Analysis. A total of 40 elasmobranch bycatch species were identified, with sharks comprising 13 species from three families, while 27 rays belonged to six families, with the majority belonging to the Myliobatiformes order (60 %). Productivity and susceptibility scores were assigned to all identified species, with values ranging from 1.27 to 2.73 and 1.50 to 2.63, respectively. The target Lakkha fish exhibited the highest susceptibility score, followed by several pelagic sharks and eagle rays. Vulnerability assessment revealed that 31.7 % (n = 13) of species were highly vulnerable, while 43.9 % (n = 18) were classified as moderate, and 24.4 % (n = 10) were considered to have low vulnerability. All the high-risk megafauna species (n = 13) are classified as threatened by the global IUCN Red List. Sensitivity analysis highlighted susceptibility as a major contributor to species' vulnerability. Alterations in susceptibility scores led to significant changes in the vulnerability status of many species. The overall data quality assessment indicated moderate data quality across species, with variability observed between productivity (76 % of species received a poor data quality score) and susceptibility attributes. However, vulnerability of these species can be reduced through adequate gear modification, shorter net deployment periods, adoption of safe discharge techniques, identification of critical habitats, and establishment of marine protected areas within this region. This study provides valuable insights into the species composition and vulnerability of elasmobranchs in the Lakkha gill net fishery, emphasizing the need for conservation measures to mitigate bycatch impacts on threatened species.

Dynamics of Gill Responses to a Natural Infection with Neoparamoeba perurans in Farmed Tasmanian Atlantic Salmon.

Gill health has become a significant global challenge for Atlantic salmon (Salmo salar) aquaculture, particularly during the marine phase of farming. The increasing prevalence of gill pathologies has been linked to rising seawater temperatures, underscoring the need to evaluate existing tools for monitoring gill health and to develop novel approaches for early detection. In this study, we investigated the gill responses of commercially farmed Atlantic salmon to natural infection with Neoparamoeba perurans during an outbreak of amoebic gill disease (AGD) in Tasmania. Our focus spanned the low AGD prevalence, high AGD prevalence, and post-freshwater treatment stages of the outbreak. Evaluations of gill tissue included assessments of the gross AGD score, histopathological score, abundance of N. perurans (measured by 18S rRNA gene expression), and expression levels of inflammation-related transcripts. We demonstrated a strong correlation between different measures of AGD-related gill pathology and significant differences between distinct stages of the N. perurans outbreak. Post-treatment, fish exhibited considerable variability in their responses to the freshwater bath, highlighting the necessity for personalized management strategies that consider genetic, environmental, and health status factors. The expression patterns of angiogenin-1 (ANG1) and complement C1q tumour necrosis factor-related protein 3-like (C1QTNF3) emphasize their potential as biomarkers for early detection of gill damage in salmon aquaculture worldwide.

Effects of ammonia nitrogen stress on the physiological, biochemical, and metabolic levels of the gill tissue of juvenile four-finger threadfin (Eleutheronema tetradactylum).

In this study, the impact of ammonia nitrogen stress on juvenile four-finger threadfin in pond culture was examined. The 96-hour median lethal concentration (LC50-96h) and safe concentration of ammonia nitrogen were assessed in juveniles with a body weight of 7.4 ± 0.6 g using ecotoxicological methods. The study design included a stress group exposed to LC50-96h levels of ammonia nitrogen and a control group without ammonia nitrogen exposure. To examine the physiological, biochemical, and metabolic effects of ammonia nitrogen on gill tissue, gill tissue samples were collected after 12, 24, 48, and 96 h of stress, with a resumption of treatment after 48 h. Compared to the control group, ammonia nitrogen adversely affected juvenile four-finger threadfin, with LC50-96h and safe concentration values of 20.70 mg/L and 2.07 mg/L, respectively. Exposure to ammonia nitrogen resulted in substantial gill damage, including fusion of lamellae, epithelial cell loss, and proliferation of chlorine-secreting cells. This tissue damage persisted even after a 48-h recovery period. Ammonia nitrogen stress triggered an increase in antioxidant enzyme activity (superoxide dismutase, catalase, and glutathione peroxidase) and malondialdehyde levels in gills, indicating oxidative stress from 12 h onwards. Although enzyme activity decreased over time, oxidative stress persisted even after recovery, suggesting an ongoing need for antioxidant defense. Metabolomics analysis showed significant alterations in 423 metabolites under ammonia nitrogen stress. Key metabolites such as L-arginine, taurine, 20-hydroxyarachidonic acid, 11,12-dihydroxy-5Z, 8Z, and 14Z eicosotrienic acid followed an increasing trend; uridine, adenosine, L-glutathione, and thymidine 5'-triphosphate followed a decreasing trend. These changes reflect metabolic adaptations to stress. In enriched metabolic pathways, the main differential pathways are membrane transport, lipid metabolism, and amino acid metabolism. After 48 h, significant differences were observed in 396 metabolites compared to the control group. Notably, L-arginine, choline, and L-histidine increased, while linoleic acid, adenosine, and glutathione decreased. Amino acid and lipid metabolism pathways were key affected pathways. Under ammonia nitrogen stress, juvenile four-finger threadfin increased the synthesis of unsaturated and saturated fatty acids to cope with low temperatures and bolster immune function by consuming spermidine. This adaptation helps to clear peroxides generated during fatty acid synthesis, thereby protecting cells from oxidative damage. This study provides insights for pond aquaculture and breeding of ammonia nitrogen-tolerant fish strains.

Effect of vitamin E on energy metabolism indicators and gill tissue structure of crucian carp (Carassius auratus) under cooling stress.

The aim of this work is to examine the effects of vitamin E addition to water on the structure of the gill tissue and energy metabolism of crucian carp (Carassius auratus) under cooling stress. The crucian carp were chilled using a cold acclimation intelligent chilling equipment from 20 °C to 5 °C. They were divided into three groups: the control group (E1), the negative control group (E2), and the 100 mg/L vitamin E (E3) solution. Three different temperature points (20 °C, 10 °C, and 5 °C) were used to collect, test, and analyze the samples. The findings demonstrated that in the E3 treatment group, phosphoenolpyruvate carboxykinase, acetyl coenzyme A carboxylase, total cholesterol, urea nitrogen, triglyceride, and fatty acid synthase contents were significantly lower under cooling stress than those in the E1 and E2 treatment groups (P < 0.05). The E3 therapy group had significantly greater blood glucose, glycogen, and glycogen synthase levels than the E1 and E2 treatment groups (P < 0.05). The levels of pyruvate kinase in the E1, E2, and E3 treatment groups did not differ significantly. Crucian carp's gill tissue changed under cooling stress, including capillary dilatation, and the E3 treatment group experienced less damage overall than the E1 and E2 treatment groups. In conclusion, supplementing water with vitamin E to treat crucian carp can decrease damage, improve the body's ability to withstand cold, and slow down the stress response brought on by cooling stress. This provides a theoretical basis for supplementing water with vitamin E to fish stress relief.

Myo-inositol: A potential game-changer in preventing gill cell death and alleviating "gill rot" in grass carp (Ctenopharyngodon idellus).

Increasing evidence shows the potential threat of gill rot in freshwater fish culture. F. columnare is wide-spread in aquatic environments, which can cause fish gill rot and result in high mortality and losses of fish. This study investigated the effects of myo-inositol (MI) on the proliferation, structural integrity, and different death modes of grass carp (Ctenopharyngodon idella) gill epithelial cells, as well as its possible mechanism. 30 mg/L MI up-regulated CCK8 OD value and the protein level of solute carrier family 5A 3 (SLC5A3), and down-regulated the reactive oxygen species (ROS) content in gill cells and lactate dehydrogenase (LDH) release in the culture medium (P < 0.05). MI up-regulated the protein level of Beclin1, the protein level and fluorescence expression of microtubule-associated protein light chain 3B (LC3B) and down-regulated the protein level of sequestosome-1 (SQSTM1, also called p62) (P < 0.05). MI down-regulated the protein levels of Cysteine aspartate protease-1 (caspase-1), Gasdermin E (GSDME) and Cleaved interleukin 1 beta (IL-1ß) (P < 0.05). MI up-regulated the protein level of caspase-8 (P < 0.05), but had no effect on apoptosis (P > 0.05). MI down-regulated the mRNA expressions and protein levels of tumor necrosis factor α (tnfα), TNF receptor 1 (tnfr1), receptor interacting protein 1 (ripk1), receptor interacting protein 3 (ripk3) and mixed lineage kinase domain-like protein (mlkl), and reduce the ratio of p-MLKL/MLKL (P < 0.05). The addition of MI or necrosulfonamide (NSA) alone, or the addition of MI after induction of necroptosis, significantly up-regulated the cell activity and the protein level of SLC5A3 in gill cells, and significantly reduced the LDH release in the culture medium and the intracellular ROS content, the number of necroptosis cells, the protein expression of TNFα, TNFR1 and RIPK1, and the ratio of p-RIPK3/RIPK3 and p-MLKL/MLKL (P < 0.05). It indicated MI induce autophagy may relate to Beclin1/LC3/p62 signaling pathway, inhibits pyroptosis may attribute to Caspase-1/GSDMD/IL-1ß signaling pathway, and inhibits necroptosis via MLKL signaling pathway. However, MI had no effect on apoptosis.

Establishment and characterization of a gill cell line from Takifugu obscurus and transcriptome analysis of its gene expression profiles upon low temperature.

Takifugu obscurus is a farmed fish of great economic importance in China. The rapid development of T. obscurus aquaculture industry has been accompanied by disease and low-temperature stress, resulting in huge economic losses. Cell lines are used extensively in teleost physiology and pathology as the most cost-effective platform for in vitro research. A novel gill cell line of T. obscurus (named TOG) was first successfully established, and passed through 52 generations. The optimal conditions for TOG growth were 20 % FBS concentration and 24 °C, TOG could be grown in both hypotonic (150 mOsmol-kg-1) and hypertonic (600 mOsmol-kg-1) environments. TOG was determined to be derived from T. obscurus by sequencing the mitochondrial COI gene. Karyotype analysis revealed that the chromosome number of TOG was 44 (2n = 44). Transfection experiment showed that TOG was able to express foreign genes. Furthermore, several immune-related genes were significantly up-regulated in TOG after LPS and poly (I:C) stimulation, including tlr3, isg15, il1ß and il10. Additionally, transcriptome analysis of TOG under low-temperature stress (24 °C, 18 °C, 12 °C, 10 °C and 8 °C) found that differentially expressed genes (DEGs) were significantly clustered in several immunological and energy metabolic pathways, and cold stress could disrupt the immune barrier and reduce immunity by downregulating the immune-related pathways. Additionally, weighted gene co-expression network analysis (WGCNA) revealed that bule module and turquoise module, which were closely correlated with low temperature and the degree of fish damage, were both predominantly found in PPAR, NOD-like receptor and Toll-like receptor signaling pathway. Hub genes were identified in these two modules, including mre11, clpb, dhx15, ddx18 and utp15. TOG cell line will become an effective experimental platform for genetic and immunological research, and our results would help us gain a deeper insight into the molecular mechanism of cold tolerance in teleost.

Three New Species of Sciadicleithrum (Monogenoidea, Dactylogyridae) Parasitizing Cichlid Fishes (Cichliformes: Cichlidae) in the Northeastern Peru.

INTRODUCTION: The present study describes three new dactylogyrid species infecting the gill filaments of cichlid fishes (Cichliformes: Cichlidae) from the Amazon basin, Peru: Sciadicleithrum amazoniensis n. sp. on Biotodoma cupido (Heckel, 1840), and Sciadicleithrum feliciajaramae n. sp. and Sciadicleithrum souzatecci n. sp. on Bujurquina peregrinabunda Kullander, 1986. MATERIALS AND METHODS: Some monogeneans were stained with Gomori's trichrome and mounted in Canada Balsam to determine internal soft structures. Others were cleared in Hoyer's medium for the study of sclerotized structures. Drawings were made using a drawing tube and a microprojector. RESULTS: Sciadicleithrum amazoniensis n. sp. is characterized by the presence of a male copulatory organ (MCO) with a coil of approximately 2 counterclockwise rings, an accessory piece articulated to base of the MCO with an expanded proximal end and a bifurcated distal end, and a sinistral vaginal aperture. Sciadicleithrum feliciajaramae n. sp. can be differentiated from all its congeners by its J-shaped MCO with about half a counterclockwise loop and a rod-shaped accessory piece articulated to the base of the MCO, with the distal end bent. Sciadicleithrum souzatecci n. sp. differs from all other members of Sciadicleithrum by having an elongated MCO with about a clockwise loop and a funnel-shaped base. Additionally, Sciadicleithrum souzatecci n. sp. is characterized by its weakly sclerotized, C-shaped accessory piece with a robust middle process. CONCLUSIONS: Present findings are added to the other 26 species previously known in Sciadicleithrum.This is the first data on the parasites of B. peregrinabunda.

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

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

Responses of RTgill-W1 cells to cyanobacterial metabolites microcystin-LR, anabaenopeptin-A, cylindrospermopsin, their binary and ternary mixtures.

The aim of our study was to investigate the effects of cyanobacterial metabolites: microcystin-LR (MC-LR) anabaenopeptin-A (ANA-A), cylindrospermopsin (CYL), their binary and ternary mixtures on rainbow trout (Oncorhynchus mykiss) gill (RTgill-W1) cell line. We determined the following cell parameters: Hoechst and propidium iodide (PI) double staining, intracellular ATP level with luminometric assay, glutathione level with ThiolTracker Violet®- glutathione detection reagent and cytoskeletal F-actin fluorescence. The results showed that although reduction of Hoechst fluorescence was observed in both binary and ternary combinations of cyanobacterial metabolites, the mixture of MC-LR + ANA-A + CYL was the most potent inhibitor (EC50 = 148 nM). PI fluorescence and ATP levels were more increased in the cells exposed to the mixtures than those exposed to the individual metabolites with synergistic toxic changes suggesting apoptosis as the mechanism of cell death. Reduced glutathione level was also decreased in cells exposed both to single metabolites and their mixtures with the highest decrease and synergistic effects at 334 nM MC-LR+334 nM ANA-A+ 334 nM CYL suggesting induction oxidative stress by the tested compounds. Reduction of F-actin fluorescence was found in the cells from all of the groups exposed to individual metabolites and their mixtures, however the highest level of inhibition showed the binary MC-LR + CYL and the ternary MC-LR + ANA-A + CYL with synergistic interactions. The study suggests that in natural conditions fish gill cells may be very sensitive to individual cyanobacterial metabolites and more prone to their binary and ternary mixtures.

Novel insights on microbiome dynamics during a gill disease outbreak in farmed rainbow trout (Oncorhynchus mykiss).

The generic term "Gill disease" refers to a wide range of disorders that affect the gills and severely impact salmonid aquaculture systems worldwide. In rainbow trout freshwater aquaculture, various etiological agents causing gill diseases have been described, particularly Flavobacterium and Amoeba species, but research studies suggest a more complex and multifactorial aetiology. Here, a cohort of rainbow trout affected by gill disease is monitored both through standard laboratory techniques and 16S rRNA Next-Generation Sequencing (NGS) analysis during a natural disease outbreak and subsequent antibiotic treatment with Oxytetracycline. NGS results show a clear clustering of the samples between pre- and post-treatment based on the microbial community of the gills. Interestingly, the three main pathogenic bacteria species in rainbow trout (Yersinia ruckeri, Flavobacterium psychrophilum, and Flavobacterium branchiophilum) appear to be weak descriptors of the diversity between pre-treatment and post-treatment groups. In this study, the dynamics of the gill microbiome during the outbreak and subsequent treatment are far more complex than previously reported in the literature, and environmental factors seem of the utmost importance in determining gill disease. These findings present a potential novel perspective on the diagnosis and management of gill diseases, showing the limitations of conventional laboratory methodologies in elucidating the complexity of this disease in rainbow trout. To the authors' knowledge, this work is the first to describe the microbiome of rainbow trout gills during a natural outbreak and subsequent antibiotic treatment. The results of this study suggest that NGS can play a critical role in the analysis and comprehension of gill pathology. Using NGS in future research is highly recommended to gain deeper insights into such diseases correlating gill's microbiome with other possible cofactors and establish strong prevention guidelines.