Effect Of Octopamine Receptor McOAR2 On Metamorphosis And Immunoreactions In Mytilus Coruscus

Mytilus coruscus is a marine economic shellfish with high nutritional value,so it has become one of the main cultured shellfish in coastal areas of China.The production of mussels is affected by the survival rate of seedlings,disease resistance of adult mussels,and other factors,especially the deterioration of the marine environment in recent years,which has brought many negative effects to the cultivation of mussels.Studies on the abnormal development mechanism of M.coruscus larvae and the immune mechanism of adult mussels under environmental stress have become an effective means to ensure the healthy cultivation of M.coruscus.Octopamine receptor（OAR）is a member of the G protein-coupled receptor family and plays an important role in the development of invertebrates and the stress response under environmental stress.Based on the M.coruscus mantle transcriptome data,the full-length octopamine receptor gene McOAR2 c DNA was cloned,and its specific function was studied.And then,we explored the immune response of octopamine and its receptors to the development of M.coruscus under environmental stress.The main results are as follows:1.The effect of McOAR2 gene in the development of the mussel M.coruscosThe octopamine receptor gene（McOAR2）was obtained from the M.coruscus transcriptome data already available in our laboratory.The full-length c DNA sequence was cloned by RACE technology.The gene was 2055 bp in length,which contained an open reading frame（ORF）of 1692 bp,encoding 563 amino acids.Multiple sequence alignment analysis of its amino acid sequence with the following species.Homologous analysis of the amino acids of McOAR2 gene in M.coruscus shared similarity with Crassostrea gigas OAR2（52.09%）,OA/TAR2（52.61%）,Crassostrea virginica（52.97%）,Pinctada fucata（52.17%）,Mizuhopecten yessoensis（53.08%）,Lottia gigantea（45.27%）,Pomacea canaliculate（44.12%）,Lymnaea stagnalis（42.68%）,Aplysia californica（55.64%）,Octopus bimaculoides（40.14%）.Real-time fluorescence quantitative PCR（RT-q PCR）was used to detect the expression of McOAR2 gene at various developmental stages of M.coruscus larvae.The McOAR2 m RNA was highest expressed in pediveliger larvae,suggesting that McOAR2 gene may participate in the process of larval metamorphosis.In order to verify this conclusion,take the pediveliger larvae exposed to octopamine receptor agonists and antagonists.The results showed that in the agonist experiment,under the conditions of 24-h exposure and continuous exposure,the agonist showed different levels of inductive activity at different concentrations,of which octopamine showed inductive activity at 10^-6 to 10^-4 mol/L.In contrast,mianserin,metoclopramide,chlorpromazine inhibited larval metamorphosis.Although octopamine can induce the larval metamorphosis,it needs to be verified whether it is regulated by McOAR2.Therefore,RNAi technology was used to knock down McOAR2 of pediveliger larvae.We found that the rate of metamorphosis was significantly reduced after RNAi.It was concluded that the octopamine receptor gene McOAR2 exists in M.coruscus,and that octopamine can regulate the larval metamorphosis via McOAR2.2.McOAR2 expression after temperature elevation and V.cyclitrophicus infectionIn order to investigate the role of octopamine and its receptor（McOAR2）in the immune process of M.coruscus,this study analyzed the expression of McOAR2 in various tissues of adult M.coruscus using RT-q PCR.V.cyclitrophicus was infected with mussels at 21 and 27°C.The expression of McOAR2 gene was detected by RT-q PCR and the content of octopamine was measured detected by liquid chromatography-mass spectrometry（LC-MS）.The results showed that the McOAR2 gene was detected in various tissues of M.coruscus and the expression levels in the foot and gills were significantly higher than those in other tissues.It is speculated that this gene may be related to the resistance of the to the external environment.McOAR2 gene quantification was per M.coruscus formed on the foot,gill,and hemolymphs after V.cyclitrophicus affected at two temperatures.It was found that the expression levels did not change significantly in the foot and gills,but significantly increased in the hemolymphs.At21℃,the expression of McOAR2 gene in hemolymph after V.cyclitrophicus infection increased from 3 h,reached the highest value at 6 h,and then stabilized;at 27℃,it was induced from 0 to 6 h,the expression of McOAR2 increased rapidly,reached the highest value at 6 hours,and then decreased and stabilized;the octopamine content in the serum showed a trend of first increase and then decrease after two temperature infections,but the 21℃treatment group,there was no significant difference in the whole（P>0.05）.In the 27℃treatment group,the octopamine content reached a maximum at 12 h with a significant difference（P<0.05）,then decreased significantly,and fell to the initial level at 48 h,and the octopamine content in the 27℃treatment group was higher than 21℃treatment group in the first 12 h;the SOD activity of the 21℃treatment group decreased first and then increased,but there was no significant change overall（P>0.05）.When the temperature was 27℃,the SOD activity increased significantly from 12 h（P<0.05）,reached a maximum of 61.87 U/ml at 24 h,and then decreased significantly to the initial state.CAT activity was not significantly different at both temperatures.The results indicate that the McOAR2 gene may participate in the immune response of M.coruscus to resist bacteria,and the immune response mediated by high temperature.3.Temperature elevation and V.cyclitrophicus infection influence the diversity of haemolymph microbiome and octopamine content of the mussel M.coruscusHemolymph is a key part of the immune response of marine invertebrates.The microbial community in hemolymph can affect the infection of the host by pathogens by improving immune function,while octopamine is a circulating hormone under pressure and plays a role in phagocytosis.Its content is affected by the severity of the immune response.This study investigated the changes in octopamine content and microbial community in M.coruscus hemolymph under the dual effects of bacterial infection and temperature.At 27°C,exposure to seawater containing V.cyclitrophicus resulted in high mortality of M.coruscus.16S r RNA gene sequencing results indicate that this is related to reducing the diversity of its microbial community,and the temperature is the key factor.During this process,the octopamine content increased significantly,indicating that environmental stress caused a decrease in hemolymph microbial diversity,and induced the release of octopamine in the serum.Susceptibility,Linear Discriminant Analysis（LEf Se）can screen differential bacteria in the hemolymph of healthy mussels and dead mussels,and use them as environmental markers to analyze the survival status of mussels.