The dynamics of N6-methyladenine RNA modification in resistant and susceptible rice varieties responding to rice stem borer damage.

N6-methyladenosine (m6A) is the most prevalent modification in cellular RNA which orchestrates diverse physiological and pathological processes during stress response. However, the differential m6A modifications that cope with herbivore stress in resistant and susceptible crop varieties remain unclear. Here, we found that rice stem borer (RSB) larvae grew better on indica rice (e.g., MH63, IR64, Nanjing 11) than on japonica rice varieties (e.g., Nipponbare, Zhonghua 11, Xiushui 11). Then, transcriptome-wide m6A profiling of representative resistant (Nipponbare) and susceptible (MH63) rice varieties were performed using a nanopore direct RNA sequencing approach, to reveal variety-specific m6A modifications against RSB. Upon RSB infestation, m6A methylation occurred in actively expressed genes in Nipponbare and MH63, but the number of methylation sites decreased across rice chromosomes. Integrative analysis showed that m6A methylation levels were closely associated with transcriptional regulation. Genes involved in herbivorous resistance related to mitogen-activated protein kinase, jasmonic acid (JA), and terpenoid biosynthesis pathways, as well as JA-mediated trypsin protease inhibitors, were heavily methylated by m6A, and their expression was more pronounced in RSB-infested Nipponbare than in RSB-infested MH63, which may have contributed to RSB resistance in Nipponbare. Therefore, dynamics of m6A modifications act as the main regulatory strategy for expression of genes involved in plant-insect interactions, which is attributed to differential responses of resistant and susceptible rice varieties to RSB infestation. These findings could contribute to developing molecular breeding strategies for controlling herbivorous pests.

Tyrosine hydroxylase plays crucial roles in larval cuticle formation and larval-pupal tanning in the rice stem borer, Chilo suppressalis.

The striped stem borer, Chilo suppressalis (Walker), a notorious pest infesting rice, has evolved a high level of resistance to many commonly used insecticides. In this study, we investigate whether tyrosine hydroxylase (TH), which is required for larval development and cuticle tanning in many insects, could be a potential target for the control of C. suppressalis. We identified and characterized the full-length cDNA (CsTH) of C. suppressalis. The complete open reading frame of CsTH (MW690914) was 1683 bp in length, encoding a protein of 560 amino acids. Within the first to the sixth larval instars, CsTH was high in the first day just after molting, and lower in the ensuing days. From the wandering stage to the adult stage, levels of CSTH began to rise and reached a peak at the pupal stage. These patterns suggested a role for the gene in larval development and larval-pupal cuticle tanning. When we injected dsCsTH or 3-iodotyrosine (3-IT) as a TH inhibitor or fed a larva diet supplemented with 3-IT, there were significant impairments in larval development and larval-pupal cuticle tanning. Adult emergence was severely impaired, and most adults died. These results suggest that CsTH might play a critical role in larval development as well as larval-pupal tanning and immunity in C. suppressalis, and this gene could form a potential novel target for pest control.

Defence and nutrition synergistically contribute to the distinct tolerance of rice subspecies to the stem borer, Chilo suppressalis.

Damage caused by the rice striped stem borer (SSB), Chilo suppressalis (Walker) (Lepidoptera: Pyralidae), is much more severe on indica/xian rice than on japonica/geng rice (Oryza sativa) which matches pest outbreak data in cropping regions of China. The mechanistic basis of this difference among rice subspecies remains unclear. Using transcriptomic, metabolomic and genetic analyses in combination with insect bioassay experiments, we showed that japonica and indica rice utilise different defence responses to repel SSB, and that SSB exploited plant nutrition deficiencies in different ways in the subspecies. The more resistant japonica rice induced patterns of accumulation of methyl jasmonate (MeJA-part of a defensive pathway) and vitamin B1 (VB1-a nutrition pathway) distinct from indica cultivars. Using gene-edited rice plants and SSB bioassays, we found that MeJA and VB1 jointly affected the performance of SSB by disrupting juvenile hormone levels. In addition, genetic variants of key biosynthesis genes in the MeJA and VB1 pathways (OsJMT and OsTH1, respectively) differed between japonica and indica rice and contributed to performance differences; in indica rice, SSB avoided the MeJA defence pathway and hijacked the VB1 nutrition-related pathway to promote development. The findings highlight important genetic and mechanistic differences between rice subspecies affecting SSB damage which could be exploited in plant breeding for resistance.

Evolution of Wolbachia reproductive and nutritional mutualism: insights from the genomes of two novel strains that double infect the pollinator of dioecious Ficus hirta.

Wolbachia is a genus of maternally inherited endosymbionts that can affect reproduction of their hosts and influence metabolic processes. The pollinator, Valisia javana, is common in the male syconium of the dioecious fig Ficus hirta. Based on a high-quality chromosome-level V. javana genome with PacBio long-read and Illumina short-read sequencing, we discovered a sizeable proportion of Wolbachia sequences and used these to assemble two novel Wolbachia strains belonging to supergroup A. We explored its phylogenetic relationship with described Wolbachia strains based on MLST sequences and the possibility of induction of CI (cytoplasmic incompatibility) in this strain by examining the presence of cif genes known to be responsible for CI in other insects. We also identified mobile genetic elements including prophages and insertion sequences, genes related to biotin synthesis and metabolism. A total of two prophages and 256 insertion sequences were found. The prophage WOjav1 is cryptic (structure incomplete) and WOjav2 is relatively intact. IS5 is the dominant transposon family. At least three pairs of type I cif genes with three copies were found which may cause strong CI although this needs experimental verification; we also considered possible nutritional effects of the Wolbachia by identifying genes related to biotin production, absorption and metabolism. This study provides a resource for further studies of Wolbachia-pollinator-host plant interactions.

Identification of a fatty acid synthase gene (FAS1) from Laodelphax striatellus planthoppers contributing to fecundity.

Fatty acid synthase (FAS) is a multifunctional enzyme that plays an important role in the formation of fatty acids. The fatty acids take part in many processes, such as cell signaling and energy metabolism, and in insects they are important in both cuticular hydrocarbon (CHC) formation and reproduction. Here we characterized the sequence structure and function of an FAS from the small brown planthopper (SBPH), Laodelphax striatellus. The full-length open reading frame (ORF) sequence of LsFAS1 was 7122 bp, encoding a predicted protein of 2373 amino acid residues. There were 7 functional domains in the LsFAS1 protein sequence. Gene expression screening by real-time quantitative polymerase chain reaction (RT-qPCR) showed that LsFAS1 was expressed in all developmental stages. Relative expression was highest at the 4th-instar and female adult stages. Among different tissues, the expression level of LsFAS1 in the ovary was the highest. Phylogenetic analysis showed that LsFAS1 clustered in a clade with 2 FASs from Nilaparvata lugens. Furthermore, these 3 FASs are related to cockroach BgFAS and locust LmFAS. After RNA interference-mediated knock-down, most treated insects died at eclosion. In addition, the lifespan of dsFAS1-treated female adults was shorter than that of the dsGFP-injected control, and offspring production decreased. Also, the expression of vitellogenin (Vg) and vitellogenin receptor (VgR) genes decreased. Virgin females dissected at days 2 and 4 post-eclosion showed many matured oocytes in planthoppers treated with dsGFP but not with dsFAS1. These data highlight the importance of LsFAS1 in SBPH, including a role in reproduction.

SoxC is Required for Ecdysteroid Induction of Neuropeptide Genes During Insect Eclosion.

In insects, the shedding of the old exoskeleton is accomplished through ecdysis which is typically followed by the expansion and tanning of the new cuticle. Four neuropeptides, eclosion hormone (EH), ecdysis triggering hormone (ETH), crustacean cardioactive peptide (CCAP) and bursicon (Bur) are known to control ecdysis. However, the regulation of these neuropeptide genes is still poorly understood. Here, we report that in the red flour beetle (RFB) Tribolium castaneum and the fall armyworm (FAW) Spodoptera frugiperda, knockdown or knockout of the SoxC gene caused eclosion defects. The expansion and tanning of wings were not complete. In both RFB and FAW, the knockdown or knockout of SoxC resulted in a decrease in the expression of EH gene. Electrophoretic mobility shift assays revealed that the SfSoxC protein directly binds to a motif present in the promoter of SfEH. The luciferase reporter assays in Sf9 cells confirmed these results. These data suggest that transcription factor SoxC plays a key role in ecdysteroid induction of genes coding for neuropeptides such as EH involved in the regulation of insect eclosion.

Terpene Synthases in Rice Pan-Genome and Their Responses to Chilo suppressalis Larvae Infesting.

Terpene synthase (TPS) catalyzes the synthesis of terpenes and plays an important role in plant defense. This study identified 45 OsTPS genes (32 core genes and 13 variable genes) based on the high-quality rice gene-based pan-genome. This indicates limitations in OsTPS gene studies based on a single reference genome. In the present study, through collinearity between multiple rice genomes, one OsTPS gene absent in the reference (Nipponbare) genome was found and two TPS genes in the reference genome were found to have atypical structures, which would have been ignored in single genome analysis. OsTPS genes were divided into five groups and TPS-b was lost according to the phylogenetic tree. OsTPSs in TPS-c and TPS-g were all core genes indicating these two groups were stable during domestication. In addition, through the analysis of transcriptome data, some structural variations were found to affect the expression of OsTPS genes. Through the Ka/Ks calculation of OsTPS genes, we found that different OsTPS genes were under different selection pressure during domestication; for example, OsTPS22 and OsTPS29 experienced stronger positive selection than the other OsTPS genes. After Chilo suppressalis larvae infesting, 25 differentially expressed OsTPS genes were identified, which are involved in the diterpene phytoalexins precursors biosynthesis and ent-kaurene biosynthesis pathways. Overall, the present study conducted a bioinformatics analysis of OsTPS genes using a high-quality rice pan-genome, which provided a basis for further study of OsTPS genes.

Variability in development of the striped rice borer, Chilo suppressalis (Lepidoptera: Pyralidae), due to instar number and last instar duration.

The striped stem borer, Chilo suppressalis (Walker), is an important insect pest of rice which shows substantial variation in developmental duration among individuals. This variation is currently poorly characterized but it is important from a control perspective because pesticides can only target early sensitive instars. It is unclear whether there are key stages that determine the length of developmental duration of individuals and/or whether variation in instar number contributes to this variation. In this study, a laboratory population and a population recently established from the field were used to test variation in development time across instar stages. The duration of developmental time of C. suppressalis started to diverge from the 5th instar onward. Individuals pupated at the 5th, 6th, 7th or even 8th instar stage. In both populations, both the instar at which the larva pupated and the duration of the last larval instar stage determined total developmental time of an individual. There was little impact of the developmental time of early instars on total developmental duration or on instar number prior to pupation. Sex influenced the number of instars but not development time within this number. The biological and applied significance of uneven development in C. suppressalis are discussed.

Transition and Transversion Mutations Are Biased towards GC in Transposons of Chilo suppressalis (Lepidoptera: Pyralidae).

Transposons are often regulated by their hosts, and as a result, there are transposons with several mutations within their host organisms. To gain insight into the patterns of the variations, nucleotide substitutions and indels of transposons were analysed in Chilo suppressalis Walker. The CsuPLE1.1 is a member of the piggyBac-like element (PLE) family, which belongs to the DNA transposons, and the Csu-Ty3 is a member of the Ty3/gypsy family, which belongs to the RNA transposons. Copies of CsuPLE1.1 and Csu-Ty3 were cloned separately from different C. suppressalis individuals, and then multiple sequence alignments were performed. There were numerous single-base substitutions in CsuPLE1.1 and Csu-Ty3, but only a few insertion and deletion mutations. Similarly, in both transposons, the occurring frequencies of transitions were significantly higher than transversions (p ≤ 0.01). In the single-base substitutions, the most frequently occurring base changes were A→G and T→C in both types of transposons. Additionally, single-base substitution frequencies occurring at positions 1, 2 or 3 (pos1, pos2 or pos3) of a given codon in the element transposase were not significantly different. Both in CsuPLE1.1 and Csu-Ty3, the patterns of nucleotide substitution had the same characteristics and nucleotide mutations were biased toward GC. This research provides a perspective on the understanding of transposon mutation patterns.

Cloning of Two Acetylcholinesterase Genes and Analysis of Point Mutations Putatively Associated with Triazophos Resistance in Chilo auricilius (Lepidoptera: Pyralidae).

Acetylcholinesterase (AChE) is the target of organophosphate (OP) and carbamate insecticides. Mutations in the AChE gene (ace) leading to decreased insecticide susceptibility is the main resistance mechanism in insects. In this study, two Chilo auricilius acetylcholinesterase genes, designated as Caace1 and Caace2, were cloned using RT-PCR and RACE. Caace1 cDNA is 2534 bp, with ORF of 2082 bp, and it encodes an acetylcholinesterase 1 (CaAChE1) protein comprising a calculated 693 amino acid (aa) residues. Caace2 cDNA contains 2280 bp, with a full-length ORF of 1917 bp, encoding acetylcholinesterase 2 (CaAChE2) comprising a calculated 638 aa residues. At the aa level, CaAChE1 displays the highest similarity (97%) with the Chilo suppressalis AChE1, and CaAChE2 shows the highest similarity with the C. suppressalis AChE2 (99%). From the restriction fragment length polymorphism (RFLP) PCR (RFLP-PCR) analysis, one mutation in Caace1, similar to the ace1 mutation associated with triazophos resistance in C. suppressalis, was detected. Detailed examination of field populations of C. auricilius indicated this resistance mutation in C. auricilius is still quite infrequent. Based on the assay of AChE activity and RFLP-PCR testing, an individual that contains resistance mutation has lower AChE activities, while the individual that does not contain the resistance mutation has higher AChE activities. This study provides a basis for future investigations into the mechanism of OP resistance in C. auricilius, as well as a guidance for C. auricilius control with reasonable choice of pesticides.

Transcriptomic response of cowpea bruchids to N-acetylglucosamine-specific lectins.

Griffonia simplicifolia lectin II (GSII) and wheat germ agglutinin (WGA) are N-acetylglucosamine-binding lectins. Previous studies demonstrated that they have anti-insect activity, a property potentially useful in pest control. To gain some insight into the insect response to dietary lectins, we performed transcriptomic analysis using the cowpea bruchid (Callosobruchus maculatus) midgut microarray platform we built. Compared to the nonnutritional cellulose treatment, dietary lectins induced more profound changes in gene expression. Ingestion of relatively high doses of lectins for 24 h resulted in alteration of gene expression involved in sugar and lipid metabolism, transport, development, defense, and stress tolerance. Metabolic genes were largely downregulated. Moreover, we observed disorganized microvilli resulting from ingestion of WGA. This morphological change is consistent with the lectin-induced changes in genes related to midgut epithelial cell repair. In addition, suboptimal nutrient conditions may serve as a stress signal to trigger senescence processes, leading to growth arrest and developmental delay.

Molecular characterization of the piggyBac-like element, a candidate marker for phylogenetic research of Chilo suppressalis (Walker) in China.

BACKGROUND: Transposable elements (TEs, transposons) are mobile genetic DNA sequences. TEs can insert copies of themselves into new genomic locations and they have the capacity to multiply. Therefore, TEs have been crucial in the shaping of hosts' current genomes. TEs can be utilized as genetic markers to study population genetic diversity. The rice stem borer Chilo suppressalis Walker is one of the most important insect pests of many subtropical and tropical paddy fields. This insect occurs in all the rice-growing areas in China. This research was carried out in order to find diversity between C. suppressalis field populations and detect the original settlement of C. suppressalis populations based on the piggyBac-like element (PLE). We also aim to provide insights into the evolution of PLEs in C. suppressalis and the phylogeography of C. suppressalis. RESULTS: Here we identify a new piggyBac-like element (PLE) in the rice stem borer Chilo suppressalis Walker, which is called CsuPLE1.1 (GenBank accession no. JX294476). CsuPLE1.1 is transcriptionally active. Additionally, the CsuPLE1.1 sequence varied slightly between field populations, with polymorphic indels (insertion/deletion) and hyper-variable regions including the identification of the 3' region outside the open reading frame (ORF). CsuPLE1.1 insertion frequency varied between field populations. Sequences variation was found between CsuPLE1 copies and varied within and among field populations. Twenty-one different insertion sites for CsuPLE1 copies were identified with at least two insertion loci found in all populations. CONCLUSIONS: Our results indicate that the initial invasion of CsuPLE1 into C. suppressalis occurred before C. suppressalis populations spread throughout China, and suggest that C. suppressalis populations have a common ancestor in China. Additionally, the lower reaches of the Yangtze River are probably the original settlement of C. suppressalis in China. Finally, the CsuPLE1 insertion site appears to be a candidate marker for phylogenetic research of C. suppressalis.

Effects of two pesticides, TZP and JGM, on reproduction of three planthopper species, Nilaparvata lugens Stål, Sogatella furcifera Horvath, and Laodelphax striatella Fallén.

Three planthopper species, the brown planthopper (BPH), Nilaparvata lugens Stål, the white-backed planthopper (WBPH), Sogatella furcifera Horvath, and the small brown planthopper (SBPH), Laodelphax striatella Fallén, often co-occur in rice grown regions of China. The present investigation examined effects of two pesticides, triazophos (TZP) and jinggangmycin (JGM) (a fungicide), on reproduction of BPH, WBPH, and SBPH. The results showed that TZP stimulated the fecundity of the three planthopper species. Interestingly, JGM stimulated the fecundity of BPH but suppressed the fecundity of WBPH. In addition, TZP and JGM had a significant effect on the preoviposition period (PVD), the oviposition period (OPD), and the longevity of adult females (LAF) of BPH and WBPH. Based on these findings, to avoid resurgence occurrence of planthoppers, we suggest that the application of TZP should be banned in rice fields, that JGM should be used to control rice sheath blight at the early growth stages of rice (with WBPH occurrence and without BPH occurrence).

Comparisons of topical and spray applications of two pesticides, triazophos and jinggangmycin, on the protein content in the ovaries and fat bodies of the brown planthopper Nilaparvata lugens Stål (Hemiptera:Delphacidae).

The pesticide-induced stimulation of reproduction in pests is one of the most important mechanisms of pest resurgence. There have been numerous reports on the insecticide-induced stimulation of reproduction. However, the relationship between pesticide application method and pest resurgence (stimulation of reproduction) has received little attention. Here, we studied the effect of two treatment methods, triazophos (TZP) and jinggangmycin (JGM), on the protein content in the ovaries and fat bodies of the brown planthopper (BPH) Nilaparvata lugens Stål. The results showed that pesticide treatment methods significantly affected the protein content in the ovaries and fat bodies of BPH. In addition, grand means (means of main effect) of the protein content at 2 and 3 days after emergence (2 and 3 DAE) for foliar sprays was significantly higher than that observed after topical treatments, which increased by 23.9% (from 1.42 to 1.76) and 8.82% (from 4.42 to 4.81), respectively. No significant differences on the protein content in the ovaries and fat bodies for the JGM topical treatment were observed compared with controls. However, the protein content for JGM foliar sprays was significantly higher than that for the controls. The protein contents in both topical and spray treatments of TZP were significantly higher than those of the controls. Ovarian protein is mainly yolk protein. There is a positive correlation between ovarian protein content and the number of eggs laid. These findings show that foliar spray of the pesticides promotes the resurgence of BPH. Therefore, the foliar spray of some pesticides, such as JGM, should be avoided for the control of pests, which is the sideeffects of the fungicide on non-target insect pests' occurrence.

Interactions between Meteorus pulchricornis and Spodoptera exigua multiple nucleopolyhedrovirus.

Baculoviruses may interact with parasitoids in the same host. A previous study has shown that infection of larvae with Spodoptera litura nucleopolyhedrovirus (SpltNPV) was deleterious to the survival and development of Meteorus pulchricornis (Wesmael) (Hymenoptera: Braconidae). In this paper, the interactions between M. pulchricornis and Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) in Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae), a permissive host of the virus and parasitoid, were investigated. The results showed that the effect of M. pulchricornis on SeMNPV and the effect of the virus on the parasitoid both depended on the concentration of the virus and the interval between viral infection and parasitism. Whether S. exigua was treated with the parasitoid and virus simultaneously or 1 day apart, the biological activities of 10(5), 10(6), and 10(7) OBs/mL SeMNPV were all significantly improved by M. pulchricornis. In contrast, the biological activity of 10(3) OBs/mL SeMNPV was significantly decreased when the host was exposed to the virus and parasitoid simultaneously. Regarding the impact of SeMNPV on M. pulchricornis, exposing the host to the parasitoid and SeMNPV with concentrations lower than 10(6) occlusion bodies (OBs)/mL produced no negative effects on the parasitoid. The results also showed that ingestion of SeMNPV by adult stage M. pulchricornis significantly increased the number of parasitoid offspring that successfully emerged from the host. Furthermore, M. pulchricornis was found to transmit SeMNPV among populations of S. exigua. Taken together, these findings indicate that M. pulchricornis integrated with an appropriate concentration of SeMNPV has the potential to improve the efficacy of biological control against S. exigua.

Enhancement of the biological activity of nucleopolyhedrovirus through disruption of the peritrophic matrix of insect larvae by chlorfluazuron.

The enhancement of Spodoptera litura (F.) nucleopolyhedrovirus (SlNPV) activity using the chitin synthesis inhibitor chlorfluazuron was investigated. When tested against fifth-instar S. litura larvae, chlorfluazuron produced synergistic effects at doses of 0.05 and 0.025 microg per insect, and additive effects at doses of 0.1 and 0.2 microg. Furthermore, the time required for SlNPV to kill larvae was significantly reduced by chlorfluazuron at all doses tested. The activity and killing speed of Autographa californica (Spey) nucleopolyhedrovirus (AcMNPV) against third-instar Spodoptera exigua (Hübner) larvae were similarly improved by chlorfluazuron at a dose of 0.05 microg per larva. Furthermore, the growth of S. exigua was significantly retarded by chlorfluazuron. Environmental scanning electron microscopy (ESEM) showed that the peritrophic matrices (PMs) of S. litura exposed to chlorfluazuron alone, or the combination treatment, were markedly disrupted. Obvious ruptures on the outer surfaces of the PM were observed, which potentially facilitated the passage of virions through the matrix.

Cloning of the Bacillus thuringiensis serovar sotto chitinase (Schi) gene and characterization of its protein

Chitinase plays a positive role in the pathogenicity of Bacillus thuringiensis to insect pests. We used touchdown PCR to clone the chitinase (Schi) gene from Bacillus thuringiensis serovar sotto (Bt sotto) chromosomal DNA. Our DNA sequencing analysis revealed that the Bt sotto Schi gene consists of an open reading frame (ORF) of 2067 nucleotides with codes for the chitinase precursor. We also found that the putative promoter consensus sequences (the -35 and -10 regions) of the Bt soto Schi gene are identical to those of the chiA71 gene from Bt Pakistani, the chiA74 gene from Bt kenyae and the ichi gene from Bt israelensis. The Schi chitinase precursor is 688 amino acids long with an estimated molecular mass of 75.75 kDa and a theoretical isoelectric point of 5.74, and contains four domains, which are, in sequence, a signal peptide, an N-terminal catalytic domain, a fibronectin type III like domain and a C-terminal chitin-binding domain. Sequence comparison and the evolutionary relationship of the Bt sotto Schi chitinase to other chitinase and chitinase-like proteins are also discussed