Promoting Effect of Choline-Phosphate Cytidylyltransferase Gene (pcyt-1) on Departure of Pinewood Nematode from Monochamus alternatus

In order to study the key gene in internal causes of pinewood nematode (PWN), Bursaphelenchus xylophilus, a departure from its vector beetle, Monochamus alternatus, we collected PWNs extracted from newly emerged M. alternatus and beetles 7 days after emergence. The total RNAs of the two groups of PWNs were extracted, transcriptomes sequencing was performed, and gene expression differences between the two groups of PWN were analyzed. It was found that the expression of the choline-phosphate cytidylyltransferase gene (pcyt-1) was markedly up-regulated. After inhibition of pcyt-1 expression by RNA interference, the rate of lipid degradation in PWN decreased significantly, and the motility of PWN also decreased significantly. The analysis identified that phosphatidylcholine could promote the emulsification and degradation of neutral lipid granules in PWN, which provides sufficient energy for PWN departure from M. alternatus. The up-regulation of the gene pcyt-1 is an important internal factor for PWN departure from its vector.

Comparative Transcriptome Analysis of Stage-Specific Changes in Gene Expression during Larval Development in Monochamus alternatus Hope

Monochamus alternatus Hope (Coleoptera: Cerambycidae) is an important trunk borer of pine trees and a major vector of pine wilt disease. Although chemicals are widely used in forest pest control, new strategies based on insect biology are offering promising approaches to manage the disease. Although there have been important research advances in this respect, there has not yet been a deep sequence analysis of M. alternatus describing the transcriptome, and no information is available about the gene function of this insect vector. We used next generation sequencing technology to provide a full transcriptome from the four larval instars of M. alternatus and successfully built an M. alternatus transcriptome database. In total, 67,456 unigenes were obtained with trinity software, information for 11,858 classified unigenes was obtained with the Clusters of Orthologous Groups (COGs) database, and 13,007 unigenes matched predicted pathways using the Kyoto Encyclopedia of Genes and Genomes (KEGG). In addition, genes related to lignocellulose, and putative Bt receptors and genes related to digestion are described. Additionally, the differential gene expression of these genes in different larval stages was analyzed. This study provides valuable information to underpin the development of new molecular tools for M. alternatus control strategies.

Mitogenome Analysis of Four Lamiinae Species (Coleoptera: Cerambycidae) and Gene Expression Responses by Monochamus alternatus When Infected with the Parasitic Nematode, Bursaphelenchus mucronatus

We determined the mitochondrial gene sequence of Monochamus alternatus and three other mitogenomes of Lamiinae (Insect: Coleoptera: Cerambycidae) belonging to three genera (Aulaconotus, Apriona and Paraglenea) to enrich the mitochondrial genome database of Lamiinae and further explore the phylogenetic relationships within the subfamily. Phylogenetic trees of the Lamiinae were built using the Bayesian inference (BI) and maximum likelihood (ML) methods and the monophyly of Monochamus, Anoplophora, and Batocera genera was supported. Anoplophora chinensis, An. glabripennis and Aristobia reticulator were closely related, suggesting they may also be potential vectors for the transmission of the pine wood pathogenic nematode (Bursaphelenchus xylophilus) in addition to M. alternatus, a well-known vector of pine wilt disease. There is a special symbiotic relationship between M. alternatus and Bursaphelenchus xylophilus. As the native sympatric sibling species of B. xylophilus, B. mucronatus also has a specific relationship that is often overlooked. The analysis of mitochondrial gene expression aimed to explore the effect of B. mucronatus on the energy metabolism of the respiratory chain of M. alternatus adults. Using RT-qPCR, we determined and analyzed the expression of eight mitochondrial protein-coding genes (COI, COII, COIII, ND1, ND4, ND5, ATP6, and Cty b) between M. alternatus infected by B. mucronatus and M. alternatus without the nematode. Expression of all the eight mitochondrial genes were up-regulated, particularly the ND4 and ND5 gene, which were up-regulated by 4–5-fold (p < 0.01). Since longicorn beetles have immune responses to nematodes, we believe that their relationship should not be viewed as symbiotic, but classed as parasitic.

Transcriptome Analysis of the Japanese Pine Sawyer Beetle, Monochamus alternatus, Infected with the Entomopathogenic Fungus Metarhizium anisopliae JEF-197

The Japanese pine sawyer (JPS) beetle, Monochamus alternatus Hope (Coleoptera: Cerambycidae), damages pine trees and transmits the pine wilt nematode, Bursaphelenchus xylophilus Nickle. Chemical agents have been used to control JPS beetle, but due to various issues, efforts are being made to replace these chemical agents with entomopathogenic fungi. We investigated the expression of immune-related genes in JPS beetle in response to infection with JEF-197, a Metarhizium anisopliae isolate, using RNA-seq. RNA samples were obtained from JEF-197, JPS adults treated with JEF-197, and non-treated JPS adults on the 8th day after fungal treatment, and RNA-seq was performed using Illumina sequencing. JPS beetle transcriptome was assembled de novo and differentially expressed gene (DEG) analysis was performed. There were 719 and 1953 up- and downregulated unigenes upon JEF-197 infection, respectively. Upregulated contigs included genes involved in RNA transport, ribosome biogenesis in eukaryotes, spliceosome-related genes, and genes involved in immune-related signaling pathways such as the Toll and Imd pathways. Forty-two fungal DEGs related to energy and protein metabolism were upregulated, and genes involved in the stress response were also upregulated in the infected JPS beetles. Together, our results indicate that infection of JPS beetles by JEF-197 induces the expression of immune-related genes.