Fgf signalling is required for gill slit formation in the skate, Leucoraja erinacea.

The gill slits of fishes develop from an iterative series of pharyngeal endodermal pouches that contact and fuse with surface ectoderm on either side of the embryonic head. We find in the skate (Leucoraja erinacea) that all gill slits form via a stereotypical sequence of epithelial interactions: 1) endodermal pouches approach overlying surface ectoderm, with 2) focal degradation of ectodermal basement membranes preceding endoderm-ectoderm contact; 3) endodermal pouches contact and intercalate with overlying surface ectoderm, and finally 4) perforation of a gill slit occurs by epithelial remodelling, without programmed cell death, at the site of endoderm-ectoderm intercalation. Skate embryos express Fgf8 and Fgf3 within developing pharyngeal epithelia during gill slit formation. When we inhibit Fgf signalling by treating skate embryos with the Fgf receptor inhibitor SU5402 we find that endodermal pouch formation, basement membrane degradation and endodermal-ectodermal intercalation are unaffected, but that epithelial remodelling and gill slit perforation fail to occur. These findings point to a role for Fgf signalling in epithelial remodelling during gill slit formation in the skate and, more broadly, to an ancestral role for Fgf signalling during pharyngeal pouch epithelial morphogenesis in vertebrate embryos.

The pseudobranch of jawed vertebrates is a mandibular arch-derived gill.

The pseudobranch is a gill-like epithelial elaboration that sits behind the jaw of most fishes. This structure was classically regarded as a vestige of the ancestral gill arch-like condition of the gnathostome jaw. However, more recently, hypotheses of jaw evolution by transformation of a gill arch have been challenged, and the pseudobranch has alternatively been considered a specialised derivative of the second (hyoid) pharyngeal arch. Here, we demonstrate in the skate (Leucoraja erinacea) that the pseudobranch does, in fact, derive from the mandibular arch, and that it shares gene expression features and cell types with gills. We also show that the skate mandibular arch pseudobranch is supported by a spiracular cartilage that is patterned by a shh-expressing epithelial signalling centre. This closely parallels the condition seen in the gill arches, where cartilaginous appendages called branchial rays, which support the respiratory lamellae of the gills, are patterned by a shh-expressing gill arch epithelial ridge. Together with similar discoveries in zebrafish, our findings support serial homology of the pseudobranch and gills, and an ancestral origin of gill arch-like anatomical features from the gnathostome mandibular arch.

Effects of pejus and pessimum zone salinity stress on gill proteome networks and energy homeostasis in Oreochromis mossambicus.

Salinity tolerance in fish involves a suite of physiological changes, but a cohesive theory leading to a mechanistic understanding at the organismal level is lacking. To examine the potential of adapting energy homeostasis theory in the context of salinity stress in teleost fish, Oreochromis mossambicus were acclimated to hypersalinity at multiple rates and durations to determine salinity ranges of tolerance and resistance. Over 3000 proteins were quantified simultaneously to analyze molecular phenotypes associated with hypersalinity. A species- and tissue-specific data-independent acquisition (DIA) assay library of MSMS spectra was created. Protein networks representing complex molecular phenotypes associated with salinity acclimation were generated. O. mossambicus has a wide "zone of resistance" from 75 g/kg salinity to 120 g/kg. Crossing into the zone of resistance resulted in marked phenotypic changes including blood osmolality over 400 mOsm/kg, reduced body condition, and cessation of feeding. Protein networks impacted by hypersalinity consist of electron transport chain (ETC) proteins and specific osmoregulatory proteins. Cytoskeletal, cell adhesion, and extracellular matrix proteins are enriched in networks that are sensitive to the critical salinity threshold. These network analyses identify specific proteome changes that are associated with distinct zones described by energy homeostasis theory and distinguish them from general hypersalinity-induced proteome changes.

Transient excited states of the metamorphic protein Mad2 and their implications for function.

The spindle checkpoint complex is a key surveillance mechanism in cell division that prevents premature separation of sister chromatids. Mad2 is an integral component of this spindle checkpoint complex that recognizes cognate substrates such as Mad1 and Cdc20 in its closed (C-Mad2) conformation by fastening a "seatbelt" around short peptide regions that bind to the substrate recognition site. Mad2 is also a metamorphic protein that adopts not only the fold found in C-Mad2, but also a structurally distinct open conformation (O-Mad2) which is incapable of binding substrates. Here, we show using chemical exchange saturation transfer (CEST) and relaxation dispersion (CPMG) NMR experiments that Mad2 transiently populates three other higher free energy states with millisecond lifetimes, two in equilibrium with C-Mad2 (E1 and E2) and one with O-Mad2 (E3). E1 is a mimic of substrate-bound C-Mad2 in which the N-terminus of one C-Mad2 molecule inserts into the seatbelt region of a second molecule of C-Mad2, providing a potential pathway for autoinhibition of C-Mad2. E2 is the "unbuckled" conformation of C-Mad2 that facilitates the triage of molecules along competing fold-switching and substrate binding pathways. The E3 conformation that coexists with O-Mad2 shows fluctuations at a hydrophobic lock that is required for stabilizing the O-Mad2 fold and we hypothesize that E3 represents an early intermediate on-pathway towards conversion to C-Mad2. Collectively, the NMR data highlight the rugged free energy landscape of Mad2 with multiple low-lying intermediates that interlink substrate-binding and fold-switching, and also emphasize the role of molecular dynamics in its function.

Identification of the effects of alkalinity exposure on the gills of oriental river prawns, Macrobrachium nipponense.

Macrobrachium nipponense is an important commercial freshwater species in China. However, the ability of alkali tolerance of M. nipponense is insufficient to culture in the major saline-alkali water source in China. Thus, it is urgently needed to perform the genetic improvement of alkali tolerance in this species. In the present study, we aimed to analyse the effects of alkali treatment on gills in this species after 96 h alkalinity exposure under the alkali concentrations of 0 mmol/L, 4 mmol/L, 8 mmol/L, and 12 mmol/L through performing the histological observations, measurement of antioxidant enzymes, metabolic profiling analysis, and transcriptome profiling analysis. The results of the present study revealed that alkali treatment stimulated the contents of malondialdehyde, glutathione, glutathione peroxidase in gills, indicating these antioxidant enzymes plays essential roles in the protection of body from the damage, caused by the alkali treatment. In addition, high concentration of alkali treatment (> 8 mmol/L) resulted in the damage of gill membrane and haemolymph vessel, affecting the normal respiratory function of gill. Metabolic profiling analysis revealed that Metabolic pathways, Biosynthesis of secondary metabolites, Biosynthesis of plant secondary metabolites, Microbial metabolism in diverse environments, Biosynthesis of amino acids were identified as the main enriched metabolic pathways of differentially expressed metabolites, which are consistent with the previous publications, treated by the various environmental factors. Transcriptome profiling analyses revealed that the alkali concentration of 12 mmol/L has more regulatory effects on the changes of gene expression than the other alkali concentrations. KEGG analysis revealed that Phagosome, Lysosome, Glycolysis/Gluconeogenesis, Purine Metabolism, Amino sugar and nucleotide sugar metabolism, and Endocytosis were identified as the main enriched metabolic pathways in the present study, predicting these metabolic pathways may be involved in the adaption of alkali treatment in M. nipponense. Phagosome, Lysosome, Purine Metabolism, and Endocytosis are immune-related metabolic pathways, while Glycolysis/Gluconeogenesis, and Amino sugar and nucleotide sugar metabolism are energy metabolism-related metabolic pathways. Quantitative PCR analyses of differentially expressed genes (DEGs) verified the accuracy of the RNA-Seq. Alkali treatment significantly stimulated the expressions of DEGs from the metabolic pathways of Phagosome and Lysosome, suggesting Phagosome and Lysosome play essential roles in the regulation of alkali tolerance in this species, as well as the genes from these metabolic pathways. The present study identified the effects of alkali treatment on gills, providing valuable evidences for the genetic improvement of alkali tolerance in M. nipponense.

Quantitative analysis of the slow exchange process by 19F NMR in the presence of scalar and dipolar couplings: applications to the ribose 2'-19F probe in nucleic acids.

Solution NMR spectroscopy is a particularly powerful technique for characterizing the functional dynamics of biomolecules, which is typically achieved through the quantitative characterization of chemical exchange processes via the measurement of spin relaxation rates. In addition to the conventional nuclei such as 15N and 13C, which are abundant in biomolecules, fluorine-19 (19F) has recently garnered attention and is being widely used as a site-specific spin probe. While 19F offers the advantages of high sensitivity and low background, it can be susceptible to artifacts in quantitative relaxation analyses due to a multitude of dipolar and scalar coupling interactions with nearby 1H spins. In this study, we focused on the ribose 2'-19F spin probe in nucleic acids and investigated the effects of 1H-19F spin interactions on the quantitative characterization of slow exchange processes on the millisecond time scale. We demonstrated that the 1H-19F dipolar coupling can significantly affect the interpretation of 19F chemical exchange saturation transfer (CEST) experiments when 1H decoupling is applied, while the 1H-19F interactions have a lesser impact on Carr-Purcell-Meiboom-Gill relaxation dispersion applications. We also proposed a modified CEST scheme to alleviate these artifacts along with experimental verifications on self-complementary RNA systems. The theoretical framework presented in this study can be widely applied to various 19F spin systems where 1H-19F interactions are operative, further expanding the utility of 19F relaxation-based NMR experiments.

Modulation of gill surface area does not correlate with oxygen loss in Chitala ornata.

Air-breathing fish risk losing aerially sourced oxygen to ambient hypoxic water since oxygenated blood from the air-breathing organ returns through the heart to the branchial basket before distribution. This loss is thought to help drive the evolutionary reduction in gill size with the advent of air-breathing. In many teleost fish, gill size is known to be highly plastic by modulation of their anatomic diffusion factor (ADF) with inter-lamellar cell mass (ILCM). In the anoxia-tolerant crucian carp, ILCM recedes with hypoxia but regrows in anoxia. The air-breathing teleost Chitala ornata has been shown to increase gill ADF from normoxic to mildly hypoxic water by reducing ILCM. Here, we test the hypothesis that ADF is modulated to minimize oxygen loss in severe aquatic hypoxia by measuring ADF, gas-exchange, and by using computed tomography scans to reveal possible trans-branchial shunt vessels. Contrary to our hypothesis, ADF does not modulate to prevent oxygen loss and despite no evident trans-branchial shunting, C. ornata loses only 3% of its aerially sourced O2 while still excreting 79% of its CO2 production to the severely hypoxic water. We propose this is achieved by ventilatory control and by compensating the minor oxygen loss by extra aerial O2 uptake.

Molecular basis of the unique osmoregulatory strategy in the inshore hagfish, Eptatretus burgeri.

Hagfishes are characterized by omo- and iono-conforming nature similar to marine invertebrates. Conventionally, hagfishes had been recognized as the most primitive living vertebrate that retains plesiomorphic features. However, some of the "ancestral" features of hagfishes, such as rudimentary eyes and the lack of vertebrae, have been proven to be deceptive. Similarly, by the principle of maximum parsimony, the unique body fluid regulatory strategy of hagfishes seems to be apomorphic, since the lamprey, another cyclostome, adopts osmo- and iono-regulatory mechanisms as in jawed vertebrates. Although hagfishes are unequivocally important in discussing the origin and evolution of the vertebrate osmoregulatory system, the molecular basis for the body fluid homeostasis in hagfishes has been poorly understood. In the present study, we explored this matter in the inshore hagfish, Eptatretus burgeri, by analyzing the transcriptomes obtained from the gill, kidney, and muscle of the animals acclimated to distinct environmental salinities. Together with the measurement of parameters in the muscular fluid compartment, our data indicate that the hagfish possesses an ability to conduct free amino acid (FAA)-based osmoregulation at a cellular level, which is in coordination with the renal and branchial FAA absorption. We also revealed that the hagfish does possess the orthologs of the known osmoregulatory genes and that the transepithelial movement of inorganic ions in the hagfish gill and kidney is more complex than previously thought. These observations pose a challenge to the conventional view that the physiological features of hagfishes have been inherited from the last common ancestor of the extant vertebrates.

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.

What frog gill resorption brings: loss of function, cell death, and metabolic reorganization.

BACKGROUND: Anuran metamorphosis, which is driven by thyroid hormone (TH)-mediated processes, orchestrates intricate morphological and functional transformations for the transition from aquatic tadpoles to terrestrial life, providing a valuable model for studying organ functionalization, remodeling, and regression. Larva-specific organ regression is one of the most striking phenomena observed during the anuran metamorphic climax. While previous studies extensively analyzed the regression mechanisms of the tail, the molecular processes governing gill resorption remain elusive. RESULTS: We employed Microhyla fissipes as a model, and utilized a comprehensive approach involving histological analysis, transmission electron microscopy, and transcriptomics to unravel gill development and resorption. The pro-metamorphic stages revealed highly developed gill structures, emphasizing their crucial role as the primary respiratory organ for tadpoles. The transcriptomic analysis highlighted the upregulation of genes associated with enhanced respiratory efficiency, such as hemoglobin and mucins. However, as metamorphosis progressed, gill filaments underwent shrinkage, decreases in blood vessel density, and structural changes that signified a decline in respiratory function. The molecular mechanisms driving gill resorption involved the TH pathway-in particular, the upregulation of thyroid hormone receptor (TR) ß, genes associated with the tumor necrosis factor pathway and matrix metalloproteinases. Two distinct pathways orchestrate gill resorption, involving apoptosis directly induced by TH and cell death through the degradation of the extracellular matrix. In addition, metabolic reorganization during metamorphosis is a complex process, with tadpoles adapting their feeding behavior and mobilizing energy storage organs. The gills, which were previously overlooked, have been unveiled as potential energy storage organs that undergo metabolic reorganization. The transcriptomic analysis revealed dynamic changes in metabolism-related genes, indicating decreased protein synthesis and energy production and enhanced substrate transport and metabolism during metamorphic climax. CONCLUSION: This study sheds light on the structural, molecular, and metabolic dynamics during gill development and resorption in M. fissipes. The findings deepen our understanding of the intricate mechanisms governing organ regression and underscore the pivotal role of the gills in facilitating the transition from aquatic to terrestrial habitats.

Identification of signalling pathways involved in gill regeneration in zebrafish.

The occurrence of regeneration of the organs involved in respiratory gas exchange amongst vertebrates is heterogeneous. In some species of amphibians and fishes, the gills regenerate completely following resection or amputation, whereas in mammals, only partial, facultative regeneration of lung tissue occurs following injury. Given the homology between gills and lungs, the capacity of gill regeneration in aquatic species is of major interest in determining the underlying molecular or signalling pathways involved in respiratory organ regeneration. In the present study, we used adult zebrafish (Danio rerio) to characterize signalling pathways involved in the early stages of gill regeneration. Regeneration of the gills was induced by resection of gill filaments and observed over a period of up to 10 days. We screened for the effects on regeneration of the drugs SU5402, dorsomorphin and LY411575, which inhibit FGF, BMP or Notch signalling pathways, respectively. Exposure to each drug for 5 days significantly reduced regrowth of filament tips in regenerating tissue, compared with unresected controls. In separate experiments under normal conditions of regeneration, we used reverse transcription quantitative PCR and observed an increased expression of genes encoding for the bone morphogenetic factor, Bmp2b, fibroblast growth factor, Fgf8a, a transcriptional regulator (Her6) involved in Notch signalling, and Sonic Hedgehog (Shha), in regenerating gills at 10 day post-resection, compared with unresected controls. In situ hybridization confirmed that all four genes were expressed in regenerating gill tissue. This study implicates BMP, FGF, Notch and Shh signalling in gill regeneration in zebrafish.

Warming, stochastic diel thermal fluctuations affect physiological performance and gill plasticity in an amphibious mangrove fish.

Natural temperature variation in many marine ecosystems is stochastic and unpredictable, and climate change models indicate that this thermal irregularity is likely to increase. Temperature acclimation may be more challenging when conditions are highly variable and stochastic, and there is a need for empirical physiological data in these thermal environments. Using the hermaphroditic, amphibious mangrove rivulus (Kryptolebias marmoratus), we hypothesized that compared with regular, warming diel thermal fluctuations, stochastic warm fluctuations would negatively affect physiological performance. To test this, we acclimated fish to: (1) non-stochastic and (2) stochastic thermal fluctuations with a similar thermal load (27-35°C), and (3) a stable/consistent control temperature at the low end of the cycle (27°C). We determined that fecundity was reduced in both cycles, with reproduction ceasing in stochastic thermal environments. Fish acclimated to non-stochastic thermal cycles had growth rates lower than those of control fish. Exposure to warm, fluctuating cycles did not affect emersion temperature, and only regular diel cycles modestly increased critical thermal tolerance. We predicted that warm diel cycling temperatures would increase gill surface area. Notably, fish acclimated to either thermal cycle had a reduced gill surface area and increased intralamellar cell mass when compared with control fish. This decreased gill surface area with warming contrasts with what is observed for exclusively aquatic fish and suggests a preparatory gill response for emersion in these amphibious fish. Collectively, our data reveal the importance of considering stochastic thermal variability when studying the effects of temperature on fishes.

Estimation of trajectory of protective efficacy in infectious disease prevention trials using recurrent event times.

In studies of infectious disease prevention, the level of protective efficacy of medicinal products such as vaccines and prophylactic drugs tends to vary over time. Many products require administration of multiple doses at scheduled times, as opposed to one-off or continual intervention. Accurate information on the trajectory of the level of protective efficacy over time facilitates informed clinical recommendations and implementation strategies, for example, with respect to the timing of administration of the doses. Based on concepts from pharmacokinetic and pharmacodynamic modeling, we propose a non-linear function for modeling the trajectory after each dose. The cumulative effect of multiple doses of the products is captured by an additive series of the function. The model has the advantages of parsimony and interpretability, while remaining flexible in capturing features of the trajectories. We incorporate this series into the Andersen-Gill model for analysis of recurrent event time data and compare it with alternative parametric and non-parametric functions. We use data on clinical malaria disease episodes from a trial of four doses of an anti-malarial drug combination for chemoprevention to illustrate, and evaluate the performance of the methods using simulation. The proposed method out-performed the alternatives in the analysis of real data in terms of Akaike and Bayesian Information Criterion. It also accurately captured the features of the protective efficacy trajectory such as the area under curve in simulations. The proposed method has strong potential to enhance the evaluation of disease prevention measures and improve their implementation strategies.

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.

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.

CBS/CSE mediated H2S production induced AMPs expression through Toll pathway in crabs with black gill syndrome.

The occurrence of black gill syndrome (BGS) is a serious threat to the healthy culture of Eriocheir sinensis. Studying the innate immune ability of E. sinensis with BGS can help develop new strategies for disease prevention and treatment. Antimicrobial peptides (AMPs) have crucial roles in crustacean humoral immunity. In this study, we found that the expression levels of two antilipopolysaccharide factor (EsALF7 and EsALF-L), one Toll receptor 3 (EsToll3), and one Pelle kinase (EsPelle) were upregulated in E. sinensis with BGS. Moreover, ALFs expressions in E. sinensis with BGS were positively regulated by EsToll3 and EsPelle. The content of hydrogen sulfide (H2S) in the gills of E. sinensis with BGS was increased. Further studies showed that the expressions of cystathionine ß-synthase (EsCBS) and cystathionine γ-lyase (EsCSE) in the gills of E. sinensis with BGS were upregulated, which positively regulate the production of H2S. Whether there was a correlation between the upregulation of ALFs expression and changes in H2S content? Further studies showed that 1) the expressions of EsToll3, EsPelle, EsALF7, and EsALF-L in the gills of E. sinensis were upregulated under H2S exposure and 2) the knockdown of EsCBS and EsCSE in E. sinensis reduced the transcriptions of EsToll3, EsPelle, EsALF7, and EsALF-L. To sum up, these findings suggest that upregulation of H2S content induced by CBS/CSE promotes the expression of ALFs through the Toll pathway in E. sinensis suffering from BGS.

Intracellular metabolome elucidates the time-of-day-dependent response to hydrogen peroxide in salmonid gill epithelial cells.

The internal timekeeping system regulates the daily cycle of physiological and behavioural changes in living organisms. This rhythmic phenomenon also influences cellular responses to reactive oxygen species, such as hydrogen peroxide (H2O2). However, the temporal interaction between H2O2 and fish mucosal cells is not well understood. This study examined the temporal variations of immunological and physiological responses to H2O2 in salmonid gill cells using the RTgill-W1 cell line. The results showed that gene expression levels varied during a 24-h cycle but did not exhibit rhythmicity. The presence of a 12-h light-dark cycle (12L:12D) signal increased gene expression levels compared to a 24-h dark cycle (0L:24D). To investigate whether the time of day affects the defences in gills, cells were exposed to H2O2 at two different times (Zeitgebertime 2, ZT2, or ZT14). Although significant expression changes were observed in genes related to stress and NF-κB signalling, only a limited time-dependent pattern of response to H2O2 was observed. The intracellular metabolome of gill cells was primarily composed of organic acid and derivatives, organoheterocyclic compounds, benzoids, organic oxygen and nitrogen compounds. Exposure to H2O2 at ZT2 led to significant changes in the metabolome compared to the control group, while no such changes were observed at ZT14. Within the control groups, the concentrations of 11 metabolites significantly varied between ZT2 and ZT14, with higher levels at ZT14. These metabolites were involved in arginine biosynthesis, amino acid metabolism, and nitrogen metabolism. In contrast, the level of 26 metabolites significantly varied between ZT2 and ZT14 in H2O2-exposed groups, with lower levels at ZT14. Comparing control and H2O2-exposed groups at ZT2, 38 metabolites were affected, primarily organic acid and derivatives and organic oxygen compounds. Functional annotation revealed that these altered metabolites were involved in 15 different pathways, with valine, leucine, and isoleucine biosynthesis being the most affected. This study reveals the presence of a time-dependent response to H2O2 in salmonid gill cells, which is reflected in the intracellular metabolome. The findings provide new insights into the temporal regulation of mucosal defences in fish.

Transcriptome analysis reveals tissue-specific responses of Mytilus unguiculatus to Vibrio alginolyticus infection.

Mytilus unguiculatus is an important economic bivalve species with wide consumption and aquaculture value. Disease is one of the primary limiting factors in mussel aquaculture, thus understanding the response of different tissues of M. unguiculatus to pathogens is crucial for disease prevention and control. In this study, we investigated the physiological and transcriptomic responses of the gills, adductor muscle, and mantle of M. unguiculatus infected with Vibrio alginolyticus. The results showed that V. alginolyticus infection caused inflammation and tissue structure changes in the gill, adductor muscle and mantle of M. unguiculatus. Meanwhile, the activities of superoxide dismutase and catalase in the three tissues increased, while the total antioxidant capacity decreased, suggesting that M. unguiculatus have an activated defense mechanism against infection-induced oxidative stress, despite a compromised total antioxidant capacity. Transcriptomic studies reveal that infected M. unguiculatus exhibits upregulation of endocytosis, lysosome activity, cellular apoptosis, and immune-related signaling pathways, indicating that M. unguiculatus responds to pathogen invasion by upregulating efferocytosis. Compared with the gill and adductor muscle, the mantle had a higher level of mytimycin, mytilin and myticin, and the three tissues also increased the expression of mytimycin to cope with the invasion of pathogens. In addition, the analysis of genes related to taste transduction pathways and muscle contraction and relaxation found that after infection with V. alginolyticus, M. unguiculatus may reduce appetite by inhibiting taste transduction in the gill, while improving muscle contraction of the adductor muscle and keeping the shell closed, to resist further invasion of pathogens and reduce the risk of pathogen transmission in the population.

Histopathology and transcriptome profiling reveal features of immune responses in gills and intestine induced by Spring viremia of carp virus.

The immune system of bony fish closely resembles that of mammals, comprising both specific (adaptive) and non-specific (innate) components. Notably, the mucosa-associated lymphoid tissue (MALT) serves as the first line of defense within the non-specific immune system, playing a critical role in protecting these aquatic organisms against invading pathogens. MALT encompasses a network of immune cells strategically distributed throughout the gills and intestines, forming an integral part of the mucosal barrier that interfaces directly with the surrounding aquatic environment. Spring Viremia of Carp Virus（SVCV）, a highly pathogenic agent causing substantial harm to common carp populations, has been designated as a Class 2 animal disease by the Ministry of Agriculture and Rural Affairs of China. Utilizing a comprehensive array of research techniques, including Hematoxylin and Eosin (HE)、Alcian Blue Periodic Acid-Schiff (AB-PAS)、transcriptome analysis for global gene expression profiling and Reverse Transcription-Polymerase Chain Reaction (RT-qPCR), this study uncovered several key findings: SVCV is capable of compromising the mucosal architecture in the gill and intestinal tissues of carp, and stimulate the proliferation of mucous cells both in gill and intestinal tissues. Critically, the study revealed that SVCV's invasion elicits a robust response from the carp's mucosal immune system, demonstrating the organism's capacity to resist SVCV invasion despite the challenges posed by the pathogen.