The complete mitochondrial genome of Choristoneura metasequoiacola Liu,1983 (Lepidoptera: Tortricidae).

Choristoneura metasequoiacola Liu, 1983 is an important caterpillar species that specifically infests the leaves and branches of Metasequoia glyptostroboides Hu & W. C. Cheng 1948 with short larval infestations, long-term dormancy, and has a limited distribution in Lichuan, Hubei, China. The complete mitochondria genome of C. metasequoiacola was determined by using Illumina NovaSeq, and analyzed based on previously annotated sibling species. In total, we obtained mitochondria genome with 15,128 bp in length, circular in shape with a double-stranded closed ring structure, including 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes, and an AT-rich region. Of which the nucleotide composition was highly A + T biased, accounting for 81.98% of the whole mitogenome. Thirteen protein-coding genes (PCGs) were 11,142 bp; Twenty-two tRNA genes and AT-rich region were 1,472 and 199 bp, respectively. Phylogenetically, the relationship between Choristoneura spp. (containing C. metasequoiacola) and Adoxophyes spp. was closer than any other two genera from Tortricidae, and the relationship between C. metasequoiacola and C.murinana was the closest among nine sibling species from that genus, which helps to explain species evolution within the family Tortricidae.

Pathogenicity virulence of Beauveria spp. and biosafety of the BbMQ strain on adult ectoparasitic beetles, Dastarcus helophoroides Fairmaire (Coleoptera: Colydiidae).

Introduction: Beauveria spp. and Dastarcus helophoroides Fairmaire adults were simultaneously released to attack elder larvae or pupae of Monochamus alternatus in pine forests in China. However, little is known about the pathogenicity virulence and biosafety of Beauveria spp. on beneficial adults of D. helophoroides, and specific Beauveria bassiana (Bb) strains should be selected for synthetic release together with D. helophoroides. Methods: A total of 17 strains of Beauveria spp. were collected, isolated, and purified, and then their mortality, cadaver rate, LT50, spore production, spore germination rate, and growth rate of D. helophoroide adults were calculated based on 0-20 days data after spore suspension and powder contact. Results and discussion: The lethality rate of BbMQ, BbFD, and BbMH-03 strains to D. helophoroides exceeded 50%, and the cadaver rate reached 70.6%, among which the mortality rate (82.22%), cadaver rate (47.78%), spore production (1.32 × 109 spores/ml), spore germination rate (94.71%), colony dimension (49.15 mm2), and LT50 (10.62 d) of the BbMQ strain were significantly higher than those of other strains (P < 0.01), and the mortality of D. helophoroides adults increased significantly with increased spore suspension concentration, with the highest mortality reaching 92.22%. This strain was identified as Beauveria bassiana by morphological and molecular methods, while the BbWYS strain had a minimum lethality of only 5.56%, which was safer compared to other strains of adult D. helophoroide. Consequently, the biological characteristics and pathogenicity of different Beauveria bassiana strains varied significantly in their effects on D. helophoroide adults, and the safety of different strains should be assessed when they are released or sprayed to control multiple pests in the forest. The BbMQ strain should not be simultaneously sprayed with releasing D. helophoroide adults in the same forest, while the BbWYS strain can be used in concert with D. helophoroide to synergize their effect.

Gut Bacterial Communities of Lymantria xylina and Their Associations with Host Development and Diet.

The gut microbiota of insects has a wide range of effects on host nutrition, physiology, and behavior. The structure of gut microbiota may also be shaped by their environment, causing them to adjust to their hosts; thus, the objective of this study was to examine variations in the morphological traits and gut microbiota of Lymantria xylina in response to natural and artificial diets using high-throughput sequencing. Regarding morphology, the head widths for larvae fed on a sterilized artificial diet were smaller than for larvae fed on a non-sterilized host-plant diet in the early instars. The gut microbiota diversity of L. xylina fed on different diets varied significantly, but did not change during different development periods. This seemed to indicate that vertical inheritance occurred in L. xylina mutualistic symbionts. Acinetobacter and Enterococcus were dominant in/on eggs. In the first instar larvae, Acinetobacter accounted for 33.52% of the sterilized artificial diet treatment, while Enterococcus (67.88%) was the predominant bacteria for the non-sterilized host-plant diet treatment. Gut microbe structures were adapted to both diets through vertical inheritance and self-regulation. This study clarified the impacts of microbial symbiosis on L. xylina and might provide new possibilities for improving the control of these bacteria.

The complete mitochondrial genome of Plagiodera versicolora (Laicharting)(Coleoptera: Chrysomelidae).

Plagiodera versicolora (Laicharting) is a leaf-eating pest widely distributed in the world. In this study, the first complete mitochondrial genome of P. Versicolora (Laicharting) was assembled and analyzed. The complete mitochondrial genome of P. Versicolora (Laicharting) is 16,857 bp with 22.39% GC containing, 13 protein-coding genes, 22 transfer RNA (tRNA), 2 ribosomal RNA (rRNA), as well as an AT-rich region. Phylogenomic analysis indicated that P. Versicolora (Laicharting) is sister to Chrysomela populiThis study provides useful information for the identification of this species and the study of genetic evolution with other species of Chrysomelidae.

The complete mitochondrial genome of Chrysolina aeruginosa Fald (Coleoptera: Chrysomelidae).

In this study, the first complete mitochondrial genome of Chrysolina aeruginosa Fald was assembled and analyzed. The total length of this mitochondrial genome is 16,335 base pairs. It consists of 13 protein-coding genes, 22 transfer RNAs, two ribosomal RNAs, and an AT-rich region. Phylogenomic analysis indicated that C. aeruginosa Fald is sister to Chrysodinopsis sp. This study provides new molecular data for the further taxonomic and phylogenetic studies of the Chrysomelidae of Coleoptera.

Identification and Characterization of Aminopeptidase-N as a Binding Protein for Cry3Aa in the Midgut of Monochamus alternatus (Coleoptera: Cerambycidae).

Bacillus thuringiensis Cry proteins have been widely used over the past decades for many different insect pests, which are safe for users and the environment. The coleopteran-specific Cry3Aa toxin from B. thuringiensis exhibits toxicity to the larvae of Monochamus alternatus. Receptors play a key role in the mechanisms underlying the toxic action of Cry. However, the binding receptor for Cry3Aa has yet to be identified in the midgut of M. alternatus larvae. Therefore, the aim of this study was to identify the receptor for Cry3Aa toxin in the brush border membrane vesicles (BBMVs) of M. alternatus larvae. Our results indicate that the Cry3Aa toxin binds to the BBMVs (Kd = 247 nM) of M. alternatus via a 107 kDa aminopeptidase N (APN) (Kd = 57 nM). In silico analysis of the APN protein predicted that an 18 amino acid sequence in the N-terminal acted as a signal peptide, and that the Asn residue, located at position 918 in the C-terminus is an anchored site for glycosyl phosphatidyl inositol. Further analysis showed that M. alternatus APN exhibits 75% homology to the APN from Anoplophora glabripenis. Our work, therefore, confirmed that APN, which is localized in the BBMVs in the midgut of M. alternatus larvae, acts as a binding protein for Cry3Aa toxins.

Comparative mitochondrial genome analysis of Dendrolimus houi (Lepidoptera: Lasiocampidae) and phylogenetic relationship among Lasiocampidae species.

Dendrolimus houi is one of the most common caterpillars infesting Gymnosperm trees, and widely distributed in several countries in Southeast Asia, and exists soley or coexists with several congeners and some Lasiocampidae species in various forest habitats. However, natural hybrids occasionally occur among some closely related species in the same habitat, and host preference, extreme climate stress, and geographic isolation probably lead to their uncertain taxonomic consensus. The mitochondrial DNA (mtDNA) of D. houi was extracted and sequenced by using high-throughput technology, and the mitogenome composition and characteristics were compared and analyzed of these species, then the phylogenetic relationship was constructed using the maximum likelihood method (ML) and the Bayesian method (BI) based on their 13 protein-coding genes (PCGs) dataset, which were combined and made available to download which were combined and made available to download among global Lasiocampidae species data. Mitogenome of D. houi was 15,373 bp in length, with 37 genes, including 13 PCGs, 22 tRNA genes (tRNAs) and 2 rRNA genes (rRNAs). The positions and sequences of genes were consistent with those of most known Lasiocampidae species. The nucleotide composition was highly A+T biased, accounting for ~80% of the whole mitogenome. All start codons of PCGs belonged to typical start codons ATN except for COI which used CGA, and most stop codons ended with standard TAA or TAG, while COI, COII, ND4 ended with incomplete T. Only tRNASer (AGN) lacked DHU arm, while the remainder formed a typical "clover-shaped" secondary structure. For Lasiocampidae species, their complete mitochondrial genomes ranged from 15,281 to 15,570 bp in length, and all first genes started from trnM in the same direction. And base composition was biased toward A and T. Finally, both two methods (ML and BI) separately revealed that the same phylogenetic relationship of D. spp. as ((((D. punctatus + D. tabulaeformis) + D. spectabilis) + D. superans) + (D. kikuchii of Hunan population + D. houi) as in previous research, but results were different in that D. kikuchii from a Yunnan population was included, indicating that different geographical populations of insects have differentiated. And the phylogenetic relationship among Lasiocampidae species was ((((Dendrolimus) + Kunugia) + Euthrix) + Trabala). This provides a better theoretical basis for Lasiocampidae evolution and classification for future research directions.

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.

Engineering of multiple trypsin/chymotrypsin sites in Cry3A to enhance its activity against Monochamus alternatus Hope larvae.

BACKGROUND: Bacillus thuringiensis Cry3 toxins exhibit specific toxicity against several coleopteran larvae. However, owing to its low toxicity to Monochamus alternatus, Cry3A toxin is not useful for managing M. alternatus larvae. Here we assessed the proteolytic activation of Cry3Aa toxin in M. alternatus larval midgut and increased its toxicity by molecular modification. RESULTS: Our results indicated that insufficient processing of Cry3Aa protoxin and non-specific enzymatic digestion of Cry3Aa toxin in the midgut of M. alternatus larvae led to low toxicity. The results of transcriptome analysis, enzymatic assay with fluorogenic substrates, and multiplex substrate profiling by mass spectrometry showed that the main digestive enzymes in M. alternatus larval midgut were trypsin-like proteases that preferentially cleaved peptides with arginine and lysine residues. Consequently, trypsin recognition sites were introduced into the Domain I of Cry3Aa protoxin in the loop regions between α-helix 3 and α-helix 4 to facilitate proteolytic activation. Multiple potential trypsin cleavage sites away from the helix sheet and functional regions in Cry3Aa proteins were also mutated to alanine to prevent non-specific enzymatic digestion. Bioassays indicated that a modified Cry3Aa-T toxin (K65A, K70A, K231A, K468A, and K596A) showed a 9.5-fold (LC50 = 12.3 µg/mL) increase in toxicity to M. alternatus larvae when compared to native Cry3Aa toxin. CONCLUSION: This study highlights an effective way to increase the toxicity of Cry3Aa toxin to M. alternatus, which may be suitable for managing the resistance of transgenic plants to other pests, including some of the most important pests in agriculture. © 2020 Society of Chemical Industry.

A new species of Glyptapanteles Ashmead (Hymenoptera, Braconidae, Microgastrinae) within Macrobrochis gigas (Lepidoptera, Arctiidae, Lithosiidae) in Fujian, China.

The south-east coastal area of Fujian, China, belongs to the Oriental Realm, and is characterized by a high insect species richness. In this work, a new species of Hymenopteran parasitoid, Glyptapanteles gigas Liang & Song, sp. nov. found in Jinjiang within hosts of caterpillars Macrobrochis gigas (Lepidoptera: Arctiidae), is described and illustrated, with differences from similar species. Additionally, we presumed that both parasitoid and host species play very important role in the coevolution and tritrophic interaction between plants, phytophagous insects, and their parasitoids, because these insects probably broke the sporangia and made contributions to their colonization, or some spores were spread for long distances by adult moths after their emergence, or some parasitoids were attracted by the eggs and larvae of these caterpillars, which was also thought to be helpful to spread of spores.

The complete mitochondrial genome of Epicauta ruficeps (Coleoptera: Meloidae).

Epicauta ruficeps is widely distributed in China and some countries in Southeast Asia, and plays an important role in medicine and biological control. The complete mitochondria genome of E. ruficeps was 15,813 bp in length, with 37 genes, including 13 PCGs, 22 tRNA genes (tRNAs), and two rRNA genes (rRNAs). The positions and sequences of genes were consistent with those of known Meloidae species. The nucleotide composition was highly A + T biased, accounting for ∼65% of the whole mitogenome. The complete mitogenome of E. ruficeps would help understand Meloidae evolution.

Characterization of Dendrolimus houi Lajonquiere (Lepidoptera: Lasiocampidae) Transcriptome across All Life Stages.

Dendrolimus houi Lajonquiere is a phytophagous caterpillar infesting many economically important coniferous tree species in China, causing serious economic and ecological environment losses. Based on previous research, it has one generation per year in South China and East China in contrast to two generations per year in Yunnan province in southwestern China. The species is potentially resilient to climatic extremes in these regions with the eggs and 1st instar larvae surviving in the winter (5 °C), older instar larvae and pupae surviving high temperatures in the summer (35 °C), suggesting some temperature stress tolerance during different developmental stages. However, little is known in this species at the genetic and genomic level. In this study, we used high throughput sequencing to obtain transcriptome data from different developmental stages (eggs, 1st-3rd instar larvae, 4th-5th instar larvae, 6th-7th instar larvae, pupae, male and female adults), which were collected from Fujian province. In total, we obtained approximately 90 Gb of data, from which 33,720 unigenes were assembled and 17,797 unigenes were annotated. We furtherly analyzed the differentially expressed genes (DGEs) across all stages, the largest number between the eggs and 1st instar larvae stage and gene expression varied significantly in different developmental stages. Furthermore, 4138 SSR genes and 114,977 SNP loci were screened from transcriptome data. This paper will be a foundation for further study towards improved integrated pest management strategies for this species.

De novo characterization of the pine aphid Cinara pinitabulaeformis Zhang et Zhang transcriptome and analysis of genes relevant to pesticides.

The pine aphid Cinara pinitabulaeformis Zhang et Zhang is the main pine pest in China, it causes pine needles to produce dense dew (honeydew) which can lead to sooty mold (black filamentous saprophytic ascomycetes). Although common chemical and physical strategies are used to prevent the disease caused by C. pinitabulaeformis Zhang et Zhang, new strategies based on biological and/or genetic approaches are promising to control and eradicate the disease. However, there is no information about genomics, proteomics or transcriptomics to allow the design of new control strategies for this pine aphid. We used next generation sequencing technology to sequence the transcriptome of C. pinitabulaeformis Zhang et Zhang and built a transcriptome database. We identified 80,259 unigenes assigned for Gene Ontology (GO) terms and information for a total of 11,609 classified unigenes was obtained in the Clusters of Orthologous Groups (COGs). A total of 10,806 annotated unigenes were analyzed to identify the represented biological pathways, among them 8,845 unigenes matched with 228 KEGG pathways. In addition, our data describe propagative viruses, nutrition-related genes, detoxification related molecules, olfactory related receptors, stressed-related protein, putative insecticide resistance genes and possible insecticide targets. Moreover, this study provides valuable information about putative insecticide resistance related genes and for the design of new genetic/biological based strategies to manage and control C. pinitabulaeformis Zhang et Zhang populations.

Identification of Genes Relevant to Pesticides and Biology from Global Transcriptome Data of Monochamus alternatus Hope (Coleoptera: Cerambycidae) Larvae.

Monochamus alternatus Hope is the main vector in China of the Pine Wilt Disease caused by the pine wood nematode Bursaphelenchus xylophilus. Although chemical control is traditionally used to prevent pine wilt disease, new strategies based in biological control are promising ways for the management of the disease. However, there is no deep sequence analysis of Monochamus alternatus Hope that describes the transcriptome and no information is available about gene function of this insect vector. We used next generation sequencing technology to sequence the whole fourth instar larva transcriptome of Monochamus alternatus Hope and successfully built a Monochamus alternatus Hope transcriptome database. In total, 105,612 unigenes were assigned for Gene Ontology (GO) terms, information for 16,730 classified unigenes was obtained in the Clusters of Orthologous Groups (COGs) database, and 13,024 unigenes matched with 224 predicted pathways in the Kyoto Encyclopedia of Genes and Genome (KEGG). In addition, genes related to putative insecticide resistance-related genes, RNAi, the Bt receptor, intestinal digestive enzymes, possible future insect control targets and immune-related molecules are described. This study provides valuable basic information that can be used as a gateway to develop new molecular tools for Monochamus alternatus Hope control strategies.