Analysis Of The Silkworm Brain Proteome And The Molecular Mechanisms Underlying BmNPV-induced Hyperactive Behavior In Bombyx Mori

Baculoviruses have been known to induce hyperactive behavior in their lepidopteran hosts for over a century. As a typical lepidopteran insect, the silkworm Bombyx mori displays enhanced locomotor activity (ELA) following infection with Bombyx mori nucleopolyhedrovirus (BmNPV). Some investigations have focused on the molecular mechanisms underlying this abnormal hyperactive wandering behavior due to the virus, and several viral genes associated with this phenomenon were identified. However, there are currently no reports about the changes at the molecular level on B. mori. Based on previous studies that have revealed that the brain is the behavior control center, we first investigated the brain proteome of B. mori applying shotgun technique to understand the protein composition of the brain and the behavior related proteins. Then the time course of BmNPV infection in the nervous system of the silkworm and histopathology and neuropathology of the brain tissue in fifth-instar larvae after BmNPV infection were analyzed, and the RNA-Seq technique was used to compare the transcriptome profiles of the brains between BmNPV-infected and non-infected silkworm larvae to reveal the changes in the BmNPV-infected brain on the transcriptional level. Based on the studies that serotonin receptors play important roles in regulating behavior of insects, the serotonin receptor gene Bm5-HT4R of B. mori was chose from those DEGs for characterizing, including its relevance with the locomotor activity of B. mori. The results are as follows:1. Total 2,210 proteins were identified using shotgun technique. Two categories of proteins were obtained according to GO and KEGG analysis, one category includes behavior-related proteins involved in the regulation of several types of behaviors, such as locomotion, reproduction and learning; the other category contains proteins related to development and function of the nervous system.2. The time course of BmNPV infection in the silkworm brain was analyzed. BmNPV entered into the brain from the periphery to the interior gradually between 48 hpi-120 hpi and filled the whole tissue finally. The abnormal morphology of the tissue and neuron occurred in the central complex of the brain infected with BmNPV.3. The results of transcriptome analysis showed that, compared with the controls, a total of 742 differentially expressed genes (DEGs), including 218 up-regulated and 524 down-regulated candidates, were identified, of which 499,117 and 144 DEGs could be classified into GO categories, KEGG pathways and COG annotations by GO, KEGG and COG analyses, respectively. We focused our attention on the DEGs that are involved in circadian rhythms, synaptic transmission and the serotonin receptor signaling pathway of B. mori. Our analyses suggested that these genes were related to the locomotor activity of B. mori and involved in the enhanced locomotor activity induced by BmNPV via their essential roles in the regulations of a variety of behaviors and the down-regulation of their expressions following BmNPV infection.4. The studies of Bm5-HT4R showed that it has seven transmembrane domains, and shares high sequence homology with the known 5-HT4 receptors in mammals. Bm5-HT4R has the highest expression level in the brain, followed by the hemocyte, ventral nerve cord and midgut, and the relative low expression level in other tissues. The RNAi results demonstrated that Bm5-HT4R plays an important role in regulating the locomotor activity of B. mori.Overall, these results provide theoretical bases for further research on the molecular mechanisms of enhanced locomotor activity in B. mori induced by BmNPV infection and the physiological functions of the silkworm brain in regulating behavior of B. mori and development of the nervous system.