Transcriptome analysis of the Japanese eel (Anguilla japonica) during larval metamorphosis.

BACKGROUND: Anguillid eels spend their larval period as leptocephalus larvae that have a unique and specialized body form with leaf-like and transparent features, and they undergo drastic metamorphosis to juvenile glass eels. Less is known about the transition of leptocephali to the glass eel stage, because it is difficult to catch the metamorphosing larvae in the open ocean. However, recent advances in rearing techniques for the Japanese eel have made it possible to study the larval metamorphosis of anguillid eels. In the present study, we investigated the dynamics of gene expression during the metamorphosis of Japanese eel leptocephali using RNA sequencing. RESULTS: During metamorphosis, Japanese eels were classified into 7 developmental stages according to their morphological characteristics, and RNA sequencing was used to collect gene expression data from each stage. A total of 354.8 million clean reads were generated from the body and 365.5 million from the head, after the processing of raw reads. For filtering of genes that characterize developmental stages, a classification model created by a Random Forest algorithm was built. Using the importance of explanatory variables feature obtained from the created model, we identified 46 genes selected in the body and 169 genes selected in the head that were defined as the "most characteristic genes" during eel metamorphosis. Next, network analysis and subsequently gene clustering were conducted using the most characteristic genes and their correlated genes, and then 6 clusters in the body and 5 clusters in the head were constructed. Then, the characteristics of the clusters were revealed by Gene Ontology (GO) enrichment analysis. The expression patterns and GO terms of each stage were consistent with previous observations and experiments during the larval metamorphosis of the Japanese eel. CONCLUSION: Genome and transcriptome resources have been generated for metamorphosing Japanese eels. Genes that characterized metamorphosis of the Japanese eel were identified through statistical modeling by a Random Forest algorithm. The functions of these genes were consistent with previous observations and experiments during the metamorphosis of anguillid eels.

RNA-seq analysis revealed the pathogenicity of Vibrio vulnificus to American eel (Anguilla rostrata) and the strategy of host anti-V. vulnificus infection.

Vibrio vulnificus is a commonly pathogenic bacterium in cultivated eels, but its pathogenicity to American eel (Anguilla rostrata) and the molecular mechanism of host anti-V. vulnificus infection remains uncertain. In this study, American eels were infected with different dose of V. vulnificus to determine the LD50. Then, bacterial load in the liver and kidney histopathology were assessed post the LD50 of V. vulnificus infection. Additionally, gene expressions of 18 immune related genes in the liver, spleen and kidney were detected. Furthermore, transcriptome sequencing and enrichment of differentially expressed genes (DEGs) were analyzed in the eel spleens between pre-infection (Con_0), post-36 h (Vv_36), and post-60 h (Vv_60) infection. The results showed that LD50 of V. vulnificus to American eels was determined to be 5.0 × 105 cfu/g body weight, and the bacterial load peaked at 24 and 12 h post the infection (hpi) in the kidney and liver, respectively. The histopathology was highlighted by necrotic hepatocytes and splenic cells, congestion blood vessels in liver and spleen, atrophied glomeruli and vacuolization of renal tubular epithelial cells. The results of RT-PCR revealed that 18 host immune-related genes showed significantly up or downregulated expression post-infection compare to that of pre-infection. Finally, results of the RNA-seq revealed 16 DEGs play essential role to the immunosuppression in American eels, and the protein-protein interactions shed light on the widespread upregulation GEGs related to metabolism and immune response maintained the host cell homeostasis post the V. vulnificus infection, shedding new light on our understanding of the V. vulnificus pathogenesis towards understudied American eel and the host anti-V. vulnificus infection strategies in gene transcript.

Δfur mutant as a potential live attenuated vaccine (LAV) candidate protects American eels (Anguilla rostrata) from Vibrio harveyi infection.

The eel farming industry is highly susceptible to Vibriosis. Although various types of vaccines against Vibriosis have been investigated, there is limited research on decreasing the virulence of Vibrions through gene knockout and utilizing it as live attenuated vaccines (LAV). In this study, we aim to develop a LAV candidate against Vibrio harveyi infection in American eels (Anguilla rostrata) using a ferric uptake regulator (fur) gene mutant strain of V. harveyi (Δfur mutant). After the eels were administrated with the Δfur mutant at the dose of 4 × 102 cfu/g body weight, the phagocytic activity of the leucocytes, plasma IgM antibody titers, activity of lysozyme and Superoxide Dismutase (SOD) enzyme, and gene expression levels of 18 immune related proteins were detected to evaluate the protection effect of the LAV. Preliminary findings suggest that the LAV achieved over 60% relative percent survival (RPS) after the American eels were challenged by a wild-type strain of V. harveyi infection on 28 and 42 days post the immunization (dpi). The protection was mainly attributed to increased plasma IgM antibody titers, higher levels of lysozyme, enhanced activity of SOD and some regulated genes encoded immune related proteins. Together, the Δfur mutant strain of V. harveyi, as a novel LAV vaccine, demonstrates promising protective effects against V. harveyi infection in American eels, thus presenting a potential candidate vaccine for fish farming.

Toll-interacting protein is activated by the transcription factor GATA1 and Sp1 to negatively regulate NF-κB and MAPK pathways in the Japanese eel (Anguilla japonica).

Toll-interacting protein (Tollip) serves as a crucial inhibitory factor in the modulation of Toll-like receptor (TLR)-mediated innate immunological responses. The structure and function of Tollip have been well documented in mammals, yet the information in teleost remained limited. This work employed in vitro overexpression and RNA interference in vivo and in vitro to comprehensively examine the regulatory effects of AjTollip on NF-κB and MAPK signaling pathways. The levels of p65, c-Fos, c-Jun, IL-1, IL-6, and TNF-α were dramatically reduced following overexpression of AjTollip, whereas knocking down AjTollip in vivo and in vitro enhanced those genes' expression. Protein molecular docking simulations showed AjTollip interacts with AjTLR2, AjIRAK4a, and AjIRAK4b. A better understanding of the transcriptional regulation of AjTollip is crucial to elucidating the role of Tollip in fish antibacterial response. Herein, we cloned and characterized a 2.2 kb AjTollip gene promoter sequence. The transcription factors GATA1 and Sp1 were determined to be associated with the activation of AjTollip expression by using promoter truncation and targeted mutagenesis techniques. Collectively, our results indicate that AjTollip suppresses the NF-κB and MAPK signaling pathways, leading to the decreased expression of the downstream inflammatory factors, and GATA1 and Sp1 play a vital role in regulating AjTollip expression.

Immune responses and protective efficacy of American eel (Anguilla rostrata) immunized with a formalin-inactivated vaccine against Anguillid herpesvirus.

Anguillid herpesvirus 1 (AngHV), the causative agent of "mucus sloughing and hemorrhagic septicemia disease", causes serious infectious diseases in farmed eel. Among the effective prevention and control strategies, vaccination is one of the most effective approaches. However, no vaccine for AngHV is available. Our study developed a formalin-inactivated AngHV vaccine and evaluated its performance in American eels. Initially, AngHV-FJ, a strain of AngHV, was inactivated completely by 0.1 % formaldehyde, mixed with adjuvant Montanide ISA 763 A VG (763A). Then, vaccines containing different amount of antigen (3 × 106 PFU, 3 × 105 PFU, 3 × 104 PFU, 3 × 103 PFU) were immunized in each American eels. The results showed that the 3 × 105 PFU/fish was the proper dose. The inactivated AngHV vaccine was proven safe for American eels by back intramuscular injection. The results of twice immunization showed that antibody production peaked in the 8th week after the first immunization, and the antibody titer was 1:64,000. Furthermore, the immunized fishes challenged with AngHV (105 PFU/ml immersion) showed a significantly lower incidence rate (33.33 %) than the control group (95.65 %). The survival of the fish in the vaccine group (94.44 %) was significantly higher than the control group (60.87 %). The relative survival rate of the vaccinated group was 85.80 %. Also, vaccine group tissue collected at 7th d post-challenge showed reduced tissue damage and a lower virus load than the control group. The expression of cytokines of IL-1ß, IFN-α, IFN-γ, Mx1, RIG-1, and IRF-3, were significantly lower in the vaccine group than the control group at the 7th and 14th d post-challenge. Overall, the formalin-inactivated AngHV vaccine was safe and had immune protective effects against AngHV infection.

Preparation of valine-curcumin conjugate and its in vitro antibacterial and antitumor activity and in vivo biological effects on American eels (Anguilla rostrata).

Curcumin (Cur) exhibits diverse natural pharmacological activities, despite its limited water solubility (hydrophobicity) and low bioavailability. In this investigation, a valine-curcumin conjugate (Val-Cur) was synthesized through amino acid side chain modification, and its solubility increased to 1.78 mg/mL. In vitro experimental findings demonstrated that the antibacterial activity of Val-Cur against Escherichia coli, Staphylococcus aureus, Aeromonas hydrophila, and Vibrio parahaemolyticus was significantly superior to that of Cur. The inhibition rate of Val-Cur against HepG2 (human hepatocellular carcinoma) cells was higher than that of Cur at low concentrations (below 25 µmol/L), although the IC50 value of Val-Cur did not differ significantly from that of Cur. In vivo biological effects of Val-Cur were assessed by adding it into the feed (150 mg/kg) of American eels (Anguilla rostrata). Val-Cur significantly improved the growth performance (↑weight gain rate, ↑specific growth rate, and ↓feed conversion rate) and activities of intestinal digestive enzymes (amylase and lipase) and antioxidant enzymes (superoxide dismutase) in American eels. Additionally, Val-Cur significantly improved serum biochemical indices (↑high-density lipoprotein cholesterol, ↓low-density lipoprotein cholesterol, ↓aspartate and alanine aminotransferases). Furthermore, Val-Cur increased intestinal microbial diversity, reduced the abundance of potentially pathogenic bacteria (Spiroplasma, Clostridium, and Pseudomonas), and elevated the abundance of beneficial digestion-promoting bacteria (Romboutsia, Phyllobacterium, Romboutsia sedimentorum, and Clostridium butyricum) conducive to glucose metabolism (P < 0.05). To the best of our knowledge, this study is the first to explore water-soluble curcumin in aquaculture, and the findings will lay the groundwork for the potential application of water-soluble curcumin in the field of aquaculture.

Effects of adding Bacillus subtilis natto NTU-18 in paste feed on growth, intestinal morphology, gastrointestinal microbiota diversity, immunity, and disease resistance of Anguilla japonica glass eels.

Japanese eel, Anguilla japonica, holds significant importance in Taiwanese aquaculture. With the intensification of eel farming, the impact of Edwardsiella tarda has become increasingly severe. Consequently, the abusive use of antibiotics has risen. Bacillus subtilis natto NTU-18, a strain of Bacillus with a high survival rate in feed processing, plays a crucial role in promoting intestinal health through competitive rejection, enhancing immune responses against bacterial pathogens, and improving intestinal health by modulating gastrointestinal microbiota to produce beneficial metabolites of mice and grass carp, Ctenopharyngodon idella. This study investigated the effects of different proportions (control, 0.25 %, 0.5 %, 1 %, and 2 %) of B. subtilis natto NTU-18 added to paste feed on the growth performance, intestinal morphology, and microbiota, expression of immune-related genes, and resistance to E. tarda in Japanese glass eel. The results indicated that the growth performance of all groups with B. subtilis natto NTU-18 added was significantly higher than that of the control group and did not impact the villi morphology. The expression of immune-related genes in the kidney, specifically HSP70 and SOD, was significantly higher from 0.5 % and above than the control; however, no significant differences were observed in CAT, POD, and HSP90. In the liver, significant differences were found in HSP70 and IgM above 0.25 % compared to the control group, with no significant differences in SOD, CAT, POD, and HSP90 among all groups. Additionally, intestinal microbiota analysis revealed that the 2 % additional group had significantly lower diversity than other groups, with Cetobacterium as the dominant species. The challenge test observed that the survival rates of the 0.5 % and 1 % groups were significantly higher. This research suggests that adding 0.5 % and 1 % of B. subtilis natto NTU-18 to the diet is beneficial for Japanese glass eel's immunity, growth performance, and disease resistance.

Transcriptome analysis of host anti-Aeromonas hydrophila infection revealed the pathogenicity of A. hydrophila to American eels (Anguilla rostrata).

Aeromonas hydrophila is a commonly pathogenic bacterium in cultivated eels, but its pathogenicity to American eel (Anguilla rostrata) and the molecular mechanism of host anti-A. hydrophila infection remains uncertain. In this study, LD50 of A. hydrophila to American eels was determined and bacterial load in the liver and kidney of eels was assessed post 2.56 doses of LD50 of A. hydrophila infection. The results showed that the LD50 of A. hydrophila to American eels was determined to be 3.9 × 105 cfu/g body weight (7.8 × 106 cfu/fish), and the bacterial load peaked at 36 h post the infection (hpi) in the liver. Then, the histopathology was highlighted by congestion in splenic blood vessels, atrophied glomeruli, and necrotic hepatocytes. Additionally, the results of qRT-PCR revealed that 18 host immune-related genes showed significantly up or downregulated post-infection compare to that of pre-infection. Finally, results of the RNA-seq revealed 10 hub DEGs and 7 encoded proteins play essential role to the anti-A. hydrophila infection in American eels. Pathogenicity of A. hydrophila to American eels and RNA-seq of host anti-A. hydrophila infection were firstly reported in this study, shedding new light on our understanding of the A. hydrophila pathogenesis and the host immune response to the A. hydrophila infection strategies in gene transcript.

The Presence of Bioactive Compounds in European Eel (Anguilla anguilla) Skin: A Comparative Study with Edible Tissue.

The presence of bioactive compounds in European eel (Anguilla anguilla) skin was studied. Proximate and lipid class compositions and analysis of the fatty acid (FA) profile (individual FAs; FA groups, i.e., saturated, monounsaturated, and polyunsaturated; FA ratios, i.e., polyunsaturated/saturated, ω3/ω6) were determined and compared to the composition of the eel muscle. As a result, higher (p < 0.05) levels of proteins (271.6 g·kg-1), lipids (38.0 g·kg-1), ash (27.7 g·kg-1), and ω6 FAs were observed in the skin tissue. Contrary, the muscle tissue showed higher (p < 0.05) moisture, ω3 FA, and ω3/ω6 ratio values. Regarding lipid classes, a higher (p < 0.05) proportion of phospholipids (111.1 g·kg-1 lipids), free sterols (104.7 g·kg-1 lipids), α-tocopherol (274.0 mg·kg-1 lipids), and free FAs (43.6 g·kg-1 lipids) was observed in the skin tissue. No differences (p > 0.05) between both tissues could be detected for triacylglycerol and FA group (saturated, monounsaturated, and polyunsaturated) values and for the polyunsaturated/saturated FA ratio. It is concluded that European eel skin, a by-product resulting from commercial processing, can be considered a valuable source for the food and pharmaceutical industries by providing value-added constituents such as proteins, lipids, ω3 FAs, phospholipids, and α-tocopherol.

Comparative phenotype and transcriptome analysis revealed the role of ferric uptake regulator (Fur) in the virulence of Vibrio harveyi isolated from diseased American eel (Anguilla rostrata).

Vibrio harveyi is commonly found in salt and brackish water and is recognized as a serious bacterial pathogen in aquaculture worldwide. In this study, we cloned the ferric uptake regulator (fur) gene from V. harveyi wild-type strain HA_1, which was isolated from diseased American eels (Anguilla rostrata) and has a length of 450 bp, encoding 149 amino acids. Then, a mutant strain, HA_1-Δfur, was constructed through homologous recombination of a suicide plasmid (pCVD442). The HA_1-Δfur mutant exhibited weaker biofilm formation and swarming motility, and 18-fold decrease (5.5%) in virulence to the American eels; compared to the wild-type strain, the mutant strain showed time and diameter differences in growth and haemolysis, respectively. Additionally, the adhesion ability of the mutant strain was significantly decreased. Moreover, there were 15 different biochemical indicators observed between the two strains. Transcriptome analysis revealed that 875 genes were differentially expressed in the Δfur mutant, with 385 up-regulated and 490 down-regulated DEGs. GO and KEGG enrichment analysis revealed that, compared to the wild-type strain, the type II and type VI secretion systems (T2SS and T6SS), amino acid synthesis and transport and energy metabolism pathways were significantly down-regulated, but the ABC transporters and biosynthesis of siderophore group non-ribosomal peptides pathways were up-regulated in the Δfur strain. The qRT-PCR results further confirmed that DEGs responsible for amino acid transport and energy metabolism were positively regulated, but DEGs involved in iron acquisition were negatively regulated in the Δfur strain. These findings suggest that the virulence of the Δfur strain was significantly decreased, which is closely related to phenotype changing and gene transcript regulation.

Superoxidase dismutases (SODs) in the European eel: Gene characterization, expression response to temperature combined with hormonal maturation and possible migratory implications.

Superoxide dismutases (SODs) are antioxidant enzymes that protect cells from oxidation. Three SODs have been identified in mammals, but there is limited information in teleosts. This study investigates SODs in the European eel and their expression patterns during testis maturation. Phylogenetic and synteny analyses revealed SODs paralogs and their evolution in vertebrates. The eel possesses one SOD1 and two SOD2/3 (a and b), indicating SOD2 and SOD3 duplication in elopomorphs. SODs expression were then evaluated in various male and female tissues. SOD1 is more expressed in females, while SOD2a and SOD2b dominate brain-pituitary-gonad tissues in both sexes. SOD3a showed predominant expression in the ovary and the male livers, whereas SOD3b was found in the pituitary and brain of both sexes. The effects of different maturation protocols (standard hormonal treatment vs. same protocol preceded with cold seawater pre-treatment) on SODs expression during testis maturation were evaluated. Salinity increase at the onset of standard treatment at 20 °C, simulating early migration, upregulated SOD1, SOD2a, and SOD2b, coinciding with spermatogonia type A differentiated cells dominance. Thereafter, SOD2a and SOD3a decreased, while SOD2b increased during hormonal treatment-induced spermatogenesis. Pre-treatment with seawater at 10 °C, mimicking the conditions at the beginning of the seawater migration, downregulated SOD1 but increased SOD3a expression. Finally, the standard hormonal treatment, replicating spawning at higher temperatures, downregulated SOD1 in eels without any pre-treatment while SOD2a expression increased in pre-treated eels. This study revealed tissue-specific, sex-dependent, and maturation-related SOD expression patterns, predicting SODs dynamic expression profiles during their reproductive migration.

Short-term induced swimming activity enhanced innate immune parameters and antioxidant status of European eel (Anguilla anguilla).

The swimming activity, although an essential trait in the life cycle of fish, is still poorly understood in farmed fish. The current study aimed to investigate the impact of short-term induced swimming on the immune and antioxidant defence systems in European eel (Anguilla anguilla). Sixteen male yellow European eels (total length: 39.9 ± 0.7 cm; body weight: 108.8 ± 6.1 g) were individually placed in swimming flumes and divided into two groups: i) no swimming (n = 8); and ii) induced-swimming (n = 8) at 0.3 body lengths (BL)·s-1 for 7 h. Swimming resulted in a 2-fold lower cortisol concentration in plasma, whereas plasma glucose, lactate, and several immune-related parameters did not present variations between groups. Interestingly, swimming led to higher lysozyme, peroxidase, and protease activities in skin mucus, whereas bactericidal activity did not show differences among groups. Additionally, the gene expression of interleukin 1 beta showed an up-regulation in the skin of fish with induced swimming, while no differences were observed in the head-kidney or gills. Furthermore, modulation of the antioxidant status was observed in the liver and posterior skeletal muscle after induced swimming. Fish subjected to swimming showed lower lipid peroxidation and higher reduced glutathione levels, increasing the reduced/oxidized glutathione ratio. However, no variations in the antioxidant status were observed between groups in the anterior skeletal muscle. This study showed modulation of immune and oxidative stress markers in European eels upon short-term induced swimming compared to non-swimming fish.

Optimizing the mass marking of fish with alizarin red S: an example with glass eels.

Fish marking is an essential tool for fisheries management, especially for evaluating the stocking of endangered fish species to support conservation and sustainable use of fish stocks. Batch marking of young European eels Anguilla anguilla (L.) prior to stocking is recommended as the benefits of stocking for the spawning stock can be evaluated by recapturing marked fish over time, therefore mass marking of young eels with substances such as alizarin red S (ARS) is becoming increasingly important. To improve the marking method and reduce marking costs when immersing glass eels in an ARS solution, eight laboratory experiments under varying conditions (e.g., temperature, ARS concentration, immersion time, osmotic induction, fish density) and with ARS from different suppliers were carried out. The results show that optimal marking of glass eels can be carried out in the field or during transport by putting approximately 50 g of glass eels per liter in 150 mg L-1 ARS solution for 3 h at 10-15°C. Lower concentrations did not result in reliable marking. Water temperatures of 5°C and below can have a stunning effect on the eels and increase mortality significantly, regardless of the concentration of ARS. Glass eel densities below 50 g L-1 in the marking bath increase marking costs unnecessarily, while a higher density of 100 g L-1 resulted in significantly higher mortality and lower marking success. A somewhat more difficult but less expensive alternative is to bathe the fish in a saline solution of 1% (10 PSU) of 80 mg L-1 ARS for 3 h at 10°C. Costs can also be significantly reduced by choice of supplier for ARS, but care should be taken as the quality of the powder appears to vary (mean percentage of sufficiently marked eels ranged from 59% to 91% among suppliers in the present study) and can lead to marking failure. The optimal marking conditions can help ensure that stocked glass eels can be reliably identified in future studies to assess stocking benefits while reducing costs.

Larger yellow-phase Japanese eels show short-range homing in a freshwater river.

The homing behavior and site fidelity to habitats in various fishes, including anguillid eels (genus Anguilla), are fascinating. However, little is known about how yellow-phase eels exhibit homing behavior and the sensory mechanisms involved. Using acoustic telemetry, we investigated the homing behavior of 18 Japanese eels, A. japonica, with total lengths ranging from 204 to 570 mm, in a narrow freshwater river in inland central Japan, where salinity gradient, tidal current, and magnetic sense cannot be used for their homing, but where olfaction could play a role. The tagged eels captured upstream and downstream were released downstream and upstream, respectively. The results showed that large eels, over approximately 400 mm in total length, exhibited homing behavior to their original sampling locations (likely to shelters and foraging sites, where they probably spent a longer time than in other locations and grew successfully) from outside their home ranges, predominantly during the dark period. Homing success was not affected by the two capture locations, indicating that eels did not use olfactory cues for short-range homing in freshwater rivers.

Unravelling the life-history patterns and habitat preferences of the Japanese eel (Anguilla japonica) in the Pearl River, China.

Eels have fascinated biologists for centuries due to their amazing long-distance migrations between freshwater habitats and very distant ocean spawning areas. The migratory life histories of the Japanese eel, Anguilla japonica, in the waters of south China are not very clear despite its ecological importance, and the need for fishery regulation and management. In this study, strontium (Sr) and calcium (Ca) microchemical profiles of the otoliths of silver eels were measured by X-ray electron probe microanalysis based on data collected from different habitats (including freshwater and brackish habitats), in the large subtropical Pearl River. The corresponding habitat preference characteristics were further analysed using redundancy analysis (RDA). A total of 195 Japanese eels were collected over 6 years. The collected individuals ranged from 180 to 771 mm in total length and from 8 to 612 g in body weight. Two-dimensional pictures of the Sr:Ca concentrations in otoliths revealed that the A. japonica in the Pearl River are almost entirely river eels, spending the majority of their lives in fresh water without exposure to salt water, while the catadromous migration time has delayed about 1 month in the Pearl River estuary in the past 20 years. RDA analysis further indicated that juveniles and adults preferred water with high salinity and high tide levels. Youth preferred habitats with high river fractals. Our findings contribute to a growing body of evidence showing that the eels are extremely scarce currently and conservation measures against them are imminent, including the protection of brackish and freshwater areas where they live in south China.

Can displaced Anguilla marmorata return to their original habitat? Daily tracking study of their homing behavior.

Anguilla marmorata collected in the Odana River lower reaches were passive integrated transponder-tagged displaced and released into the upper river reaches (54 up-transported eels), and eels collected in the upper reaches were tagged and released downstream (52 down-transported eels). Their movements were detected once per day for 10 days using a portable radio-frequency identification (RFID) system. The homing rate of the down-transported eels was 38.9%, compared to 3.7% for the up-transported eels, suggesting that eels inhabiting upstream areas have relatively high fidelity to their habitats and downstream eels have less fidelity.

Effect of seawater temperature and pH on the sperm motility of the European eel.

The current climate change situation could bring critical effects for marine species, especially those already considered endangered. Although some species can adapt fast to the environmental changes, it is necessary to get into the worst scenario and develop tools to anticipatedly assess the physiological effects of such environmental change. With this purpose, our study aims to determine the effect of a range of seawater temperatures and pHs on sperm motility in the European eel (Anguilla anguilla). Low seawater pH (6.5-7.4) decreased the eel sperm motility in comparison to the control (pH = 8.2). We also studied the combined effect of the pH of the artificial seminal plasma (the plasma where the sperm cells are suspended) with the pH of Artificial Sea Water (ASW, pH 7.8 or and 8.2). We did not find statistical differences in sperm motility and kinetic parameters caused by the artificial seminal plasma pH. However, seawater pH induced significantly higher values of total sperm motility, and the percentage of fast spermatozoa with a pH of 8.2 in comparison with a pH of 7.8. In contrast, the seawater temperature did not affect sperm motility parameters or sperm longevity. To study the effect of the interaction between seawater temperature and pH on sperm motility, two temperatures: 4 and 24 °C, and two pHs 7.8 and 8.2, were tested. There were significant differences between temperature and pH in several kinetic parameters and, in general, the lowest values were observed in the samples activated at low temperature and low pH (4 °C, pH 7.8). This work suggest that eel sperm motility and kinetics will not be affected by the expected changes in pH or temperature due to the climate change.

Aureispira anguillae sp. nov., isolated from Japanese eel Anguilla japonica leptocephali.

A novel filamentous eel-leptocephalus pathogenic marine bacterium, designated strain EL160426T, was isolated from Japanese eel, Anguilla japonica, leptocephali reared at a laboratory in Mie, Japan. In experimental infection studies on eel larvae, the strain EL160426T caused massive larval mortality and was reisolated from moribund leptocephali. Characteristically, observations of infected larvae found that EL160426T forms columnar colonies on the cranial surface of larvae. The novel isolate exhibited growth at 15-30 °C, pH 7-9, and seawater concentrations of 60-150% (W/V). Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain EL160426T was most closely related to Aureispira maritima 59SAT with 97.7% sequence similarity. The whole genome sequence analysis of the strain EL160426T showed that the strain maintained a circular chromosome with a size of approximately 7.58 Mbp and the DNA G + C content was 36.2%. The major respiratory quinone was MK-7 and the predominant cellular fatty acids were 16:0, 20:4 w6c (arachidonic acid), 17:0 iso and 16:0 N alcohol. DNA relatedness between the closest phylogenetic neighbor strain EL160426T and A. maritima (JCM23207T) was less than 13%. On the basis of the polyphasic taxonomic data, the strain represents a novel species of the genus Aureispira, for which the name Aureispira anguillae sp. nov. is proposed. The type strain is EL160426T (= JCM 35024 T = TSD-286 T).

Temperature and pressure dependency of oxygen consumption during long-term sustained swimming of European eels.

Many aspects of the typically 5000-10,000 km spawning migration of the European eel (Anguilla anguilla) remain unknown. As part of this migration, eels undertake extensive diurnal vertical migrations to depths below 1000 m, being exposed to a wide range of temperatures and hydrostatic pressures. In this experimental study, we exposed eels to different combinations of temperature (12-20°C) and pressure (100--800 kPa) during long-term sustained swimming (32-47 days). Both temperature and pressure affected oxygen consumption rate, such that there was a significant increase of metabolic rate with temperature, whereas pressure reduced oxygen consumption, albeit only at higher temperatures. Average oxygen consumption rates ranged between 15 mg kg-1 h-1 (12°C, 100 kPa) and 30.2 mg kg-1 h-1 (20°C, 100 kPa), highlighting the remarkably high swimming efficiency of this species and, more importantly, indicating that past evaluations of the cost of transport are potentially overestimates as they are often based on experiments conducted at atmospheric pressure at higher temperatures.

Inoculation of Freund's adjuvant in European eel (Anguilla anguilla) revealed key KEGG pathways and DEGs of host anti-Edwardsiella anguillarum infection.

Freund's complete (FCA) and incomplete adjuvants (FIA), generally applied in subunit fishery vaccine, have not been explored on the molecular mechanism of the nonspecific immune enhancement. In this study, we examined the RNA-seq in the spleen of European eel (Anguilla anguilla) inoculated with FCA and FIA (FCIA group) to elucidate the key KEGG pathways and differential expressed genes (DEGs) in the process of Edwardsiella anguillarum infection and A. anguilla anti-E. anguillarum infection using genome-wide transcriptome. After eels were challenged by E. anguillarum at 28 d post the first inoculation (dpi), compared to the control uninfected eels (Con group), the control infected eels (Con_inf group) showed severe pathological changes in the liver, kidney and spleen, although infected eels post the inoculation of FCIA (FCIA_inf group) also formed slight bleeding. Compared to the FCIA_inf group, there was more than 10 times colony forming unit (cfu) in the Con_inf group per 100 µg spleen, kidney or blood, and the relative percent survival (RPS) of eels was 44.4% in FCIA_inf vs Con_inf. Compared to the Con group, the SOD activity in the FCIA group increased significantly in the liver and spleen. Using high-throughput transcriptomics, DEGs were identified and 29 genes were verified using fluorescence real-time polymerase chain reaction (qRT-PCR). The result of DEGs clustering showed 9 samples in 3 groups of Con, FCIA and FCIA_inf were similar, contrast to distinct differences of 3 samples in the Con_inf group. We found 3795 up and 3548 down regulated DEGs in the compare of FCIA_inf vs Con_inf, of which 5 enriched KEGG pathways of "Lysosome", "Autophagy", "Apoptosis", "C-type lectin receptor signaling" and "Insulin signaling" were ascertained, and 26 of 30 top GO terms in the compare were significantly enriched. Finally, protein-protein interactions between the DEGs of the 5 KEGG pathways and other DEGs were explored using Cytoscape 3.9.1. The compare of FCIA_inf vs Con_inf showed 110 DEGs from the 5 pathways and 718 DEGs from other pathways formed total of 9747° in a network, of which 9 hub DEGs play vital roles in anti-infection or apoptosis. Together, the interaction networks revealed that 9 DEGs involved in the 5 pathways underlies the key process of A. anguilla anti-E. anguillarum infection or host cell apoptosis.