Abstract:Aquatic environments support a diverse range of microbial communities that have mutualistic associations with aquatic plants and farm animals. Under certain conditions, some microorganisms undergo transition to potential pathogenic bacteria in response to the influence of internal and external factors within the water environment, which can contribute to bacterial diseases and influence the survival and growth of farm animals. The fat innkeeper worm (Urechis unicinctus), which is the only Xenopneusta worm distributed along the coast of China, has rich nutritional and medicinal values. U. unicinctus is a filter-feeding marine invertebrate that inhabits muddy and sandy substrates and is susceptible to attack by environmental pathogens. As a means of defense, these worms are primarily dependent on physical barriers and cellular and humoral immune factors within the coelomic fluid that constitute a non-specific type of immunity. The coelomic fluid of U. unicinctus is equivalent to the blood of vertebrates, in which the internal organs are bathed, and plays a particularly important role in immunity. To date, studies on the coelomic fluid of U. unicinctus have mainly focused on the isolation and purification of fibrinolytic enzymes and the effects of environmental factors, such as sulfide, hexavalent chromium, and bisphenol A stress, whereas comparatively few studies have sought to examine the effects of bacteria on U. unicinctus. In this study, we assessed the patterns of antioxidant and immune activities in the coelomic fluid of U. unicinctus in response to bacterial infection. On the basis of our findings that the coelomic fluid has certain inhibitory effects on aquatic pathogens, we sought to determine the number and phagocytotic rates of coelomocytes, the antioxidant and immune indices of the coelomic fluid, and the expression of immune-related genes in response bacterial attack, with the aim of providing a reference for further studies on the immune defense mechanisms of U. unicinctus for countering bacterial infections. In this study, Aeromonas veronica and Micrococcus luteus were selected as experimental bacterial pathogens, to which U. unicinctus was exposed, and coelomic fluid samples were collected at 0.5, 1, 3, 6, 12, and 24 h post-infection. Initially, 100 μL samples were obtained to determine coelomocyte cell numbers and the rate of phagocytosis. The remaining samples were centrifuged for 10 min at 3,000 r/min and 4℃, and the coelomic fluid supernatant and cell precipitates were collected, respectively. The cell precipitates were suspended in sterile saline, and the supernatants containing coelomocytes were collected by centrifugation after fragmentation, and we subsequently determined the total antioxidant capacity, catalase activity, NO content, and lysozyme activity in the two supernatants. In addition, freshly obtained coelomic fluid was centrifuged for 15 min at 3,000 r/min and 4℃, and the resulting coelomocyte precipitate was washed three times with sterile saline, after which, the coelomocytes were resuspended in L-15 cell culture medium, with the concentration being adjusted to 1×106 cells/mL to obtain a coelomocyte suspension. This suspension was subsequently mixed with A. veronica, M. luteus, or A. veronica +lipopolysaccharides, and thereafter, samples were collected following 0, 5, 10, 20, 30, 40, 50, and 60 min of treatment. These samples were centrifuged at 1,500 r/min for 5 min, and the absorbance values of the supernatants were measured at 404 nm for analysis of the hemolysis of coelomocytes. On the basis of previous transcriptome sequencing of U. unicinctus in response to Vibrio anguillarum treatment, we screened five immunity-related genes, namely, cbl-b, gck, actr3, lhpp, and cyp450, and assessed the expression of these genes in response to treatment with the four pathogens A. veronica, M. luteus, V. anguillarum, and Staphylococcus aureus, and a bacterial attack toxicity test was performed separately on U. unicinctus using sterile saline as a control. Coelomic fluid was collected with a sterile syringe at 3, 6, 12, 24, 48, and 96 h post-treatment. The coelomocytes were obtained by centrifugation for 15 min at 3,000 r/min and 4℃, and changes in the expression patterns of immune-related genes in coelomocytes were analyzed using real-time fluorescence quantitative PCR, with the β-actin and 18S rRNA genes being used as internal reference genes. The results revealed that in response to the different bacterial treatments, the number and rate of phagocytosis of coelomocytes were characterized by a trend of an initial decline, followed by a subsequent increase, after which there was a further decline, and we found that the intensity of hemolytic reactions could be ordered as follows: A. veronica+lipopolysaccharide group > A. veronica group > M. luteus group. Similar patterns of initial decline, subsequent increase, and further decline were also observed for the total antioxidant capacity, catalase activity and NO content of the coelomic fluid supernatant and fragmented coelomocyte supernatant, whereas lysozyme activity was characterized by an increase-decline-increase trend, with a prolongation of the bacterial treatment time. Furthermore, the expression of cbl-b, actr3, gck, and lhpp in the coelomocytes of U. unicinctus initially increased and subsequently declined, whereas the expression of cyp450 generally showed a trend of an initial decline, subsequent increase, and further decline In conclusion, different pathogens can have significant effects on the number of coelomocytes and the rates of coelomocyte phagocytosis in U. unicinctus, as well as the antioxidant capacity and immune indices of the coelomic fluid. Our comparisons revealed that in response to bacterial stimulation, the antioxidant and immunological capacities of coelomic fluid supernatant obtained from U. unicinctus were stronger than those of a fragmented coelomocyte supernatant. Furthermore, lipopolysaccharide was found to induce cellular immunity and thereby enhance the antioxidant capacity of U. unicinctus. Our findings in this study may provide important insights into the innate immune mechanisms of U. unicinctus in response to bacterial infections.
