Biological Characteristics Of Disease-Resistance Of Rabbitfish (Siganus Oramin) To Cryptocaryon Irritans Infection

Cryptocaryon irritans is an important protozoan parasite which infects almost all kinds of marine teleosts,causing a massive death of cultured fish.Previous studies have found that rabbitfish?Siganus oramin?displayed high resistance to C.irritans infection,the serum that was lethal to C.irritans,and an anti-parasitic protein,L-amino acid oxidase?LAAO?was identified from the serum.However,little is known about the biological characteristics of cisease resistance of rabbitfish to C.irritans infection.Such as the biochemical and immune responses of rabbitfish post C.irritans infection?PI?,the development of C.irritans in rabbitfish.In addition,the source of C.irritans in cage culture needs to be further studied.Therefore,study on the biological characteristics of rabbitfish against C.irritans infection,it not only further clarifed the mechanism of rabbitfish against C.irritans infection,but also provide ideas for the prevention and control of C.irritans infection.The main result of this study was as follows.?1?Biochemical responses of rabbitfish after exposure to C.irritansIn order to reveal the biochemical responses of rabbitfish PI.In this study,survival rate and feeding intake of rabbitfish and susceptible host large yellow croaker?Larimichthys crocea?PI were compared.In addition,the activity of enzymes(SOD,CAT,Na⁺/K⁺-ATPase and Ca²⁺/Mg²⁺-ATPase)and the LAAO mRNA expression pattern of rabbitfish PI were measured.The results indicated that the food intake of rabbitfish decreased by 14.6%at day 1 PI,but immediately returned to normal food intake levels by day 2 PI,and was not affected by secondary infection.However,large yellow croaker stopped feeding at day 3 PI,and all fish died following secondary infection with C.irritans.The SOD and CAT activities in the liver was significantly higher in the infection group than in the control group by 48 h and 6 h PI,respectively,while no significant difference was detected at the other time points or gill throughout the experiment.In addition,the Na⁺/K⁺-ATPase and Ca²⁺/Mg²⁺-ATPase activities in the gill were decreased by 12 and 24 h after infection,no significant difference was detected at liver.Together,these results suggest that biochemical responses of rabbitfish are relatively mild and transitory PI.Tissue distribution analysis showed that LAAO were distributed in various organizations and most pronounced in the immune tissue.After infection with C.irritans,the LAAO was up-regulated early post infection in both gill and spleen,but then returned to normal levels,implying that LAAO may play an important role in the host's early immune response,which suggested the LAAO may play an important role in the host's defense against C.irritans.?2?Immune response characteristics of rabbitfish post C.irritans infectionThere were most trophonts lefting the rabbitfish during early infected stages,whether they can induce an immune response to rabbtitfish still remained unclear.Here,comparative transcriptome analysis of rabbitfish skin was investigaete and assessed the immune protective effect of rabbitfish PI.The transcriptomes contained 238,504,124clean reads were obtained and then assembled into 258,869 unigenes with an average length of 621 bp and an N50 of 833 bp.Among them,we obtained 418 differentially expressed genes?DEGs?in the skin of rabbitfish under C.irritans infection and control conditions,including 336 up-regulated genes and 82 down-regulated genes.32differentially expressed immune genes were obtained using GO and KEGG analysis.DEGs included innate immune molecules?such as LAAO,antimicrobial peptide and lysozyme?,as well as complement components,chemokines and chemokine receptors,NOD-like/Toll-like receptor signaling pathway molecules,antigen processing and T/B cell activation and proliferation molecules.These results indicated that the C.irritans not only induces the innate immune response,and acquired immunity further strengthens the resistance against the parasite.?3?Molecular cloning and prokaryotic expression of LZMg and NK-lysinLZMg and NK-lysin of rabbitfish were significantly up-regulated in the transcriptome analysis.In order to study the gene sequence characteristics and expression profiles of LZMg and NK-lysin in rabbitfish PI.In this study,LZMg and NK-lysin were cloned and characterized from rabbitfish.The ORF sequence of LZMg was567 bp,coding 188 amino acides,it had a Pfam motif and theree conserved catalytic sites.Multiple sequence alignment showed that the LZMg protein shared high sequence identity with sea bass and were clustered into the same subgroup with large yellow croaker in the phylogenetic tree.The ORF sequence of NK-lysin was 450 bp,encoding149 amino acids,it possessed a Saposin B domain of members of saposin-like protein?SAPLIP?family.The NK-lysin shared highest identity with rock bream.Tissue expression analysis indicated that LZMg and NK-lysin were widely distributed in different tissues,especially in liver,blood,heart and spleen.LZMg and NK-lysin expression were up-regulated in the gill and spleen post C.irritans infection.We obtained the recombinant protein of LZMg and NK-lysin as inclusion bodieds in Escherichia coli.?4?The development of C.irritans in a less susceptible host rabbitfishAt present,little is known about the development of C.irritans in rabbitfish.Thus,in the present study,rabbitfish were artificially infected with C.irritans,using large yellow croaker as a control.The number of trophonts in gill,relative number of tomonts,reproductive rate,characteristics of C.irritans tomonts,and the microscopic of C.irritans at different stages were measured.The trophonts in the rabbitfish gill exhibited a significant downward trend at 6 h PI,and could not form tomonts until parasitic more18 h;in contrast,the trophonts of large yellow croaker was not significantly different from 0 to 72 h PI.The tomonts from rabbitfish could divide normally and the virulence of theronts remained unchanged,and scanning electron microscopy showed that the trophonts,protomonts and tomonts had normal cell morphology.However,over the same time frame,the diameter of tomonts from rabbitfish was smaller compared with those from large yellow croaker.The reproductive rate of C.irritans reached to 59.05-fold in large yellow croaker,but only 8%in rabbitfish.These results indicated that the rabbitfish were somehow able to limit the development of C.irritans,such that most trophonts left the host during early developmental stages,whereas those that survive could only develop into small tomonts.?5?Study on the source of C.irritans in cage culturePrevious studies have shown that C.irritans tomonts were difficult to hatch in the seabed,resulting in a large accumulation of tomonts in the seabed,which can erupt when they were involved in the shallow sea under the action of typhoons,etc.In this study,the tomonts were placed at depths of 2 m,10 m and 30 m in sea water and silt,respectively,and determined the hatching rate of tomonts on day 3 and 6.The study indicated that the silt and depth of 30 m delayed the tomont division,however,there were no significant effect on the survival and hatching rate of tomont.It implied that the tomonts were difficult to accumulate on the seabed.In addition,to explore whether the C.irritans tomonts can adhere on the net.Net was placed at the bottom of the tank,with large yellow croaker which infected C.irritans,and detected tomont's adhesion rate on the netting.The results showed that the tomonts could be attached to the netting,and the adhesion rate of tomont was higher with the weight of biofouling of the netting,up to 17.3%.These results suggested that the main source of C.irritans in the cage were from the net.