MicroRNA expression profiling of Spodoptera frugiperda under Chlorantraniliprole, Indoxacarb and Thiamethoxam exposure

Jun Won Shin,Rashmi Manohar Mahalle,Keon Mook Seong 한국응용곤충학회 2024 한국응용곤충학회 학술대회논문집 Vol.2024 No.04

The fall armyworm, Spodoptera frugiperda, has developed extremely high levels of resistance to chlorantraniliprole and other classes of insecticides in the field. As microRNAs (miRNAs) play important roles in various biological processes through gene regulation. we examined the miRNA profile of S. frugiperda in response to Chlorantraniliprole, Indoxacarb and Thiamethoxam. Transcriptome analysis showed significant changes in the abundance of some miRNAs after treatment of S. frugiperda larvae with LC20 concentrations of three insecticides. A total of 197 miRNAs were systematically identified from S. frugiperda, and 16, 9, 2 miRNAs were differentially expressed after treatments of three insecticides. Importantly, three miRNAs were significantly downregulated and three were upregulated by RT-qPCR after treatment the LC50 of three insecticides with S. frugiperda larvae. Microinjection of agomirs of these six miRNAs into S. frugiperda larvae resulted in significant changes in mortality rates when exposed to three insecticides. Additionally, we also screened potential target genes for some of differentially expressed miRNAs, which may play important roles in insecticide resistance development. These findings provide valuable insights into the molecular mechanisms of insecticide resistance and underscore the potential of miRNAs as targets for the development of novel pest control strategies in S. frugiperda.

한국에서 열대거세미나방 알기생벌 Telenomus remus Nixon (가칭: 밤나방검정알벌) [검정알벌과]의 첫 보고 및 생물적 특성 연구

조점래,서보윤,최준열,이관석,서미자,김정환 한국환경생물학회 2022 환경생물 : 환경생물학회지 Vol.40 No.2

In this study, Telenomus remus Nixon (Hymenoptera: Scelionidae) was first reported as a natural enemy of the fall armyworm Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) egg collected from corn fields in Korea, and its biological characteristics was studied. Based on morphological and molecular analysis, the parasitoid emerged from S. frugiperda eggs was identified as T. remus. We found that T. remus can attack the eggs of S. frugiperda, Spodoptera litra and Spodoptera exigua under a laboratory condition. The longevity of T. remus female adult was longer than that of male adult. The egg-to-adult period of T. remus was not affected by the host age and sex. T. remus female adult laid at least 1-3 eggs a day to a maximum of 37 or more eggs, and the most oviposited on the 3rd to 4th day after emergence. The host preference for oviposition of T. remus adult was high in the order of S. litura>S. exigua>S. frugiperda. T. remus preferred to parasitize 1- and 2-day-old host egg rather than 3-day-old host egg. When compared to the sex ratio of T. remus progeny, the rate of female progeny was higher at the initiation time of oviposition, while the proportion of male progeny increased significantly with female adult age, especially after 8-day-old adult. This information may be useful for improving T. remus mass rearing system and developing a biological control program to control S. frugiperda. 본 연구에서는 국내 옥수수 포장에서 채집한 열대거세미나방 알덩어리로부터 우화한 알기생벌을 형태 및 분자동정하여 검정알벌과 (Scelionidae)의 Telenomus remus Nixon (1937) [(가칭)밤나방검정알벌]로 최초로 확인되었다. T. remus 성충의 수명 및 발육기간 (알-성충까지)은 기주 종류 및 성별에 따른 차이가 없었다. T. remus 암컷 성충은 우화 후 바로 산란하고, 하루에 1~3개에서부터 최고 37개까지 산란하였으며, 우화 후 1~2일째 가장 많이 산란하였다. 총 산란수는 기주로 열대거세미나방 알을 사용하였을 경우 평균 118.4 (7.0~352.0)개, 담배거세미나방의 알에 서는 평균 164.9 (5.0~372.0)개로 담배거세미나방 알을 사용하였을 때 산란수가 많았으나, 유의미한 차이는 없었다. T. remus의 산란 기주 선호도는 담배거세미나방>파밤나방>열대거세미나방 순으로 높았다. 기주 알의 나이에 따라 T. remus의 선호도가 달랐는데, 1~2일 된 알을 선호하였다. T. remus 자손의 암수 비율은 기주와 상관없이 암컷의 비율이 수컷보다 높았으며, 산란 초기에는 암컷 산자의 비율이 높다가 나이가 들수록 수컷 산자의 비율이 현저하게 높았다. 교미하지 않은 T. remus 암컷이 산란하여 부화한 경우, 100% 수컷 성충으로 arrhenotoky type의 단위생식을 보여주었으며, 교미하여 산란한 경우, 암수의 비율은 8.0 : 2.0로 암컷의 비율이 높았다. T. remus는 중복기생자 (gregarious egg parasitoid)가 아닌 단독기생자 (solitary egg parasitoid)로 판단된다. 본 연구에서 보고한 T. remus의 생물적 특성에 관한 연구 결과는 실험실 조건에서 대량생산을 위한 정보로 활용하거나 생물적방제 프로그램을 개발할 때 활용될 수 있을 것이다.

Promising entomopathogenic fungal isolates for biological control of Spodoptera frugiperda

In Soo Jeon,So Eun Park,Ye Ram Im,Yu Lim Park,Yu Jin Jeong,Ki Jung Kim,Sue Yeon Lee,Jae Su Kim 한국농약과학회 2021 한국농약과학회 학술발표대회 논문집 Vol.2021 No.11

Spodoptera frugiperda is an invasive polyphagous pest derived from America. It has annually invaded to Korea at summer season since it was firstly reported in 2019. To minimize the damages by S. frugiperda, appropriate control methods should be studied and developed. This study aimed to assess the potential ability of entomopathogenic fungi as biological control agents against S. frugiperda. Ninety-three isolates of entomopathogenic fungi were selected to screen insecticidal activity against 2nd instar larvae of S. frugiperda. Among them, thirty-four isolates showed 100% mortality at 8 days after fungal treatments in laboratory condition. Conidial productivity and thermotolerance of conidia cultured on 1/4 SDA were evaluated to characterize the possibility for field application. Based on the characterization, about ten isolates were selected to determine the high virulence against S. frugiperda.

Preliminary study on the effect of using unmanned aerial vehicles (UAVs) to control Spodoptera frugiperda

Lu Hui,Li Fen,Zhu Xiaoming,Tang Jihong,Lyu Baoqian,Wu Shaoying 한국곤충학회 2021 Entomological Research Vol.51 No.9

Spraying insecticides by unmanned aerial vehicle (UAV) is an effective method to prevent and control Spodoptera frugiperda. This study investigated the influences of different UAV types, spray adjuvants, spray times, maize growth periods, and insecticides on S. frugiperda in three cities, Hainan Province, China. The results showed that at least two times are needed to spray insecticides using UAVs when controlling the S. frugiperda. Both UAVs had a substantially higher control effect and lower cost than artificial spray methods. Oil spray adjuvant Refei was more suitable for both UAVs than Tiancheng to control pests. Chlorantraniliprole had the best control effect of up to 90%. The control effects at the seedling stage and small bell stage of S. frugiperda were 94.3% and 91.3%, respectively. Spraying at 7:00 am and 7:00 pm by UAVof T20 had good control effect on S. frugiperda in three cities.

Evaluation of the expression stability of potential reference genes for RT-qPCR in Spodoptera frugipreda larvae exposed to camptothecin

Huang Yuting,Yu Xuanyue,Xie Xinyi,Liu Cuiting,Zhang Huijiao,Yuan Jiaxiang,Lin Jintian,Shu Benshui,Zhang Jingjing 한국응용곤충학회 2024 Journal of Asia-Pacific Entomology Vol.27 No.3

Camptothecin, a quinoline alkaloid, has strong action against Spodoptera frugiperda, a polyphagous pest found globally. Real-time quantitative PCR (RT-qPCR) is a popular and dependable technique for analyzing the mRNA expression of target genes. While RT-qPCR normalization requires reference genes with consistent expression. Many investigations have discovered that pesticides can change the expression patterns of reference genes. To date, the effects of CPT treatments on the expression stability of reference genes in S. frugiperda larvae are un clear. This study chose eight candidate reference genes, including alpha-tubulin (α-TUB), beta-1-tubulin (β-1-TUB), Actin, elongation factor 1 alpha (EF1α), elongation factor 2 (EF2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal protein L3 (RPL3), and ribosomal protein L13 (RPL13). Five approaches were used to inves tigate expression stability in S. frugiperda larval samples treated with CPT: ΔCt, BestKeeper, geNorm, Norm Finder, and RefFinder, respectively. Furthermore, the ideal number of reference genes was determined using GeNorm. Our findings revealed that two reference genes were sufficient to normalize RT-qPCR in samples treated with CPT. The recommended reference gene combinations for different samples are as follows: α-TUB and β-1-TUB for CPT-treated larval samples; β-1-TUB and RPL13 for samples of larval cuticle tissues; RPL3 and RPL13 for the larval fat body samples; EF1α and RPL3 for the larval malpighian tube samples; and EF2 and Actin for the larval midgut samples. Our results laid the groundwork for the mRNA expression analysis of target genes in S. frugiperda impacted by CPT exposure, contributing to the research of the molecular action mechanism of CPT in S. frugiperda.

Plant-derived metabolites as juvenile hormone disruptor in Spodoptera frugiperda larvae

Jun Hyoung Jeon,Seon-Ah Jeong,Sang-Woon Shin,Doo-Sang Park,Ji-ae Kim,Hong-Hyun Park,Hyun-Woo Oh 한국응용곤충학회 2023 한국응용곤충학회 학술대회논문집 Vol.2023 No.10

During the larval development process of insects, juvenile hormone (JH) is essential for regulating various aspects of larval life, including growth, reproduction, and behavior, throughout their larval stage. The larval stage of Spodoptera frugiperda, when it consumes plant-derived metabolites, develops into pupae, but these pupae are unable to molt successfully. In this way, plant-derived metabolites contain or produce inhibitors of juvenile hormone, thereby disrupting the development of insect larvae and making them vulnerable to harm. Therefore, in this study, we established an in vitro screening system using yeast cells transformed with the Met-SRC juvenile hormone receptor of S. frugiperda. Through this system, we were able to identify juvenile hormone disruptors from plant-derived metabolites and confirm their developmental inhibitory effects on the larvae of S. frugiperda.

miRNA-Targ et mRNA Interactions Reveal Novel Insights into Detoxification Mechanisms in Spodoptera frugiperda

Rashmi Manohar Mahalle,Barry R. Pittendrigh,Keon Mook Seong 한국응용곤충학회 2024 한국응용곤충학회 학술대회논문집 Vol.2024 No.04

Spodoptera frugiperda, commonly known as the fall armyworm (FAW), is a major pest across the globe due to its broad host range and distribution worldwide. We investigated the function of microRNAs (miRNAs) in the detoxification of insecticides, with a specific focus on its susceptibility to chlorantraniliprole which is widely utilized insecticide for its management. miRNAs are small non-coding RNA molecules, crucial for post-transcriptional regulation of gene expression. This study aims to elucidate the impact of these miRNAs on the expression of cytochrome P450 genes, which play a significant role in conferring insecticide resistance. We identified notable changes in the abundance of two specific miRNAs, sfr-miR-10465-5p and sfr-miR- 10476-5p through RNA sequencing, after chlorantraniliprole exposure. These miRNAs exhibited significantly high expression in the fat body tissue, while showing relatively lower expression in the head, midgut, and malpighian tubules. Further analysis suggested that these miRNAs might target specific cytochrome P450 genes, like CYP4C1 and CYP4C21, which are known to play a role in insecticide resistance development. Experimentation with miRNA mimics through microinjection revealed a notable increase in the survival rates of S. frugiperda larvae when subjected to chlorantraniliprole exposure, with a significant reduction in CYP4C1 and CYP4C21 gene expression levels. This suggests a direct connection between the miRNAs and the increased tolerance of Spodoptera larvae to the insecticide. Our research presents the complex function of miRNAs in gene expression regulation related to insecticide resistance, offering valuable insights into the molecular mechanisms of chlorantraniliprole resistance in S. frugiperda. These findings pave the way for further investigations into miRNA roles and their potential in managing pesticide resistance in agricultural pests.

Seed treatment with diamides provides protection against early and mid-stage larvae of the fall armyworm, Spodoptera frugiperda (Lepidoptera: Noctuidae), in maize

Kumar Behera Rakesh,Muralimohan K. 한국응용곤충학회 2024 Journal of Asia-Pacific Entomology Vol.27 No.1

The fall armyworm, Spodoptera frugiperda (J. E. Smith) is one of the major pests of maize worldwide. Farmers apply multiple rounds of pesticides within a single cropping season to control the fall armyworm. This leads to increased costs for plant protection and accelerates the selection pressure for evolution of resistance to various insecticide molecules in a short period. Hence, it is crucial to minimize the frequency of insecticide sprays on the crop. In this study we assessed the efficacy of seed treatment chemicals viz.- cyantraniliprole 19.8 % + thia methoxam 19.8 % FS, chlorantraniliprole 62.5 % FS, and thiamethoxam 70 % WS against neonates and third instar larvae of S. frugiperda. Lab bioassays using leaves from seed treated plants were conducted. Results showed that thiamethoxam 70 % WS was completely ineffective. It was thus presumed that only cyantraniliprole contributed to the observed larval mortality in the combination chemical tested here. Although both cyan traniliprole and chlorantraniliprole were effective for up to a month, the latter consistently recorded greater larval mortality. As seed treatment is easy to practice, poses lower risk and economical, we recommend this application method, especially using chlorantraniliprole 62.5 % FS, as a part of the integrated pest management strategy to combat the fall armyworm in maize.

In Vitro Transcription Analyses of Autographa californica Nuclear Polyhedrosis Virus Genes

Huh, Nam-Eung The Korean Society for Microbiology and Biotechnol 1994 Journal of microbiology and biotechnology Vol.4 No.3

Cell-free extracts prepared from cultured insect cells, Spodoptera. frugiperda, were analyzed for activation of early gene transcription of an insect baculovirus, Autographa californica nuclear polyhedrosis virus (AcNPV). The template DNA used for in vitro transcription assays contained promoter sites for the baculovirus genes that have been classified as immediate early ($\alpha$) or early genes. These genes are located in the HindIII-K/Q region of the AcNPV genome. Nuclei isolated from the AcNPV-infected Spodoptera frugiperda cells were also used for in vitro transcription analysis by RNase-mapping the labeled RNA synthesized from in vitro run-on reaction in the isolated nuclei. The genes studied by this technique were p26 and pl0 genes which were classified as delayed early and late gene, respectively. We found that transcription of the genes from the HindIII-K region was accurately initiated and unique in the whole cell extract obtained from uninfected cells, although abundance of the in vitro transcripts was reverse to that of in vivo RNA. With isolated nuclei transcription of the p26 gene was inhibited by $\alpha$-amanitin suggesting that the p26 gene was transcribed by host RNA polymerase II. However, transcription of the pl0 gene in isolated nuclei was not inhibited by $\alpha$-amanitin, but rather stimulated by the inhibitor. We also found that the synthesis of $\alpha$-amanitin-resistant RNA polymerase was begun before 6 hr p.i., the time point at which the onset of viral DNA replication as well as the appearance of a-amanitin-resistant viral transcripts were detected. These studies give us strong evidence to support the previous data that early genes of AcNPV were transcribed by host RNA polymerease III, while transcription of late genes was mediated at least by a novel $\alpha$-amanitin-resistant RNA polymerase.

열대거세미나방의 환경친화적 방제를 위한 성페로몬의 합성

최선희,손혜란,김재근 강원대학교 농업생명과학연구원 2022 강원 농업생명환경연구 Vol.34 No.3

In this study, using an economical method, we synthesized the (Z)-9-tetradecenyl acetate and (Z)-7-dodecenyl acetate components of the sex pheromone of Spodoptera frugiperda, a major pest of corn and rice crops. Given the expensive starting materials, the method previously developed for the synthesis of these chemicals is unsuitable for bulk production at industrial scales. Moreover, this method necessitates the use of the highly explosive reducing agent lithium aluminum hydride. To reduce production costs, as starting materials, we used the comparatively inexpensive chemicals 1,8-octanediol and 1,6-hexanediol. In addition, for conversion of triple bonds to Z-double bonds, we used Ni(OAc)2 instead of the more expensive Pt or Pd. The production cost of mass-synthesized products was approximately 50,000 won/g, which represents a considerable saving on the existing method, and can be distributed at a low price. As these products will be available to consumers at economical prices, this method will contribute to the environmentally friendly control of S. frugiperda.