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.

Influence of ultrasound on juvenile hormone titers in Monochamus alternatus Hope (Coleoptera: Cerambycidae)

Abiotic stress factors can significantly affect insects. In particular, the stressful effects of exposure to ultrasound on insects are considered important. In the present study, we investigated the effects of ultrasound on the important global pest Monochamus alternatus (Coleoptera: Cerambycidae), which is the main vector of the pinewood nematode. We exposed M. alternatus adults (aged 1 day, 3 days, and 5 days) to ultrasound at different frequencies (using two ultrasonic devices, i.e., LHC20 with a mixture of frequencies at 35 kHz, 70 kHz, and 105 kHz; and GFG-8016G at two separate frequencies of 30 kHz and 60 kHz) for different periods of time (1 h, 12 h, and 24 h), before evaluating the juvenile hormone III (JHIII) titers. All of the ultrasound treatments significantly decreased the JHIII titers in M. alternatus adults. The decreases in the JHIII titers due to ultrasound exposure did not differ according to sex, but the effects on beetles of different ages differed significantly depending on the duration of exposure. The decreases in the JHIII titers were highest in male and female beetles after exposure to ultrasound for 12 h. Following exposure to ultrasound for any time period, the decreases in the JHIII titers were lower in adults aged 3 days than those aged 1 day and 5 days. The different ultrasonic frequencies led to variable decreases in the JHIII titers in M. alternatus adults, where the greatest decreases occurred in beetles exposed to ultrasound at 60 kHz. Our results indicate that ultrasound can negatively affect the normal JHIII levels and it may further disrupt sexual maturation by M. alternatus adults.

Characterization of bacterial communities associated with the pinewood nematode insect vector Monochamus alternatus Hope and the host tree Pinus massoniana

Background Monochamus alternatus Hope is one of the insect vectors of pinewood nematode ( Bursaphelenchus xylophilus ), which causes the destructive pine wilt disease. The microorganisms within the ecosystem, comprising plants, their environment, and insect vectors, form complex networks. This study presents a systematic analysis of the bacterial microbiota in the M. alternatus midgut and its habitat niche. Methods Total DNA was extracted from 20 types of samples (with three replicates each) from M. alternatus and various tissues of healthy and infected P. massoniana (pines). 16S rDNA amplicon sequencing was conducted to determine the composition and diversity of the bacterial microbiota in each sample. Moreover, the relative abundances of bacteria in the midgut of M. alternatus larvae were verified by counting the colony-forming units. Results Pinewood nematode infection increased the microbial diversity in pines. Bradyrhizobium , Burkholderia , Dyella , Mycobacterium , and Mucilaginibacter were the dominant bacterial genera in the soil and infected pines. These results indicate that the bacterial community in infected pines may be associated with the soil microbiota. Interestingly, the abundance of the genus Gryllotalpicola was highest in the bark of infected pines. The genus Cellulomonas was not found in the midgut of M. alternatus , but it peaked in the phloem of infected pines, followed by the phloem of heathy pines. Moreover, the genus Serratia was not only present in the habitat niche, but it was also enriched in the M. alternatus midgut. The colony-forming unit assays showed that the relative abundance of Serratia sp. peaked in the midgut of instar II larvae (81%). Conclusions Overall, the results indicate that the bacterial microbiota in the soil and in infected pines are correlated. The Gryllotalpicola sp. and Cellulomonas sp. are potential microbial markers of pine wilt disease. Additionally, Serratia sp. could be an ideal agent for expressing insecticidal protein in the insect midgut by genetic engineering, which represents a new use of microbes to control M. alternatus .