Biocidal efficacy of tutin and its influence on immune cells and expression of growth-blocking and neuroglian peptides in Mythimna separata (Lepidoptera: Noctuidae).

Mythimna separata Walker (Lepidoptera: Noctuidae) is one of the major pests that can cause severe damage to grain crops. The development of low-toxicity and high-performance botanical insecticides is becoming the focus of new pesticide research to control M. separata. Tutin, a sesquiterpene lactone compound obtained from Coriaria sinica Maxim, a native Chinese poisonous plant, has antifeedant, absorption, and stomach poisoning against a variety of pests. To understand the toxic effect of tutin on M. separata larvae, we set out to determine their antifeedant, mortality, paralysis, weight change, and to examine the spreading of M. separata hemocytes under different concentrations of tutin treatment. Tissue distribution of the immune-associated gene growth-blocking peptide (GBP) and neuroglian peptide (Nrg) was detected by reverse transcription polymerase chain reaction (PCR). Furthermore, real-time quantitative PCR was carried out to determine the expression profiles of GBP and Nrg after different concentrations of tutin stimulation. Our results revealed that tutin exhibited significant antifeedant and insecticidal activities, paralysis, weight loss to M. separata. Besides, tutin significantly influenced on the morphology of hemocytes and enhanced the expression of GBP and Nrg in M. separata.

Author Correction: Bacterial and fungal gut communities of Agrilus mali at different developmental stages and fed different diets.

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Bacterial and fungal gut communities of Agrilus mali at different developmental stages and fed different diets.

Agrilus mali (Coleoptera: Buprestidae) is an invasive wood borer pest that has caused considerable damage to the Xinjiang wild fruit forest. In this study, we investigated the bacterial and fungal intestinal microbial communities of A. mali during different developmental stages, including larvae, pupae and newly eclosed adults or fed different diets (leaves of Malus halliana and Malus pumila) using Illumina MiSeq high-throughput sequencing technology. The results showed that microbial alpha diversity first increased and then decreased during the developmental stages, with the most dominant bacteria and fungi exhibiting the dynamic patterns "Decrease", "Increase" and "Fluctuation". With respect to the different diets, the bacterial communities were similar between the newly eclosed adults and adults fed M. pumila leaves, while the structure of the fungal communities showed great differences between newly eclosed adults and adults fed different diets. Through a co-correlation network analysis, we observed complex microbial interactions among bacterial and fungal taxa that were associated with potential diverse functions and intricate biological processes in the intestinal microbiota of A. mali. Overall, the results of this study demonstrated that the invasive insect A. mali harbours diverse, dynamic, and presumably multifunctional microbial communities, an understanding of which could improve our ability to develop more effective management approaches to control A. mali.

Genetic Population Structure of Dastarcus helophoroides (Coleoptera: Bothrideridae) From Different Long-Horned Beetle Hosts Based on Complete Sequences of Mitochondrial COI.

Dastarcus helophoroides (Fairmaire) (Coleoptera: Bothrideridae) is an important natural enemy of long-horned beetles in China, Japan, and Korea. In this study, the genetic sequence of cytochrome oxidase subunit Ι was used to investigate the genetics and relationships within and among D. helophoroides populations collected from five different geographic locations. We used principal component analysis, heatmap, and Venn diagram results to determine the relationship between haplotypes and populations. In total, 26 haplotypes with 51 nucleotide polymorphic sites were defined, and low genetic diversity was found among the different populations. Significant genetic variations were observed mainly within populations, and no correlation was found between genetic distribution and geographical distance. Low pairwise fixation index values (-0.01424 to 0.04896) and high gene flows show that there was high gene exchange between populations. The codistributed haplotype DH01 was suggested to be the most ancestral haplotype, and other haplotypes were thought to have evolved from it through several mutations. In four of the populations, both common haplotypes (DH01, DH03, and DH22) and unique haplotypes were found. Low genetic diversity among different populations is related to a relatively high flight capacity, host movement, and human-aided dispersal of D. helophoroides. The high gene exchange and typically weak population genetic structure among five populations, especially among populations of Anoplophora glabripennis (Motschulsky), Monochamus alternatus (Hope), and Massicus raddei (Blessig), may suggest that these populations cross naturally in the field.

Identification, characterization and expression analysis of superoxide dismutase genes in Dastarcus helophoroides (Coleoptera: Bothrideridae).

Dastarcus helophoroides, a predatory natural enemy of longhorned beetles, has a relatively longer lifespan compared to other insects. To determine the potential physiological roles of antioxidant enzymes superoxide dismutase (SOD) in longevity and aging of D. helophoroides, analyses including molecular information, bioinformatics research, phylogenetic relationship and expression patterns were combined for investigation. Four D.hSODs were classified into three groups: one cytoplasmic Cu/Zn-SOD, two extracellular Cu/Zn-SOD and one Mn-SOD, were identified and characterized by multiple alignments. All D.hSODs were highly homologous to SODs of Tribolium castaneum, and closely clustered together with SOD genes from insects in phylogenetic analyses. Comparison of the D.hSODs expression in different tissues, stages and age groups showed that the SOD transcripts could be detected in all examined specimens. The expression of D.hSODs revealed tissue-specificity with relatively high levels in the male reproductive system and head and low levels in female reproductive systems and midgut. Expression analyses of D.hSODs in different development stages demonstrated that D.hSOD1 and D.hSOD2 increased in 2nd and 5th instar larvae, whereas two extracellular Cu/Zn-SOD genes (D.hSOD3-a and D.hSOD3-b) were much more expressed in newly emerged adults. The expression fluctuations of D.hSODs during aging seemed to be less significant than during development, and exhibited relatively stable expression with an initial decline and then increased in older groups. The relatively stable and increased expression of D.hSODs may indicate a strong ability of SODs to eliminate oxidative damage products accumulated during aging and possibly retard aging. The research provides molecular biology and in vivo expression levels for future analysis of the SOD family in D. helophoroides and other insects, and provides a basis for further study about the SOD genes contribution on longevity of D. helophoroides.

Identification, Characterization and Expression of Methuselah-Like Genes in Dastarcus helophoroides (Coleoptera: Bothrideridae).

Dastarcus helophoroides, which has a relatively longer lifespan compared to other insects, is one of the most effective natural enemies of many large-body long-horned beetles. Methuselah (Mth) is associated with the lifespan, stress resistance, and reproduction in Drosophila melanogaster, but Mth is not present in non-drosophiline insects. A number of methuselah-like genes (mth-likes, mthls) have been identified in non-drosophiline insects, but it is still unknown whether they are present in Dastarcus helophoroides. We identified three novel mth-like genes in D. helophoroides: mth-like1, mth-like2, and mth-like5, and carried out bioinformatic analysis based on the full-length nucleic acid sequences and deduced amino acid sequences. Real-time quantitative polymerase chain reaction (RT-qPCR) showed variations in expression patterns of mth-like genes in different tissues (highly expressed in reproductive systems) and at different developmental stages, indicating that mth-likes were likely be involved in reproduction and development. The altered mRNA expression in aging adults and under oxidation, high temperature, and starvation stress, indicated that mth-like genes were likely to be involved in aging and the resistance of oxidation, high temperature, and starvation. These results characterize, for the first time, the basic properties of three mth-like genes from D. helophoroides that probably play important roles in development, aging, reproduction, and stress resistance.

The mitochondrial genome of Dastarcus helophoroides (Coleoptera: Bothrideridae) and related phylogenetic analyses.

The complete mitochondrial genome of Dastarcus helophoroides (Coleoptera: Bothrideridae) which consists of 13 PCGs, 22 tRNA genes, two rRNA genes and a non-coding region (D-loop), is sequenced for its nucleotide sequence of 15,878 bp (GenBank: KF811054.1). The genome has a typical gene order which is identical to other Coleoptera species. Except for COI gene generally starts with non-canonical initial codon, all protein-coding genes start with ATN codon and terminate with the stop codon TA(A) or TAG. The secondary structure of rrnL and rrnS consists of 48 helices (contains four newly proposed helices) and 35 helices (contains two newly proposed helices) respectively. All 22 tRNAs in D. helophoroides are predicted to fold into typical cloverleaf secondary structure, except trnS1 (AGN), in which the dihydrouracil arm (DHU arm) could not form stable stem-loop structure. Thirteen protein-coding genes (nucleotide dataset and nucleic acid dataset) of the available species (29 taxa) have been used to infer the phylogenetic relationships among these orders. Tenebrionoidea and Cucujoidea form a sister group, and D. helophoroides is classified into Cucujoidea (Bothrideridae). The study first research on the phylogenetic analyses involving to the D. helophoroides mitogenome, and the results strongly bolster the current morphology-based hypothesis.

Molecular cloning and characterization of a novel P450 gene encoding CYP6BK18 from Dastarcus helophoroides (Coleoptera: Bothrideridae).

A novel cDNA clone encoding a cytochrome P450 gene, named CYP6BK18 (GenBank KC683905), was isolated by reverse transcription PCR from Dastarcus helophoroides (Fairmaire) (Coleoptera: Bothrideridae), a natural enemy of beetles. The full-length cDNA sequence is 1,659 bp, containing a 1,533 bp open reading frame predicting a 510-amino acid protein possessing a transmembrane domain with a calculated molecular weight of 59.4 kDa and a theoretical pI of 8.94. The deduced amino acid sequence of CYP6BK18 showed a 59% identity with CYP6BK17 (GenBank XP_970481.1) from Tribolium castaneum. Phylogenetic analysis indicated that CYP6BK18 was most closely related to CYP6BK17 and CYP6BK14 (GenBank EFA05731.1) from T. castaneum. Expression patterns of CYP6BK18 in different tissues (head, oviduct, midgut, fat bodies, and Malpighian tubules), developmental stages (first- to sixth-instar larvae and adult) and 10 age groups of adult were analyzed by real-time quantitative PCR (RT-qPCR). The results showed that CYP6BK18 was highly expressed in adulthood. Also, RT-qPCR analysis among different age groups of adult showed that CYP6BK18 transcripts were abundant in the spawning period and peaked at the early stage of the adult development. Moreover, the tissue-specific expression levels of CYP6BK18 were in the order of midgut, Malpighian tubes and fatty body from high to low. These results suggest that cytochrome P450 CYP6BK18 may play a role in regulating the development and aging of D. helophoroides.

Molecular cloning and sequence analysis of novel cytochrome P450 cDNA fragments from Dastarcus helophoroides.

The predatory beetle Dastarcus helophoroides (Fairmaire) (Coleoptera: Bothrideridae) is a natural enemy of many longhorned beetles and is mainly distributed in both China and Japan. To date, no research on D. helophoroides P450 enzymes has been reported. In our study, for the better understanding of P450 enzymes in D. helophoroides, 100 novel cDNA fragments encoding cytochrome P450 were amplified from the total RNA of adult D. helophoroides abdomens using five pairs of degenerate primers designed according to the conserved amino acid sequences of the CYP6 family genes in insects through RT-PCR. The obtained nucleotide sequences were 250 bp, 270 bp, and 420 bp in length depending on different primers. Ninety-six fragments were determined to represent CYP6 genes, mainly from CYP6BK, CYP6BQ, and CYP6BR subfamilies, and four fragments were determined to represent CYP9 genes. Twenty-two fragments, submitted to GenBank, were selected for further homologous analysis, which revealed that some fragments of different sizes might be parts of the same P450 gene.

Comparative analysis of the composition of intestinal bacterial communities in Dastarcus helophoroides fed different diets.

The diversity of the intestinal bacterial communities in Dastarcus helophoroides (Fairmaire) (Coleoptera: Bothrideridae) larvae and adults was assayed by PCR-DGGE to determine whether different artificial diets could influence these bacterial communities. Two diets were used for feeding the larvae and four for the adults. Escherichia, Desemzia, Staphylococcus, Asticcacaulis, Cellvibrio, Aurantimonas, and Planomicrobium were isolated from the gut of the adults, with Escherichia and Staphylococcus being the main bacterial communities, and the quantities of intestinal bacterial were different in the adults fed different diets. Specifically, the amount of intestinal bacteria from the adults fed different diets had the following ranking according to the major component of the diet: ant powder > darkling beetle pupa powder > cricket powder > silkworm pupa powder. Escherichia, Bacillus, Staphylococcus, Kurthia, Planococcaceae, Ralstonia, Leptothrix, Acinetobacter, and Pseudomonas were isolated from the gut of the larvae. The quantity of intestinal bacteria from the larvae fed the darkling beetle pupae was greater than that from the larvae fed other artificial diets. This study, for the first time, investigated the effect of artificial diets on the bacterial community and the intestinal microbial diversity of D. helophoroides.

Transcriptome analysis of Dastarcus helophoroides (Coleoptera: Bothrideridae) using Illumina HiSeq sequencing.

BACKGROUND: Dastarcus helophoroides is known as the most valuable natural enemy insect against many large-body longhorned beetles. The molecular mechanism of its long lifespan and reproduction makes it a unique resource for genomic research. However, molecular biological studies on this parasitic beetle are scarce, and genomic information for D. helophoroides is not currently available. Thus, transcriptome information for this species is an important resource that is required for a better understanding of the molecular mechanisms of D. helophoroides. In this study, we obtained transcriptome information of D. helophoroides using high-throughput RNA sequencing. RESULTS: Using Illumina HiSeq 2000 sequencing, 27,543,746 clean reads corresponding to a total of 2.48 Gb nucleotides were obtained from a single run. These reads were assembled into 42,810 unigenes with a mean length of 683 bp. Using a sequence similarity search against the five public databases (NR, Swiss-Prot, GO, COG, KEGG) with a cut-off E-value of 10(-5) using Blastx, a total of 31,293 unigenes were annotated with gene description, gene ontology terms, or metabolic pathways. CONCLUSIONS: To the best of our knowledge, this is the first study on the transcriptome information of D. helophoroides. The transcriptome data presented in this study provide comprehensive information for future studies in D. helophoroides, particularly for functional genomic studies in this parasitic beetle.

Semisynthesis of N-acyl homoserinelactone derivatives and the antifeedant activity against Mythimna separata.

Seven homoserine-lactone (HL) acylated derivatives (HL1-HL7) were synthesized to determine the differences in antifeedant affects. The differences between these derivatives and tutin against Mythimna separata were tested. The structural assignments of these semisynthetic compounds were examined based on their infrared radiaion (IR), electrospray ionization mass spectrometry (ESIMS), and ¹H- and ¹³C-nuclear magnetic resonance (¹³C-NMR) spectral data. Compound HL1 (N-(4-nitrobenzoyl)-homoserinelactone) is the optimized insecticidal agent among these compounds. In addition, the antifeedant activities between homoserinelactone and 7-hydroxycoumarin, tutin derivatives with the same acidylated substitutions were compared, which could help design and synthesize stronger novel botanical insecticides.

Composition and diversity analysis of the gut bacterial community of the Oriental armyworm, Mythimna separata, determined by culture-independent and culture-dependent techniques.

The intestinal bacteria community structure and diversity of the Oriental armyworm, Mythimna separata (Walker) (Lepidoptera: Noctuidae), was studied by analysis of a 16S rDNA clone library, denaturing gradient gel electrophoresis,and culture-dependent techniques. The 16S rDNA clone library revealed a bacterial community diversity comprising Cyanobacteria, Firmicutes, Actinobacteria, Gracilicutes and Proteobacteria, among which Escherichia coli (Migula) (Enterobacteriales: Enterobacteriaceae) was the dominant bacteria. The intestinal bacteria isolated by PCR-denaturing gradient gel electrophoresis were classified to Firmicutes, Proteobacteria, and Gracilicutes, and E. coli was again the dominant bacteria. The culture-dependent technique showed that the intestinal bacteria belonged to Firmicutes and Actinobacteria, and Staphylococcus was the dominant bacteria. The intestinal bacteria of M. separata were widely distributed among the groups Cyanobacteria, Firmicutes, Actinobacteria, Gracilicutes, Proteobacteria, and Gracilicutes. 16S rDNA clone library, denaturing gradient gel electrophoresis, and culture-dependent techniques should be integrated to obtain precise results in terms of the microbial community and its diversity.