Skin Ulceration Syndrome Research Articles

Bacillus coagulans restores pathogen-induced intestinal dysfunction via acetate-FFAR2-NF-κB-MLCK-MLC axis in Apostichopus japonicus.

Skin ulceration syndrome (SUS) is currently the main disease threatening Apostichopus japonicus aquaculture due to its higher mortality rate and infectivity, which is caused by Vibrio splendidus. Our previous studies have demonstrated that SUS is accompanied by intestinal microbiota (IM) dysbiosis, alteration of short-chain fatty acids (SCFAs) content and the damage to the intestinal barrier. However, the mediating effect of IM on intestine dysfunction is largely unknown. Herein, we conducted comprehensive intestinal microbiota transplantation (IMT) to explore the link between IM and SUS development. Furthermore, we isolated and identified a Bacillus coagulans strain with an ability to produce acetic acid from both healthy individual and SUS individual with IM from healthy donors. We found that dysbiotic IM and intestinal barrier function in SUS recipients A. japonicus could be restored by IM from healthy donors. The B. coagulans strain could restore IM community and intestinal barrier function. Consistently, acetate supply also restores intestinal homeostasis of SUS-diseased and V. splendidus-infected A. japonicus. Mechanically, acetate was found to specifically bind to its receptor-free fatty acid receptor 2 (FFAR2) to mediate IM structure community and intestinal barrier function. Knockdown of FFAR2 by transfection of specific FFAR2 siRNA could hamper acetate-mediated intestinal homeostasis in vivo. Furthermore, we confirmed that acetate/FFAR2 could inhibit V. splendidus-activated NF-κB-MLCK-MLC signaling pathway to restore intestinal epithelium integrity and upregulated the expression of ZO-1 and Occludin. Our findings provide the first evidence that B. coagulans restores pathogen-induced intestinal barrier dysfunction via acetate/FFAR2-NF-κB-MLCK-MLC axis, which provides new insights into the control and prevention of SUS outbreak from an ecological perspective.IMPORTANCESkin ulceration syndrome (SUS) as a main disease in Apostichopus japonicus aquaculture has severely restricted the developmental A. japonicus aquaculture industry. Intestinal microbiota (IM) has been studied extensively due to its immunomodulatory properties. Short-chain fatty acids (SCFAs) as an essential signal molecule for microbial regulation of host health also have attracted wide attention. Therefore, it is beneficial to explore the link between IM and SUS for prevention and control of SUS. In the study, the contribution of IM to SUS development has been examined. Additionally, our research further validated the restoration of SCFAs on intestinal barrier dysfunction caused by SUS via isolating SCFAs-producing bacteria. Notably, this restoration might be achieved by inhibition of NF-κB-MLCK-MLC signal pathway, which could be activated by V. splendidus. These findings may have important implications for exploration of the role of IM in SUS occurrence and provide insight into the SUS treatment.

Multi-object detection and behavior tracking of sea cucumbers with skin ulceration syndrome based on deep learning

Skin ulceration syndrome of sea cucumbers is one of the most serious diseases in intensive aquaculture, and it is the most effective way of preventing the spread of this disease to detect the abnormal behavior of sea cucumbers in time and take corresponding measures. However, the detection and tracking of multi-object is a hard problem in sea cucumber behavior analysis. To solve this problem, this paper first proposes a novel one-stage algorithm SUS-YOLOv5 for multi-object detection and tracking of sea cucumbers. The proposed SUS-YOLOv5 optimizes the maximum suppression algorithm in the overlapping region of the object detection box. Next, the SE-BiFPN feature fusion structure is proposed to enhance the transmission efficiency of feature information between deep and shallow layers of the network. Then, a MO-Tracking algorithm is proposed integrated with DeepSORT to achieve real-time multi-object tracking. Experimental results show that the mAP@0.5 and mAP@0.5:0.95 of the proposed object detector reach 95.40% and 83.80%, respectively, which are 3.30% and 4.10% higher than the original YOLOv5s. Compared with the traditional SSD, YOLOv3, and YOLOv4, the mAP of SUS-YOLOv5 is improved by 5.49%, 1.57%, and 3.76%, respectively. This research can realize the multi-object detection and tracking, which lays the foundation for the prediction of skin ulceration syndrome in sea cucumbers and has a certain practical application value for improving the intelligence level of aquaculture.

Effect of probiotic feed supplementation on skin ulceration syndrome in Apostichopus japonicus

Skin ulceration syndrome is among the most harmful diseases in sea cucumber (Apostichopus japonicus). The annual loss due to this disease is more than 1 billion dollars, which seriously affected the healthy and sustainable development of A. japonicus. In this paper, animals were divided into 4 experimental groups: a control group given basic feed, experimental groups with 1% Lactobacillus, 1% Bacillus, 1% mixture with 0.5% Lactobacillus and 0.5% Bacillus added to the basic feed. After feeding for 60 days, the samples were taken to analyze the growth performance, digestive enzyme activity, and immune indexes of A. japonicus. Furthermore, the resistance to the skin ulceration syndrome of A. japonicus was analyzed. The results showed that the probiotics promoted the growth performance of A. japonicus, the weight gain rate of the control group was 120.08±12.07%, the weight gain rate of the Lactobacillus group was 152.11±10.06%, the weight gain rate of the Bacillus group was 148.03±9.88%, and the weight gain rate of the mixed bacteria group was as high as 210.11±19.41%. The digestive enzyme activities in the intestinal tract of A. japonicus increased significantly in the probiotic-added groups, and the mixed bacteria were more effective in enhancing the digestive enzyme activities than single Bacillus and Lactobacillus. In addition, the probiotics could significantly increase the phagocytosis rate of the body cavity cells of A. japonicus. The phagocytic rate of Lactobacillus and Bacillus subtilis was comparable; the mixed probiotics were significantly higher than those of single probiotics, and this phagocytic rate gradually increased over time. After Vibrio splendidus and Pseudoalteromonas infection, the mortality rate of A. japonicus in the probiotic-added group was reduced, which indicated that probiotics could improve the immunity of A. japonicus, and the ability to resist the infection of Vibrio splendidus and Pseudoalteromonas was enhanced. Bacillus exhibits superior antimicrobial ability compared to Lactobacillus, and the mixed strain displays the most effective antimicrobial properties. This study provided a scientific basis for effectively preventing and controlling skin ulceration syndrome in the aquaculture of A. japonicus.

Characterization of microbiota and histology of cultured sea cucumber Isostichopus badionotus juveniles during an outbreak of skin ulceration syndrome.

Due to the dramatic reduction of sea cucumber Isostichopus badionotus populations in the Yucatan Peninsula by overfishing and poaching, aquaculture has been encouraged as an alternative to commercial catching and restoring wild populations. However, the scarcity of broodstock, the emergence of a new disease in the auricularia larvae stage, and the development of skin ulceration syndrome (SUS) in the culture have limited aquaculture development. This study presents the changes in the intestine and skin microbiota observed in early and advanced stages of SUS disease in cultured juvenile I. badionotus obtained during an outbreak in experimental culture through 16S rRNA gene sequencing and histological evidence. Our results showed inflammation in the intestines of juveniles at both stages of SUS. However, more severe tissue damage and the presence of bacterial clusters were detected only in the advanced stages of SUS. Differences in the composition and structure of the intestinal and skin bacterial community from early and advanced stages of SUS were detected, with more evident changes in the intestinal microbial communities. These findings suggest that SUS was not induced by a single pathogenic bacterium. Nevertheless, a decrease in the abundance of Vibrio and an increase in Halarcobacter (syn. Arcobacter) was observed, suggesting that these two bacterial groups could be keystone genera involved in SUS disease.

Cloning and characterization of an aerolysin gene from a marine pathogen Vibrio splendidus

Vibrio splendidus is one of the main pathogens caused diseases with a diversity of marine cultured animals, especially the skin ulcer syndrome in Apostichopus japonicus. However, limited virulence factors have been identified in V. splendidus. In this study, one aerAVs gene coding an aerolysin of V. splendidus was cloned and conditionally expressed in Escherichia coli. The haemolytic activity of the recombinant AerAVs was analyzed. Western blotting was used to study of the secretion pathway of proaerolysin, and it showed that the proaerolysin was secreted via both outer membrane vehicles and classical secretion pathways. Since no active protein of aerolysin was obtained, one aerolysin surface displayed bacterium DH5α/pAT-aerA was constructed, and its haemolytic activity and virulence were determined. The results showed that the AerAVs displayed on the surface showed obvious haemolytic activity and cytotoxic to the coelomocyte of A. japonicus. Artificial immerse infection separately using the DH5α/pAT or DH5α/pAT-aerA was conducted. The result showed that the mortality percent of sea cucumber A. japonicus challenged with DH5α/pAT-aerA was 38.89 % higher than that challenged with the control strain DH5α/pAT, and earlier death occurred. Combined all the results indicates that aerolysin with the haemolytic activity and cytotoxic activity is a virulence factor of V. splendidus.

Comparative proteomic analysis of the protein profile in the cuttlefish Sepia pharaonis associated with skin ulceration syndrome

Skin ulceration syndrome (SUS) is becoming a severe problem in the breeding and culturing process of the cuttlefish Sepia pharaonis. However, limited knowledge is available about the occurrence of this devastating disease. In this study, proteomic analysis was used to identify the differentially expressed proteins (DEPs) and the biological pathways enriched in SUS-diseased S. pharaonis. Both the healthy group and diseased group were analyzed in triplicate, with 4 cuttlefish in each replicate. The results showed that 85 DEPs were identified between the two groups, including 36 upregulated proteins and 49 downregulated proteins in the diseased group compared to the healthy group. GO enrichment analysis revealed that the DEPs were mainly enriched in cellular component organization or biogenesis, nucleus and ion binding processes. The results of the KEGG pathway analysis indicated that extracellular matrix (ECM)-receptor interaction was the most enriched upregulated pathway. Real-time reverse transcriptase PCR was used to identify the expression of two differentially expressed matrix metalloproteinases (MMPs), and the results showed that the mRNA expression of MMP14 and MMP19 was significantly upregulated in the skin tissue of the diseased group. Furthermore, the protease activity of the diseased group was higher than that of the healthy group. Our results offer basic knowledge on the changes in protein profiles during the occurrence of SUS in the cuttlefish S. pharaonis.

Midgut microbiota affects the intestinal barrier by producing short-chain fatty acids in Apostichopus japonicus.

The intestinal microbiota participates in host physiology and pathology through metabolites, in which short-chain fatty acids (SCFAs) are considered principal products and have extensive influence on intestine homeostasis. It has been reported that skin ulceration syndrome (SUS), the disease of Apostichopus japonicus caused by Vibrio splendidus, is associated with the alteration of the intestinal microbiota composition. To investigate whether the intestinal microbiota affects A. japonicus health via SCFAs, in this study, we focus on the SCFA profiling and intestinal barrier function in A. japonicus treated with V. splendidus. We found that V. splendidus could destroy the mid-intestine integrity and downregulate the expression of tight junction proteins ZO-1 and occludin in A. japonicus, which further dramatically decreased microorganism abundance and altered SCFAs contents. Specifically, acetic acid is associated with the largest number of microorganisms and has a significant correlation with occludin and ZO-1 among the seven SCFAs. Furthermore, our findings showed that acetic acid could maintain the intestinal barrier function by increasing the expression of tight junction proteins and rearranging the tight junction structure by regulating F-actin in mid-intestine epithelial cells. Thus, our results provide insights into the effects of the gut microbiome and SCFAs on intestine barrier homeostasis and provide essential knowledge for intervening in SUS by targeting metabolites or the gut microbiota.

Bacillus licheniformis reduces mortality, improves symptoms, and controls residual pathogenicity in juvenile sea cucumbers Apostichopus japonicus infected with skin ulceration syndrome

Mass mortality caused by skin ulceration syndrome (SUS) is the bottle-neck for the sustainable aquaculture of the sea cucumber Apostichopus japonicus. In the present study, probiotic Bacillus licheniformis (0.25 × 109 CFU/g) was used as the treatment for A. japonicus infected with the SUS that caused by Vibrio harveyi. We found that B. licheniformis significantly reduced the number of infected sea cucumbers 5 days and 7 days after the treatment (group B), compared to those without B. licheniformis treatment (group C) (P < 0.001; P < 0.001). Further, the sea cucumbers fed B. licheniformis had significantly lower mortality at the end of the experiment (<10%) than that of those without the B. licheniformis treatment (>60%) (P < 0.001). These results suggest that the treatment of B. licheniformis is an effective method to reduce the mass mortality resulted from SUS in sea cucumber aquaculture. Further, 3–5 days of treatment significantly improved the adverse symptoms of SUS on the physiology and behavior of sea cucumbers, including the righting behavior, adhesion behavior, food consumption, fecal output and mobility. This indicates B. licheniformis treatment has the advantage in the recovery of sea cucumbers after SUS. Moreover, there was no significant difference observed in the physiology and behavior of sea cucumbers between the SUS infected sea cucumbers after the 7-day treatment of B. licheniformis and the healthy individuals. SUS infected sea cucumbers effectively returned to a stage of normalcy. Further, we found a significantly lower infected rate in sea cucumbers exposed to the culture water of group B (∼5%) than that of those in exposure to the culture water of group C (∼60%). This indicates that the treatment of B. licheniformis efficiently controls the residual pathogenicity of SUS in culture water. The present study demonstrated the effectiveness of B. licheniformis treatment as an environmentally friendly approach to reducing mortality, improving symptoms, and controlling residual pathogenicity in sea cucumber aquaculture.

CircRNA254 functions as the miR-375 sponge to inhibit coelomocyte apoptosis via targeting BAG2 in V. splendidus-challenged Apostichopus japonicus

Circular RNAs (circRNAs) function as immune regulators in many biological processes in mammals, while their function and underlying mechanisms in invertebrates are largely unexplored. In this study, the competing endogenous RNA (ceRNA) mechanism of circRNA that sponges miR-375 and thus regulates AjBAG2-mediated coelomocyte apoptosis was evaluated in Apostichopus japonicus. The results showed that circRNA254 (circ254) was significantly down-regulated in the intestines and coelomocytes after Vibrio splendidus challenge or Lipopolysaccharide exposure, which matched the RNA-seq results in A. japonicus within skin ulceration syndrome. Dual-luciferase and RNA FISH assays indicated that circ254 could directly combine with miR-375, in which circ254 possesses three binding sites of miR-375. Moreover, circ254 knockdown significantly promoted the coelomocyte apoptosis levels upon pathogen infection in vivo and in vitro. Furthermore, circ254 silencing could also down-regulate AjBAG2 expression and thereby promoting the levels of coelomocyte apoptosis levels and the expression of caspase 3, which the phenomenon could be reversed by treatment with miR-375 inhibitors. Taken together, our results confirmed that circ254 functions as a ceRNA of AjBAG2 by sponging miR-375, resulting in the inhibition of coelomocyte apoptosis in A. japonicus.

Vibrio splendidus Fur regulates virulence gene expression, swarming motility, and biofilm formation, affecting its pathogenicity in Apostichopus japonicus.

Vibrio splendidus is an opportunistic pathogen that causes skin ulcer syndrome and results in huge losses to the Apostichopus japonicus breeding industry. Ferric uptake regulator (Fur) is a global transcription factor that affects varieties of virulence-related functions in pathogenic bacteria. However, the role of the V. splendidus fur (Vsfur) gene in the pathogenesis of V. splendidus remains unclear. Hence, we constructed a Vsfur knock-down mutant of the V. splendidus strain (MTVs) to investigate the role of the gene in the effect of biofilm, swarming motility, and virulence on A. japonicus. The result showed that the growth curves of the wild-type V. splendidus strain (WTVs) and MTVs were almost consistent. Compared with WTVs, the significant increases in the transcription of the virulence-related gene Vshppd mRNA were 3.54- and 7.33-fold in MTVs at the OD600 of 1.0 and 1.5, respectively. Similarly, compared with WTVs, the significant increases in the transcription of Vsm mRNA were 2.10- and 15.92-fold in MTVs at the OD600 of 1.0 and 1.5, respectively. On the contrary, the mRNA level of the flagellum assembly gene Vsflic was downregulated 0.56-fold in MTVs at the OD600 of 1.0 compared with the WTVs. MTVs caused delayed disease onset time and reduced A. japonicus mortality. The median lethal doses of WTVs and MTVs were 9.116 × 106 and 1.658 × 1011 CFU·ml-1, respectively. Compared with WTVs, the colonization abilities of MTVs to the muscle, intestine, tentacle, and coelomic fluid of A. japonicus were significantly reduced. Correspondingly, the swarming motility and biofilm formation in normal and iron-replete conditions were remarkably decreased compared with those of WTVs. Overall, these results demonstrate that Vsfur contributes to the pathogenesis of V. splendidus by regulating virulence-related gene expression and affecting its swarming and biofilm formation abilities.

CircRNA432 enhances the coelomocyte phagocytosis via regulating the miR-2008-ELMO1 axis in Vibrio splendidus-challenged Apostichopus japonicus

Circular RNAs (circRNAs) are a kind of extensive and diverse covalently closed circular endogenous RNA, which exert crucial functions in immune regulation in mammals. However, the functions and mechanisms of circRNAs in invertebrates are largely unclarified. In our previous work, 261 differentially expressed circRNAs including circRNA432 (circ432) were identified from skin ulcer syndrome (SUS) diseased sea cucumber Apostichopus japonicus by RNA-seq. To better address the functional role of sea cucumber circRNAs, circ432 was first found to be significantly induced by Vibrio splendidus challenge and LPS exposure in this study. Knock-down circ432 could depress the V. splendidus-induced coelomocytes phagocytosis. Moreover, circ432 is validated to serve as the sponge of miR-2008, a differential expressed miRNA in SUS-diseased sea cucumbers, by Argonaute 2-RNA immunoprecipitation (AGO2-RIP) assay, luciferase reporter assay and RNA fluorescence in situ hybridization (FISH) in vitro. Engulfment and cell motility protein 1 (AjELMO1) is further demonstrated to be the target of miR-2008, and silencing AjELMO1 inhibits the V. splendidus-induced coelomocytes phagocytosis, and this phenomenon could be further suppressed by supplementing with miR-2008 mimics, suggesting that circ432 might regulate coelomocytes phagocytosis via miR-2008-AjELMO1 axis. We further confirm that the depressed coelomocytes’ phagocytosis by circ432 silencing is consistent with the decreased abundance of AjELMO1, and could be recovered by miR-2008 inhibitors transfection. All our results provide the evidence that circ432 is involved in regulating pathogen-induced coelomocyte phagocytosis via sponge miR-2008 and promotes the abundance of AjELMO1. These findings will enrich the regulatory mechanism of phagocytosis in echinoderm and provide theoretical data for SUS disease prevention and control in sea cucumbers.

Gut Microbiota Mediates Skin Ulceration Syndrome Outbreak by Readjusting Lipid Metabolism in Apostichopus japonicus.

The intestinal tract is the most important location for symbiotes and pathogens, and the microbiota plays a crucial role in affecting the health of the gut and other host organs. Dysbacteriosis in the intestinal system has been proven to be significant in skin ulceration syndrome (SUS) in sea cucumbers. This study investigates whether the gut microbiota and lipid metabolites are relevant to the initiation and progression of SUS in a Vibrio-splendidus-infected sea cucumber model. The tight junction genes were downregulated and the inflammatory factor gene transcriptions were upregulated after V. splendidus infection in the intestinal tissue of the sea cucumber. V. splendidus infection modulated the gut microbiota by interacting with Psychromonas macrocephali, Propionigenium maris, Bacillus cereus, Lutibacter flavus, and Hoeflea halophila. Meanwhile, the metabolites of the long-chain fatty acids in the intestinal tissue, including triglycerides (TG), phosphatidylethanolamines (PE), and phosphatidylglycerols (PG), were altered after V. splendidus infection. V. splendidus engaged in positive interactions with PG and PE and negative interactions with specific TG. These results related to gut microbiota and metabolites can offer practical assistance in the identification of the inflammatory mechanisms related to SUS, and this study may serve as a reference for predicting the disease.

CircRNA1149 from Apostichopus japonicus suppresses coelomocyte apoptosis act as miR-92a sponge to regulate Bax expression in response to Vibrio splendidus infection

Circular RNAs (circRNAs) are a class of widespread and diverse covalently closed circular endogenous RNAs that play pivotal roles in mediating immune regulation in mammals. However, the function and regulatory mechanisms of circRNAs in lower invertebrates are still far unidentified. In this study, we discovered a circRNA derived from AJAPscaffold1149: 133758|203,802 of Apostichopus japonicus genome, termed circRNA1149 (circ1149), that is closely related to the occurrence of skin ulcer syndrome (SUS) in sea cucumber A. japonicus. Results revealed that circ1149 plays a significant role in pathogen Vibrio splendidus-induced coelomocyte apoptosis in sea cucumbers. Our study also found a microRNA miR-92a, which could promote coelomocyte apoptosis caused by V. splendidus via targeting Bcl-2-associated X protein (Bax). And, Bax is the first identified pro-apoptotic member of the Bcl-2 protein family. Moreover, we detected that the increased coelomocyte apoptosis level and Bax protein level by circ1149 silencing in response to pathogen infection could be partly recovered by miR-92a inhibitors in vivo and in vitro. Our data indicated that circ1149 functions as a competing endogenous RNA (ceRNA) of Bax by competing for adsorbing miR-92a, thereby suppressing the coelomocyte apoptosis levels induced by V. splendidus. The overall results of this study suggested that circ1149 plays a crucial role in regulating the innate immune response of sea cucumbers, and provides a new insight for understanding the functions of circRNAs in lower invertebrates in response to bacterial infection.

Genome-wide identification m6A modified circRNAs revealed their key roles in skin ulceration syndrome disease development in Apostichopus japonicus

Circular RNAs (circRNAs) are novel endogenous non-coding RNAs (ncRNAs) and can be acted as competing endogenous RNAs (ceRNAs) to regulate microRNA (miRNA) and downstream gene expression. Recently, m6A modification has been found in circRNA, and m6A circRNAs also play important roles in various biological processes and a variety of diseases. Our previous study had been demonstrated that circRNAs were differentially expressed in skin ulceration syndrome (SUS) diseased sea cucumber Apostichopus japonicus. However, whether the function of circRNAs are dependent on m6A levels are largely unknown. Here, we firstly investigated the genome-wide map of m6A circRNAs in sea cucumbers with different stages of Vibrio splendidus challenge, that's Control group, SUS-diseased group, and SUS-resistant group. MeRIP-seq revealed that m6A abundances were enriched in circRNAs in all three groups, especially for SUS-resistant group. Among them, more than 62% of modified circRNAs harbor only a single m6A peak and about 55% of m6A sites in circRNAs were derived from sense overlapping in each group. After V. splendidus infection, we found that most of m6A peaks in circRNAs were upregulated and less were downregulated in both SUS-diseased and SUS-resistant groups when compared with Control. Furthermore, GO analysis indicated that the host genes of circRNAs with dysregulated m6A peaks in SUS-diseased and SUS-resistant groups were both mainly enriched in the adhesion pathway. More importantly, we discovered that more than 50% m6A circRNAs showed a positive correlation between the circRNAs expression and m6A methylation levels both in SUS-diseased and SUS-resistant groups. Therefore, a core circRNA-miRNA-mRNA (ceRNA) network whether influenced by m6A modification was constructed based on conjoint analysis. Our results indicated that several selected m6A circRNAs bind with miRNAs were mainly targeting to ubiquitylation system and adhesion pathway. What's more, three candidate m6A circRNAs and three target genes were validated by MeRIP-qPCR and qPCR, whose m6A levels in circRNA and mRNA expressions were consistent with disease occurrence or disease resistance. All of our current findings suggested that m6A circRNAs could play important roles during pathogen infection and might be served as a new molecular biomarker in SUS disease diagnose of A. japonicus.

Dynamic N6-methyladenosine modification of lncRNA modulated by METTL3 during bacterial disease development in an echinoderm

Long non-coding RNAs (lncRNAs) are novel functional non-coding RNAs which engaged in many aspects of biological processes. N6-methyladenosine (m6A) as a kind of abundant epitranscriptomic modification in eukaryotes, plays important roles in regulation of gene expression for various physiological functions. Our previous study demonstrated that sea cucumber lncRNAs were differentially expressed during bacterial infection. However, whether the post-transcriptional regulation of lncRNAs influenced by m6A modification in sea cucumbers with different stages of skin ulceration syndrome (SUS) are largely unknown. Here, we generated the genome-wide map of m6A lncRNAs in SUS-diseased and SUS-resistant sea cucumbers for the first time, revealed that m6A levels in lncRNAs were mainly upregulated in SUS-resistant group. Intriguingly, most of the m6A lncRNAs showed a positive correlation between the expression levels and m6A levels based on conjoint analysis, suggesting that m6A modification on a lncRNA may contribute to its RNA stability. Furthermore, the host genes of lncRNAs with dysregulated m6A peaks were enriched in immune pathway. More importantly, methyltransferase METTL3 was required for m6A methylation modification and played positive roles in lncRNA expression. Collectively, this study presents the comprehensive characters of m6A lncRNAs in marine invertebrate. These m6A modified lncRNAs may be served as potential regulators associated with SUS and provide a promising avenue for disease therapy through targeting METTL3.

Vibrio splendidus virulence to Apostichopus japonicus is mediated by hppD through glutamate metabolism and flagellum assembly

ABSTRACT Vibrio splendidus is the main opportunistic pathogen that causes skin ulcer syndrome in Apostichopus japonicus. hppDIn the present study, mutant V. splendidus with an in-frame deletion of hppDV.s. (MTVs) was constructed. The median lethal doses of wild-type V. splendidus (WTVs) and MTVs were 5.129 × 106 and 2.606 × 1010 CFU mL−1, respectively. RNA-Seq was performed using WTVs and MTVs cells at different growth stages to explore the mechanisms of the pathogenesis mediated by hppDV.s. Gene Ontology analysis showed that the expression levels of 105 genes involved in amino acid metabolism and protein binding were remarkably different between MTVs and WTVs. Kyoto Encyclopedia of Genes and Genomes analysis showed that the pathways of glutamate metabolism and flagellum assembly involved in biofilm formation and swarming motility were suppressed in MTVs. Correspondingly, the swarming motility, biofilm formation and colonisation of MTVs were remarkably decreased compared with those of WTVs. The results showed that 4-hppD catalyses tyrosine into fumarate, which could enhance glutamate metabolism and ATP production; promote flagellum assembly through the TCA cycle and lead to higher swarming, biofilm formation and colonisation abilities, to contribute to the pathogenesis of V. splendidus.

AMPK-mediated glutaminolysis maintains coelomocytes redox homeostasis in Vibrio splendidus-challenged Apostichopus japonicus

Glutaminolysis has been proved to play an irreplaceable role in vertebrate immunity, including effects on cytokine production, bacterial killing, and redox homeostasis maintenance. Our previous metabolomics analysis indicated that glutaminolysis metabolic substrates glutamine (Gln) and metabolites glutamate (Glu) were significantly lower in Skin ulceration syndrome (SUS)-diseased Apostichopus japonicus. To further delineate the role of glutaminolysis, we assayed the levels of Gln and Glu. We found that their contents in coelomocytes were decreased, accompanied by an increase in glutathione (GSH) in pathogen-challenged Apostichopus japonicus. Consistently, the mRNA transcripts of three key genes in glutaminolysis (AjASCT2, AjGOT, and AjGCS) were significantly induced. Moreover, the increased MDA and NADPH/NADP + levels in response to pathogen infection indicated that oxidative stress occurs during the immune response. The metabolic regulator AMPKβ could regulate glutaminolysis in vertebrates by inducing cells to take up extracellular Gln. To explore the underlying regulatory mechanism behind glutaminolysis that occurred in coelomocytes, the full-length cDNA of AMPKβ was identified from A. japonicus (designated as AjAMPKβ). AjAMPKβ expression was significantly induced in the coelomocytes after pathogen challenge, which was consistent with the expression of key genes of glutaminolysis. A functional assay indicated that AjAMPKβ silencing by siRNA transfection could increase the levels of Gln and Glu and depress the production of GSH. Moreover, the expression of glutaminolysis-related genes was significantly inhibited, and the reduction of redox homeostasis indexes (MDA and NADPH/NADP+) was also observed. Contrastingly, AjAMPKβ overexpression promoted redox homeostasis balance. Intracellular ROS is mostly responsible for breaking redox homeostasis and leading to oxidative stress, contributing to cell fate changes in immune cells. Exogenous Gln and GSH treatments could significantly reduce ROS level while the AjAMPKβ silencing induced the level of ROS and accelerated the necrosis rate. All these results collectively revealed that AjAMPKβ could modulate cellular redox homeostasis by affecting the glutaminolysis in A. japonicus.

CircRNA75 and CircRNA72 Function as the Sponge of MicroRNA-200 to Suppress Coelomocyte Apoptosis Via Targeting Tollip in Apostichopus japonicus

Circular RNAs (circRNAs) act as essential regulators in many biological processes, especially in mammalian immune response. Nonetheless, the functions and mechanisms of circRNAs in the invertebrate immune system are largely unclarified. In our previous work, 261 differentially expressed circRNAs potentially related to the development of Apostichopus japonicus skin ulceration syndrome (SUS), which is a major problem restricting the sea cucumber breeding industry, were identified by genome-wide screening. In this study, via miRanda analysis, both circRNA75 and circrRNA72 were shown to share the miR-200 binding site, a key microRNA in the SUS. The two circRNAs were verified to be increased significantly in LPS-exposed primary coelomocytes, similar to the results of circRNA-seq in sea cucumber under Vibrio splendidus-challenged conditions. A dual-luciferase assay indicated that both circRNA75 and circRNA72 could bind miR-200 in vivo, in which circRNA75 had four binding sites of miR-200 and only one for circRNA72. Furthermore, we found that miR-200 could bind the 3’-UTR of Toll interacting protein (Tollip) to negatively mediate the expression of Tollip. Silencing Tollip increased primary coelomocyte apoptosis. Consistently, inference of circRNA75 and circRNA72 could also downregulate Tollip expression, thereby increasing the apoptosis of primary coelomocytes, which could be blocked by miR-200 inhibitor treatment. Moreover, the rate of si-circRNA75-downregulated Tollip expression was higher than that of si-circRNA72 under an equivalent amount. CircRNA75 and circRNA72 suppressed coelomocyte apoptosis by sponging miR-200 to promote Tollip expression. The ability of circRNA to adsorb miRNA might be positively related to the number of binding sites for miRNA.

Tissue inhibitor of metalloproteinases 1 is involved in ROS-mediated inflammation via regulating matrix metalloproteinase 1 expression in the sea cucumber Apostichopus japonicus

Tissue inhibitors of metalloproteinases (TIMPs) serve as matrix metalloproteinase (MMP) inhibitors in the pathogenesis of inflammatory diseases in vertebrates. We cloned and characterised the TIMP1 gene from Apostichopus japonicus using RACE approaches (designated as AjTIMP1). For Vibrio splendidus-challenged sea cucumbers, the peak expression of AjTIMP1 mRNAs in coelomocytes was detected at 24 h (23.44-fold) and remained at high levels (4.01-fold) until 72 h. Similarly, AjTIMP1 expression was upregulated in primary coelomocytes exposed to 10 μg mL−1 LPS. AjTIMP1 was expressed in all tissues, and the highest expression was observed in the body wall. Functional investigation revealed an imbalance in the ratio of AjMMP1/AjTIMP1 in the skin ulceration syndrome (SUS) diseased group; it was sharply up-regulated to 3.97:1 compared with the healthy group. Furthermore, when AjTIMP1 was knocked down using small interfering RNA (siRNA-KD) to 0.4-fold, AjMMP1 and AjMMP19 were upregulated to 1.99- and 1.85-fold, respectively. AjTIMP1 siRNA-KD can promote ROS production by 26.2%, whereas AjMMP1 siRNA-KD can eliminate the increase in ROS. In inflamed tissues, collagen I and III levels were decreased by 33.1% and 33.6%, respectively, in the AjTIMP1 siRNA group at 24 h AjTIMP1 was involved in the inflammatory response by mediating ROS formation and collagen degradation.

Kültürü yapılan Holothuria poli (Delle Chiaje, 1823) türü deniz hıyarının deri ülserlerinden Vibrio mediterranei (Pujalte ve Garay 1986) / Vibrio shiloi (Kushmaro vd. 2001) bakterisinin gözlenmesi

Skin ulcer syndrome is frequently reported as a serious disease affecting the health, growth and mortality of stocks in sea cucumber aquaculture. In this study, bacteria isolated predominantly from skin ulcers of sea cucumber Holothuria poli (Delle Chiaje, 1823), a new candidate for aquaculture in the Mediterranean, were investigated. Morphological and biochemical tests, and molecular analysis methods were used to examine the dominant bacteria in the lesions of H. poli showing skin ulceration, peristome tumour and visceral ejection symptoms in rearing tanks. Present study is the first report for isolation and identification of Vibrio mediterranei (Pujalte and Garay 1986) (called also Vibrio shiloi Kushmaro et al. 2001) as a predominant gram-negative bacterium in the skin ulcers of H. poli. Reference data provided from the present study would lead to understand possible major pathogens causing skin ulceration syndrome and is crucial for the prophylaxis and treatment of such disease in holothuriculture.