Genome comparison of long-circulating field CnmeGV isolates from the same region.

Cnaphalocrocis medinalis granulovirus (CnmeGV), belonging to Betabaculovirus cnamedinalis, can infect the rice pest, the rice leaf roller. In 1979, a CnmeGV isolate, CnmeGV-EP, was collected from Enping County, China. In 2014, we collected another CnmeGV isolate, CnmeGV-EPDH3, at the same location and obtained the complete virus genome sequence using Illumina and ONT sequencing technologies. By combining these two virus isolates, we updated the genome annotation of CnmeGV and conducted an in-depth analysis of its genome features. CnmeGV genome contains abundant tandem repeat sequences, and the repeating units in the homologous regions (hrs) exhibit overlapping and nested patterns. The genetic variations within EPDH3 population show the high stability of CnmeGV genome, and tandem repeats are the only region of high genetic variation in CnmeGV genome replication. Some defective viral genomes formed by recombination were found within the population. Comparison analysis of the two virus isolates collected from Enping showed that the proteins encoded by the CnmeGV-specific genes were less conserved relative to the baculovirus core genes. At the genomic level, there are a large number of SNPs and InDels between the two virus isolates, especially in and around the bro genes and hrs. Additionally, we discovered that CnmeGV acquired a segment of non-ORF sequence from its host, which does not provide any new proteins but rather serves as redundant genetic material integrated into the viral genome. Furthermore, we observed that the host's transposon piggyBac has inserted into some virus genes. Together, dsDNA viruses could acquire non-coding genetic material from their hosts to expand the size of their genomes. These findings provide new insights into the evolution of dsDNA viruses.

Metagenomic Analyses Reveal Gut Microbial Profiles of Cnaphalocrocis medinalis Driven by the Infection of Baculovirus CnmeGV.

The composition of microbiota in the digestive tract gut is essential for insect physiology, homeostasis, and pathogen infection. Little is known about the interactions between microbiota load and oral infection with baculoviruses. CnmeGV is an obligative baculovirus to Cnaphalocrocis medinalis. We investigated the impact of CnmeGV infection on the structure of intestinal microbes of C. medinalis during the initial infection stage. The results revealed that the gut microbiota profiles were dynamically driven by pathogen infection of CnmeGV. The numbers of all the OTU counts were relatively higher at the early and later stages, while the microbial diversity significantly increased early but dropped sharply following the infection. The compositional abundance of domain bacteria Firmicutes developed substantially higher. The significantly enriched and depleted species can be divided into four groups at the species level. Fifteen of these species were ultimately predicted as the biomarkers of CnmeGV infection. CnmeGV infection induces significant enrichment of alterations in functional genes related to metabolism and the immune system, encompassing processes such as carbohydrate, amino acid, cofactor, and vitamin metabolism. Finally, the study may provide an in-depth analysis of the relationship between host microbiota, baculovirus infection, and pest control of C. medinalis.

Molecular Characterization of Plant Volatile Compound Interactions with Cnaphalocrocis medinalis Odorant-Binding Proteins.

Odorant-binding proteins (OBPs) play important roles in the insect olfactory system since they bind external odor molecules to trigger insect olfactory responses. Previous studies have identified some plant-derived volatiles that attract the pervasive insect pest Cnaphalocrocis medinalis (Lepidoptera: Pyralidae), such as phenylacetaldehyde, benzyl acetate, 1-heptanol, and hexanal. To characterize the roles of CmedOBPs in the recognition of these four volatiles, we analyzed the binding abilities of selected CmedOBPs to each of the four compounds, as well as the expression patterns of CmedOBPs in different developmental stages of C. medinalis adult. Antennaes of C. medinalis adults were sensitive to the studied plant volatile combinations. Expression levels of multiple CmedOBPs were significantly increased in the antennae of 2-day-old adults after exposure to volatiles. CmedOBP1, CmedOBP6, CmedPBP1, CmedPBP2, and CmedGOBP2 were significantly up-regulated in the antennae of volatile-stimulated female and male adults when compared to untreated controls. Fluorescence competition assays confirmed that CmedOBP1 could strongly bind 1-heptanol, hexanal, and phenylacetaldehyde; CmedOBP15 strongly bound benzyl acetate and phenylacetaldehyde; and CmedOBP26 could weakly bind 1-heptanol. This study lays a theoretical foundation for further analysis of the mechanisms by which plant volatiles can attract C. medinalis. It also provides a technical basis for the future development of efficient plant volatile attractants of C. medinalis.

Defense Responses of Different Rice Varieties Affect Growth Performance and Food Utilization of Cnaphalocrocis medinalis Larvae.

Rice leaf folder, Cnaphalocrocis medinalis (Guenée), is one of the most serious pests on rice. At present, chemical control is the main method for controlling this pest. However, the indiscriminate use of chemical insecticides has non-target effects and may cause environmental pollution. Besides, leaf curling behavior by C. medinalis may indirectly reduce the efficacy of chemical spray. Therefore, it is crucial to cultivate efficient rice varieties resistant to this pest. Previous studies have found that three different rice varieties, Zhongzao39 (ZZ39), Xiushui134 (XS134), and Yongyou1540 (YY1540), had varying degrees of infestation by C. medinalis. However, it is currently unclear whether the reason for this difference is related to the difference in defense ability of the three rice varieties against the infestation of C. medinalis. To explore this issue, the current study investigated the effects of three rice varieties on the growth performance and food utilization capability of the 4th instar C. medinalis. Further, it elucidated the differences in defense responses among different rice varieties based on the differences in leaf physiological and biochemical indicators and their impact on population occurrence. The results showed that the larval survival rate was the lowest, and the development period was significantly prolonged after feeding on YY1540. This was not related to the differences in leaf wax, pigments, and nutritional components among the three rice varieties nor to the feeding preferences of the larvae. The rate of superoxide anion production, hydrogen peroxide content, and the activity of three protective enzymes were negatively correlated with larval survival rate, and they all showed the highest in YY1540 leaves. Compared to other tested varieties, although the larvae feeding on YY1540 had higher conversion efficiency of ingested food and lower relative consumption rate, their relative growth was faster, indicating stronger food utilization capability. However, they had a lower accumulation of protein. This suggests that different rice varieties had different levels of oxidative stress after infestation by C. medinalis. The defense response of YY1540 was more intense, which was not conducive to the development of the larvae population. These results will provide new insights into the interaction mechanism between different rice varieties and C. medinalis and provide a theoretical basis for cultivating rice varieties resistant to this pest.

Comparative Transcriptomic Assessment of Chemosensory Genes in Adult and Larval Olfactory Organs of Cnaphalocrocis medinalis.

The rice leaf folder, Cnaphalocrocis medinalis (Lepidoptera: Pyralidae), is a notorious pest of rice in Asia. The larvae and adults of C. medinalis utilize specialized chemosensory systems to adapt to different environmental odors and physiological behaviors. However, the differences in chemosensory genes between the olfactory organs of these two different developmental stages remain unclear. Here, we conducted a transcriptome analysis of larvae heads, male antennae, and female antennae in C. medinalis and identified 131 putative chemosensory genes, including 32 OBPs (8 novel OBPs), 23 CSPs (2 novel CSPs), 55 ORs (17 novel ORs), 19 IRs (5 novel IRs) and 2 SNMPs. Comparisons between larvae and adults of C. medinalis by transcriptome and RT-qPCR analysis revealed that the number and expression of chemosensory genes in larval heads were less than that of adult antennae. Only 17 chemosensory genes (7 OBPs and 10 CSPs) were specifically or preferentially expressed in the larval heads, while a total of 101 chemosensory genes (21 OBPs, 9 CSPs, 51 ORs, 18 IRs, and 2 SNMPs) were specifically or preferentially expressed in adult antennae. Our study found differences in chemosensory gene expression between larvae and adults, suggesting their specialized functions at different developmental stages of C. medinalis. These results provide a theoretical basis for screening chemosensory genes as potential molecular targets and developing novel management strategies to control C. medinalis.

Functional Identification of Olfactory Receptors of Cnaphalocrocis medinalis (Lepidoptera: Crambidae) for Plant Odor.

Cnaphalocrocis medinalis (Lepidoptera: Crambidae) is a migratory insect pest on rice crops. The migratory C. medinalis population in a particular location may be immigrants, local populations, emigrants, or a mix of these. Immigrants are strongly attracted to plant odor. We conducted research to identify the olfactory receptors in a floral scent mixture that is strongly attractive to C. medinalis. Through gene cloning, 12 olfactory receptor (OR) genes were amplified and expressed in Xenopus oocytes in vitro, and three of them were found to be responsive to plant foliar and floral volatiles. These were CmedOR31, a specific receptor for geraniol; CmedOR32, a broad-spectrum OR gene that responded to both foliar and floral odors; and CmedOR1, which strongly responded to 10-4 M phenylacetaldehyde. The electrophysiological response to phenylacetaldehyde was extremely high, with a current of 3200 ± 86 nA and an extremely high sensitivity. We compared the phylogenetic tree and sequence similarity of CmedOR genes and found that CmedOR1 belonged to a uniquely conserved OR pedigree in the evolution of Glossata species, and the ORs of this pedigree strongly responded to phenylacetaldehyde. The expression of OR1 was significantly higher in the females than in the males. Localization of CmedOR1 in the antennae of C. medinalis by fluorescence in situ hybridization showed that CmedOR1 was expressed in both males and females. CmedOR1 may be an odor receptor used by females to locate food sources. The function of these ORs and their role in pest monitoring were discussed.

CmHem, a hemolin-like gene identified from Cnaphalocrocis medinalis, involved in metamorphosis and baculovirus infection.

Background: As a member of the immunoglobulin superfamily, hemolins play a vital role in insect development and defense against pathogens. However, the innate immune response of hemolin to baculovirus infection varies among different insects. Methods and results: In this study, the hemolin-like gene from a Crambidae insect, Cnaphalocrocis medinalis, CmHem was cloned, and its role in insect development and baculovirus infection was analyzed. A 1,528 bp contig as potential hemolin-like gene of C. medinalis was reassembled from the transcriptome. Further, the complete hemolin sequence of C. medinalis (CmHem) was cloned and sequenced. The cDNA of CmHem was 1,515 bp in length and encoded 408 amino acids. The deduced amino acid of CmHem has relatively low identities (41.9-62.3%) to various insect hemolins. However, it contains four Ig domains similarity to other insect hemolins. The expression level of CmHem was the highest in eggs, followed by pupae and adults, and maintained a low expression level at larval stage. The synthesized siRNAs were injected into mature larvae, and the CmHem transcription decreased by 51.7%. Moreover, the abdominal somites of larvae became straightened, could not pupate normally, and then died. Infection with a baculovirus, C. medinalis granulovirus (CnmeGV), the expression levels of CmHem in the midgut and fat body of C. medinalis significantly increased at 12 and 24 h, respectively, and then soon returned to normal levels. Conclusions: Our results suggested that hemolin may be related to the metamorphosis of C. medinalis. Exposure to baculovirus induced the phased expression of hemolin gene in the midgut and fat body of C. medinalis, indicated that hemolin involved in the immune recognition of Crambidae insects to baculovirus.

CMRD-Net: a deep learning-based Cnaphalocrocis medinalis damage symptom rotated detection framework for in-field survey.

The damage symptoms of Cnaphalocrocis medinalis (C.medinalis) is an important evaluation index for pest prevention and control. However, due to various shapes, arbitrary-oriented directions and heavy overlaps of C.medinalis damage symptoms under complex field conditions, generic object detection methods based on horizontal bounding box cannot achieve satisfactory results. To address this problem, we develop a Cnaphalocrocis medinalis damage symptom rotated detection framework called CMRD-Net. It mainly consists of a Horizontal-to-Rotated region proposal network (H2R-RPN) and a Rotated-to-Rotated region convolutional neural network (R2R-RCNN). First, the H2R-RPN is utilized to extract rotated region proposals, combined with adaptive positive sample selection that solves the hard definition of positive samples caused by oriented instances. Second, the R2R-RCNN performs feature alignment based on rotated proposals, and exploits oriented-aligned features to detect the damage symptoms. The experimental results on our constructed dataset show that our proposed method outperforms those state-of-the-art rotated object detection algorithms achieving 73.7% average precision (AP). Additionally, the results demonstrate that our method is more suitable than horizontal detection methods for in-field survey of C.medinalis.

Glucose Dehydrogenases-Mediated Acclimation of an Important Rice Pest to Global Warming.

Global warming is posing a threat to animals. As a large group of widely distributed poikilothermal animals, insects are liable to heat stress. How insects deal with heat stress is worth highlighting. Acclimation may improve the heat tolerance of insects, but the underlying mechanism remains vague. In this study, the high temperature of 39 °C was used to select the third instar larvae of the rice leaf folder Cnaphalocrocis medinalis, an important insect pest of rice, for successive generations to establish the heat-acclimated strain (HA39). The molecular mechanism of heat acclimation was explored using this strain. The HA39 larvae showed stronger tolerance to 43 °C than the unacclimated strain (HA27) persistently reared at 27 °C. The HA39 larvae upregulated a glucose dehydrogenase gene, CmGMC10, to decrease the reactive oxygen species (ROS) level and increase the survival rate under heat stress. The HA39 larvae maintained a higher activity of antioxidases than the HA27 when confronted with an exogenous oxidant. Heat acclimation decreased the H2O2 level in larvae under heat stress which was associated with the upregulation of CmGMC10. The rice leaf folder larvae may acclimate to global warming via upregulating CmGMC10 to increase the activity of antioxidases and alleviate the oxidative damage of heat stress.

Insecticide resistance monitoring of Cnaphalocrocis medinalis (Lepidoptera: Pyralidae) and its mechanism to chlorantraniliprole.

BACKGROUND: The rice leaffolder, Cnaphalocrocis medinalis (Guenée), has become an increasingly occurring pest in Asia in recent years. Chemical control remains the most efficient and primary tool for controlling this pest. In this study, we report the resistance status of C. medinalis in China to multiple insecticides including chlorantraniliprole and the main resistance mechanism. RESULTS: Significant variations among field populations of C. medinalis in their resistance to 10 insecticides were observed during 2019-2022. Most of the tested field populations have developed low-to-moderate levels of resistance to abamectin (RR = 2.4-22.2), emamectin benzoate (RR = 1.9-40.3) and spinetoram (RR = 4.2-24.8). Some field populations have developed low resistance to chlorpyrifos (RR = 0.9-6.8). Indoxacarb, metaflumizone, methoxenozide and Bacillus thuringiensis (Bt) potency against all tested populations remained similar. For diamides, significantly higher levels of resistance to chlorantraniliprole (RR = 64.9-113.7) were observed in 2022, whereas all tested field populations in 2019-2021 exhibited susceptible or moderate resistance level to chlorantraniliprole (RR = 1.3-22.1). Cross-resistance between chlorantraniliprole and tetraniliprole was significant. Analysis of ryanodine receptor (RyR) mutations showed that mutation of I4712M was present in resistant populations of C. medinalis with different levels of chlorantraniliprole resistance and was the main mechanism conferring diamide resistance. Mutation of Y4621D also was detected in one tested population. Resistance management strategies for the control of C. medinalis are discussed. CONCLUSION: C. medinalis has developed high level of resistance to chlorantraniliprole. RyR mutations were deemed as the mechanism. © 2023 Society of Chemical Industry.

Processing Properties and Potency of Bacillus thuringiensis Cry Toxins in the Rice Leaffolder Cnaphalocrocis medinalis (Guenée).

Different Cry toxins derived from Bacillus thuringiensis (Bt) possess different insecticidal spectra, whereas insects show variations in their susceptibilities to different Cry toxins. Degradation of Cry toxins by insect midgut extracts was involved in the action of toxins. In this study, we explored the processing patterns of different Cry toxins in Cnaphalocrocis medinalis (Lepidoptera: Crambidae) midgut extracts and evaluated the impact of Cry toxins degradation on their potency against C. medinalis to better understand the function of midgut extracts in the action of different Cry toxins. The results indicated that Cry1Ac, Cry1Aa, and Cry1C toxins could be degraded by C. medinalis midgut extracts, and degradation of Cry toxins by midgut extracts differed among time or concentration effects. Bioassays demonstrated that the toxicity of Cry1Ac, Cry1Aa, and Cry1C toxins decreased after digestion by midgut extracts of C. medinalis. Our findings in this study suggested that midgut extracts play an important role in the action of Cry toxins against C. medinalis, and the degradation of Cry toxins by C. medinalis midgut extracts could reduce their toxicities to C. medinalis. They will provide insights into the action of Cry toxins and the application of Cry toxins in C. medinalis management in paddy fields.

Moth sex pheromones affect interspecific competition among sympatric species and possibly population distribution by modulating pre-mating behavior.

Premating behaviors mediated by pheromones play pivotal roles in animal mating choices. In natural populations of the striped stem borer Chilo suppressalis and the rice leaf roller Cnaphalocrocis medinalis in the rice field habitat, we discovered that Z11-16:Ald, a major component of the C. suppressalis pheromone, modulated the premating behavior of C. medinalis. Z11-16:Ald evoked a strong olfactory response in male antennae and strongly inhibited the sex pheromone trapping of male C. medinalis in the field. The functions of three C. medinalis sex pheromone receptor genes (CmedPR1-3) were verified through heterologous expression in Xenopus oocytes. CmedPR1 responded to Z11-18:OH and Z11-18:Ald, as well as the interspecific pheromone compound Z11-16:Ac of sympatric species; CmedPR2 responded to Z13-18:OH and Z13-18:Ald, as well as the sex pheromone compounds Z11-16:Ald and Z9-16:Ald of sympatric species; and CmedPR3 responded to Z11-18:OH and Z13-18:OH, as well as the interspecific pheromones Z11-16:OH, Z9-16:Ald, Z11-16:Ac, and Z11-16:Ald of sympatric species. Thus, CmedPR2 and CmedPR3 share the ligand Z11-16:Ald, which is not a component of the C. medinalis sex pheromone. Therefore, the sex pheromones of interspecific species affected the input of neural signals by stimulating the sex pheromone receptors on the antennae of male C. medinalis moths, thereby inhibiting the olfactory responses of the male moths to the sex pheromones. Our results demonstrate chemical communication among sympatric species in the rice field habitat, the recognition of intra- and interspecific sex pheromones by olfactory receptors, and how insect premating behaviors are modulated to possibly affect resource partitioning.

Reference Genes for Expression Analysis Using RT-qPCR in Cnaphalocrocis medinalis (Lepidoptera: Pyralidae).

Cnaphalocrocis medinalis is a destructive migratory rice pest. Although many studies have investigated its behavioral and physiological responses to environmental changes and migration-inducing factors, little is known about its molecular mechanisms. This study was conducted to select suitable RT-qPCR reference genes to facilitate future gene expression studies. Here, thirteen candidate housekeeping genes (EF1α, AK, EF1ß, GAPDH, PGK, RPL13, RPL18, RPS3, 18S rRNA, TBP1, TBP2, ACT, and UCCR) were selected to evaluate their stabilities under different conditions using the ∆CT method; the geNorm, NormFinder, BestKeeper algorithms; and the online tool RefFinder. The results showed that the most stable reference genes were EF1ß, PGK, and RPL18, related to developmental stages; RPS3 and RPL18 in larval tissues; EF1ß and PGK in larvae feeding on different rice varieties; EF1α, EF1ß, and PGK in larvae temperature treatments; PGK and RPL13, related to different adult ages; PGK, EF1α, and ACT, related to adult nutritional conditions; RPL18 and PGK, related to adult mating status; and, RPS3 and PGK, related to different adult take-off characteristics. Our results reveal reference genes that apply to various experimental conditions and will greatly improve the reliability of RT-qPCR analysis for the further study of gene function in this pest.

Identification of Key Headspace Volatile Compounds Signaling Preference for Rice over Corn in Adult Females of the Rice Leaf Folder Cnaphalocrocis medinalis.

Volatile organic compounds are important for herbivorous insects in locating their host plants. The rice leaf folder, Cnaphalocrocis medinalis (Guenée), is a devastating migratory insect pest of rice in Asian countries. Although C. medinalis can develop even better on corn than on rice plants in insectaries, it rarely occurs on corn plants in the field. We hypothesized that plant volatile-mediated oviposition preference for rice over corn in adult females may be the reason for the observed rare field occurrence of the pest on corn plants. The present study was conducted to identify the olfactory active volatile compounds (OAVCs) that enable C. medinalis females to discriminate rice from corn plants. In cage tests, rice plants were highly preferred for oviposition over corn plants by C. medinalis females. From headspace, chemical analyses identified 15 rice unique, 8 corn unique, and 28 common volatile compounds. Fourteen OAVCs, including seven common, five rice unique, and two corn unique, were determined. In electroantennogram tests, the rice unique and common OAVCs activated the antennal responses in C. medinalis. In Y-tube olfactometer tests, (E)-2-hexenal and 3-hexanol(common OAVCs) and (Z)-3-hexenyl acetate and (E)-2-hexen-1-ol (rice unique OAVCs) attracted more C. medinalis females than the control, and only blends with both rice unique and common OAVCs were highly preferred over the control. Our results provide insights into the chemical cues used by C. medinalis adult females in host location, which may aid the development of novel crop protection strategies based on the manipulation of host-finding behaviors of C. medinalis.

The Gut Microbiota Composition of Cnaphalocrocis medinalis and Their Predicted Contribution to Larval Nutrition.

Intestinal bacterial flora plays an important role in the nutrition, physiology, and behavior of herbivorous insects. The composition of gut microbiota may also be affected by the food consumed. Cnaphalocrocis medinalis is an oligophagous pest, feeds on rice leaves almost exclusively and causes serious damage to rice in Asian countries. Using antibiotic treatment and metagenome sequencing, we investigated the influence of the food sources (rice and maize seedlings) on the structure and functions of intestinal bacteria of C. medinalis. Firstly, food utilization indices, relative growth rate (RGR), relative consumption rate (RCR), efficiency of conversion of ingested food (ECI), and efficiency of conversion of digested food (ECD), were all significantly adversely affected in the antibiotic treatment eliminating gut bacteria, showing that the microbiota loading in the gut were essential for the larva growth and development of C. medinalis. Further, metagenome sequencing revealed that different diets caused a variation in gut microbiota composition of C. medinalis, indicating that the gut microbiota were in part driven by the diet provided. However, the larvae of C. medinalis hosted a core microbial community in the gut, which was independent from the diets changing. The dominant bacteria in the two feeding groups were highly consistent in the gut of C. medinalis larvae, with the gut bacterial community dominated by Firmicutes at the phylum level, Enterococcus at the genus level, Enterococcus sp. FDAARGOS-375, E. casseliflavus, E. gallinarum, and E. sp. CR-Ec1 accounted for more than 96% of the gut microbiota. Functional prediction analysis demonstrated that gut bacteria encoded a series of metabolism-related enzymes involved in carbohydrate metabolism and amino acid synthesis. Carbohydrate metabolism was the most enriched function in both groups and was more abundant in rice feeding group than in maize feeding group. The core dominant Enterococcus species possessed complete pathways of 14 carbohydrates metabolism, 11 amino acids biosynthesis, and two vitamins synthesize, implied to contribute an essential role to the nutrition intake and development of C. medinalis. Finally, the study may provide an in-depth analysis of the symbiont-host co-adaptation and new insights into the management of C. medinalis.

Cuticular protein genes involve heat acclimation of insect larvae under global warming.

Cuticular proteins (CPs) play important roles in insect growth and development. However, it is unknown whether CPs are related to heat tolerance. Cnaphalocrocis medinalis, a serious pest of rice, occurs in summer and exhibits strong adaptability to high temperature, but the underlying mechanism is unclear. Here, the role of CP genes in heat acclimation was studied. Heat tolerance of the heat-acclimated larvae was significantly stronger than the unacclimated larvae. The cuticular protein content in the heat-acclimated larvae was higher than that of the unacclimated larvae. 191 presumed CP genes of C. medinalis (CmCPs) were identified. Expression patterns of 14 CmCPs were different between the heat acclimated (S39) and unacclimated (S27) larvae under heat stress. CmCPs were specifically expressed in epidermis and the head except CmCPR20 mainly expressed in Malpighian tubules. CmCPR20 was upregulated in S39 while downregulated in S27, but CmTweedle1 and CmCPG1 were upregulated in S27 and downregulated in S39. RNAi CmTweedle1 or CmCPG1 remarkably decreased heat tolerance and cuticular protein content of the heat-acclimated larvae but not the unacclimated larvae. RNAi CmCPR20 decreased heat tolerance and cuticular protein content of the unacclimated larvae but not the heat-acclimated larvae. CmTweedle1 and CmCPG1 genes involve heat acclimation of C. medinalis.

Identification and Characterization of a Double-Stranded RNA Degrading Nuclease Influencing RNAi Efficiency in the Rice Leaf Folder Cnaphalocrocis medinalis.

Rice leaf folder Cnaphalocrocis medinalis is one of the most serious pests of rice in rice-planting regions worldwide. DsRNA-degrading nucleases (dsRNases) are important factors in reducing the efficiency of RNA interference (RNAi) in different insects. In this study, a dsRNase gene from C. medinalis (CmdsRNase) was cloned and characterized. The CmdsRNase cDNA was 1395 bp in length, encoding 464 amino acids. The CmdsRNase zymoprotein contains a signal peptide and an endonuclease NS domain that comprises six active sites, three substrate-binding sites, and one Mg2+-binding site. The mature CmdsRNase forms a homodimer with a total of 16 α-helices and 20 ß-pleated sheets. Homology and phylogenetic analyses revealed that CmdsRNase is closely related to dsRNase2 in Ostrinia nubilalis. Expression pattern analysis by droplet digital PCR indicated that the expression levels of CmdsRNase varied throughout the developmental stages of C. medinalis and in different adult tissues, with the highest expression levels in the fourth-instar larvae and the hemolymph. CmdsRNase can degrade dsRNA to reduce the efficiency of RNAi in C. medinalis. Co-silencing of CmCHS (chitin synthase from C. medinalis) and CmdsRNase affected significantly the growth and development of C. medinalis and thus improved RNAi efficacy, which increased by 27.17%. These findings will be helpful for green control of C. medinalis and other lepidopteran pests by RNAi.

Evidence of a shared binding site for Bacillus thuringiensis Cry1Ac and Cry2Aa toxins in Cnaphalocrocis medinalis cadherin.

Insect midgut cadherins function as receptors and play critical roles as protein receptors of insecticidal Bacillus thuringiensis (Bt) toxins used as biopesticides and in Bt transgenic crops worldwide. Here, we cloned and characterized the full-length midgut cadherin (CmCad) cDNA from the rice leaffolder (Cnaphalocrocis medinalis), a destructive pest of rice in many Asian countries. Expression of recombinant proteins corresponding to the extracellular domain of CmCad allowed testing binding of Cry proteins. Results from in vitro ligand blotting and enzyme-linked immunosorbent assays supported that the extracellular domain of CmCad contains regions recognized by both Cry1Ac and Cry2Aa. Molecular modelling and docking simulations indicated that binding to both Cry1Ac and Cry2Aa is localized primarily within a CmCad motif corresponding to residues T1417-D1435. A recombinant CmCad protein produced without residues T1417-D1435 lacked binding to Cry1Ac and Cry2Aa, confirmed our modelling predictions that CmCad has a shared Cry1Ac and Cry2Aa binding site. The potential existence of a shared binding region in CmCad suggests that caution should be taken when using combinations of Cry1Ac and Cry2Aa in pyramided transgenic rice, as their combined use could speed the evolution of resistance to both toxins.

Male nutritional status does not impact the reproductive potential of female Cnaphalocrocis medinalis moths under conditions of nutrient shortage.

In addition to sperm, some accessory substances transferred to females during copulation act as nuptial gifts by passing on valuable nutrients in many insect species. The nutritional status of the males can thus have a great effect on the mating behavior, fecundity and even the longevity of females. However, little is known about the effect of male nutritional status on the female reproductive traits in migratory insect species, particularly when females experience nutrient shortage and have to choose between reproduction and migration. Here, Cnaphalocrocis medinalis, a migratory rice pest in Asia, was studied to explore this issue. Our results showed that in male moths fed with honey solution, their gonads had higher energy content than gonads of starved males, resulting in increased energy content of the bursa copulatrix of females after mating with fed males. Such females showed increased mating frequency, fecundity and longevity compared to females mating with starved males, indicating that male moths deliver nuptial gifts to females and improve their reproductive performance. However, when females were starved, only about 45% mated, with just a single copulation, regardless of male nutritional status. Starved females showed lower fecundity, and a longer pre-oviposition period (indicating a greater propensity to migrate), compared to fed females. However, copulation still significantly extended their longevity. These results suggest that starved females invest in migration to escape deteriorating habitats, rather than investing the nuptial gift to increased fecundity. Our results further our understanding of the reproductive adaptability of migratory insects under conditions of food stress.

Transcriptomics identifies key defense mechanisms in rice resistant to both leaf-feeding and phloem feeding herbivores.

BACKGROUND: Outbreaks of insect pests in paddy fields cause heavy losses in global rice yield annually, a threat projected to be aggravated by ongoing climate warming. Although significant progress has been made in the screening and cloning of insect resistance genes in rice germplasm and their introgression into modern cultivars, improved rice resistance is only effective against either chewing or phloem-feeding insects. RESULTS: In this study, the results from standard and modified seedbox screening, settlement preference and honeydew excretion tests consistently showed that Qingliu, a previously known leaffolder-resistant rice variety, is also moderately resistant to brown planthopper (BPH). High-throughput RNA sequencing showed a higher number of differentially expressed genes (DEGs) at the infestation site, with 2720 DEGs in leaves vs 181 DEGs in sheaths for leaffolder herbivory and 450 DEGs in sheaths vs 212 DEGs in leaves for BPH infestation. The leaf-specific transcriptome revealed that Qingliu responds to leaffolder feeding by activating jasmonic acid biosynthesis genes and genes regulating the shikimate and phenylpropanoid pathways that are essential for the biosynthesis of salicylic acid, melatonin, flavonoids and lignin defensive compounds. The sheath-specific transcriptome revealed that Qingliu responds to BPH infestation by inducing salicylic acid-responsive genes and those controlling cellular signaling cascades. Taken together these genes could play a role in triggering defense mechanisms such as cell wall modifications and cuticular wax formation. CONCLUSIONS: This study highlighted the key defensive responses of a rarely observed rice variety Qingliu that has resistance to attacks by two different feeding guilds of herbivores. The leaffolders are leaf-feeder while the BPHs are phloem feeders, consequently Qingliu is considered to have dual resistance. Although the defense responses of Qingliu to both insect pest types appear largely dissimilar, the phenylpropanoid pathway (or more specifically phenylalanine ammonia-lyase genes) could be a convergent upstream pathway. However, this possibility requires further studies. This information is valuable for breeding programs aiming to generate broad spectrum insect resistance in rice cultivars.