Transcriptomic Analysis On The Larval Density Dependent Immune Of Mythimna Separate And RNA Interference Of Key Gene Tyramine ?-hydroxylase

The population density-dependent prophylaxis to pathogens of insect has been well established,however,the molecular mechanisms of immune function underlying high density prophylaxis remain unclear.Biogenic amines are neurohormone released from the insect nervous system,implicating profoundly in insect immune system.Prelimitary results showed that octopamine play an important role in regulating larval density-dependent prophylaxis in Mythimina seperata.To elucidate the molecular mechanisms of larval density-dependent prophylaxis in M.seperata,the transcriptomic analysis of the head,haemocytes,and fat body of larvae reared at high and low population densities was performed,and the genes related to immunity and its regulation were screened.The mainly results are as follows:The inhibition ability of haemolymph to different bacteria were investigated in the larvae reared at high and low population density(10 and 1 larvae per jar,respectively)under laboratory conditions(23 ± 1?,RH 70%± 5%,photoperiod 16L:8D).The results showed that the inhibition ability to Staphylococcus aureus,Bacillus subtilis,Vibrio and Edwardsiella were significantly different between high and low density larvae.The hemolymph inhibition activity to Staphylococcus aureus,Bacillus subtilis,Vibrio and Edwardsiella was significantly higher at the ten larvae per jar than that of one larva per jar and the control group Within 36 hour,the inhibition ability of larvae increased with time delayed.The results suggested that the antibacterial ability of larvae from high density was significantly higher than that of larvae from low density.To further explore the molecular mechanisms underlying larval density-dependent prophylaxis,transcriptomic analysis of heads,haemocyte,and fat body of larvae from high and low density were investigated.The resulted showed that nineteen associated with pattern recognition protein,twenty-five antimicrobial peptide genes,fifteen genes involved in the phenoloxidase cascade reaction,nine genes related to cellular immunity and six biogenic amine synthetase and biogenic amine receptor genes were significantly highly expressed in the larvae from 10 larvae per jar than that of 1 larva per jar.differential expressed.Four antimicrobial peptide genes were randomly selected for RT-PCR validation.The results demonstrated that the transcription levels of four antimicrobial peptide genes of larvae from 10 larvae per jar was obviously higher than that of 1 larva per jar,the results was consistent with our transcriptome results.One of the differentially expressed key genes regulating density-dependent larval immunity,tyramine ?-hydroxylase(T?H),was selected for further study.The total haemocyte count,phenoloxidase activity,lysozyme activity and the resistance activity to Bacillus thuringiensis of larvae from 10 larvae per jar after silencing T?H gene were determined.The results showed that the T?H relative expression level of larvae injected with T ? HdsRNA decreased significantly.Meanwhile,the total hemocyte count,lysozyme activity,and phenoloxidase activity of dsT?H-treated larvae reduced by 55.77%,88.48%,and 82%,respectively.In addition,the resistance to Bacillus thuringiensis of larvae after silencing T?H gene dramatically reduced,and the calibrated survival rate of larvae dropped by 55.77%.The results showed that tyramine ?-hydroxylase play an important role in the density-dependent immunity of Mythimna seperata.