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Simple Summary Many bark beetles are destructive pests in coniferous forests and cause extensive ecological and economic losses worldwide. Comparative studies of the structural characteristics of mitogenomes and phylogenetic relationships of bark beetles can improve our understanding of mitogenome evolution. In this study, we sequenced eight mitogenomes of bark beetles. Our results show that the use of start and stop codons, the abundance of amino acids, and the relative frequency of codon use are conserved among the eight bark beetles. Different regions of tRNA exhibit different degrees of conservatism. Together with the analysis of evolutionary rates and genetic distance among bark beetle species, our results reveal phylogenetic relationships among bark beetles of the subfamily Scolytinae. Abstract Many bark beetles of the subfamily Scolytinae are the most economically important insect pests of coniferous forests worldwide. In this study, we sequenced the mitochondrial genomes of eight bark beetle species, including Dendroctonus micans, Orthotomicus erosus, Polygraphus poligraphus, Dryocoetes hectographus, Ips nitidus, Ips typographus, Ips subelongatus, and Ips hauseri, to examine their structural characteristics and determine their phylogenetic relationships. We also used previously published mitochondrial genome sequence data from other Scolytinae species to identify and localize the eight species studied within the bark beetle phylogeny. Their gene arrangement matched the presumed ancestral pattern of these bark beetles. Start and stop codon usage, amino acid abundance, and the relative codon usage frequencies were conserved among bark beetles. Genetic distances between species ranged from 0.037 to 0.418, and evolutionary rates of protein-coding genes ranged from 0.07 for COI to 0.69 for ND2. Our results shed light on the phylogenetic relationships and taxonomic status of several bark beetles in the subfamily Scolytinae and highlight the need for further sequencing analyses and taxonomic revisions in additional bark beetle species.

Chitinases, which are crucial enzymes required for chitin degradation and reconstruction, are often selectively considered to be effective molecular targets for pest control due to their critical roles in insect development. Although the Hyphantria cunea chitinase gene has been reported previously, its sequence characteristics, gene function, and feasibility as a potential target for pest management were absent. In the present study, we characterized the H. cunea chitinase gene and designated it HcCht5. Phylogenic and domain structure analysis suggested that HcCht5 contained the typical chitinase features and was clustered into chitinase group I. Tissue-specific and developmental expression pattern analysis with Real-Time Quantitative PCR (RT-qPCR) showed that HcCht5 was mainly expressed in the integument tissues and that the transcript levels peaked during molting. RNA interference (RNAi)-mediated silencing of HcCht5 caused 33.3% (2 ug) and 66.7% (4 ug) mortality rates after double-stranded RNA (dsRNA) injection. Importantly, the interference efficiency of HcCht5 depended on the injection time of double-stranded RNA (dsRNA), as the pre-molting treatment achieved molt arrest more effectively. In addition, transcriptome sequencing (RNA-seq) analysis of RNAi samples demonstrated silencing of the down-regulated HcCht5 genes related to chitin metabolism and molting hormone signaling, as well as genes related to detoxification metabolism. Our results indicate the essential role of HcCht5 in H. cunea development and detail the involvement of its gene function in the larval molting process.

The association between insects and fungi has evolved over millions of years and is ubiquitous in nature. This symbiotic relationship holds critical implications for both partners, the insects and the associated microbes. Numerous fungi are externally allied with bark beetles and form a close symbiosis, but the community structures of these fungi are largely unknown. In Yunnan Province in southwestern China, the beetles Tomicus yunnanensis, T. minor, and T. brevipilosus are major forest pests that cause large losses of two indigenous pines, Pinus yunnanensis and P. kesiya. In this study, we used the Illumina MiSeq PE300 platform to process 48 samples of epibiotic fungal communities pooled from 1348 beetles, the beetles were collected during both the branch- and trunk-infection sections from five locations across Yunnan Province. Considerably greater species richness was detected using high-throughput sequencing of amplified internal transcribed spacer 1 (ITS1) ribosomal DNA (rDNA) libraries than previously documented by using culture-dependent methods. In total, 1,413,600 reads were generated, and a 97% sequence-similarity cutoff produced eight phyla, 31 classes, 83 orders, 181 families, 331 genera, 471 species, and 1157 operational taxonomic units (OTUs), with 659, 621, and 609 OTUs being confined to T. yunnanensis, T. minor, and T. brevipilosus, respectively. Tomicus yunnanensis, T. minor, and T. brevipilosus had the similar OTUs richness and evenness of fungal communities in Yunnan Province, nevertheless, the two fungal community compositions associated with T. yunnanensis and T. minor were structurally similar to each other but distinct from that associated with T. brevipilosus. Lastly, the results of principal co-ordinates analysis suggested that epibiotic fungal community structures of the three Tomicus spp. were conditioned strongly by the locations and pine hosts but weakly by beetle species and infection sections. Our findings provide baseline knowledge regarding the epibiotic fungal communities of three major Tomicus spp. in southwestern China.