Initial detections and spread of invasive Spodoptera frugiperda in China and comparisons with other noctuid larvae in cornfields using molecular techniques

The fall armyworm, Spodoptera frugiperda, is a species native to the Americas and has spread to many countries in Africa and Asia in recent years. Proactive actions for potential invasion of S. frugiperda to China coordinated by government agencies and agricultural extension systems resulted in timely detection in January 2019 in Yunnan province neighboring onto Myanmar. The extensive monitoring in southern provinces of China since February 2019 resulted in dynamic tracking of S. frugiperda spreading to 13 provincial regions in China within 4 months by May 10, 2019, which is crucial for timely management actions in the fields. The first detections of S. frugiperda (corn strain) in China were confirmed using cytochrome oxidase subunit 1 (CO1) and triosephosphate isomerase (Tpi) genes molecular marker method. In addition to S. frugiperda, larvae of three other noctuid species with similar morphological appearance (S. litura, S. exigua and Mythimna separata) can occur simultaneously and cause similar damage in cornfields in southern China. Thus, we can use both morphological and molecular marker methods to compare larval stages of four noctuid species. Further, we discuss the risk of potential spread of invasive S. frugiperda to other regions and impact on corn production in China.     

Functional characterization of sex pheromone receptors in Spodoptera frugiperda,S. exigua,and S. litura moths

Moths possess an extremely sensitive and diverse sex pheromone processing system, in which pheromone receptors (PRs) are essential to ensure communication between mating partners. Functional properties of some PRs are conserved among species, which is important for reproduction. However, functional differentiation has occurred in some homologous PR genes, which may drive species divergence. Here, using genome analysis, 17 PR genes were identified from Spodoptera frugiperda, S. exigua, and S. litura, which belong to 6 homologous groups (odorant receptor [OR]6, 11, 13, 16, 56, and 62); of which 6 PR genes (OR6, OR11, OR13, OR16, OR56, and OR62) were identified in S. frugiperda and S. exigua, and 5 PR genes were identified in S. litura, excluding OR62. Using heterologous expression in Xenopus oocytes, we characterized the functions of PR orthologs including OR6, OR56, and OR62, which have not been clarified in previous studies. OR6 orthologs were specifically tuned to (Z,E)-9,12-tetradecadienyl acetate (Z9,E12-14:OAc), and OR62 orthologs were robustly tuned to Z7-12:OAc in S. frugiperda and S. exigua. The optimal ligand for OR56 was Z7-12:OAc in S. frugiperda, but responses were minimal in S. exigua and S. litura. In addition, SfruOR6 was male antennae-specific, whereas SfruOR56 and SfruOR62 were male antennae-biased. Our study further clarified the functional properties of PRs in 3 Spodoptera moth species, providing a comprehensive understanding of the mechanisms of intraspecific communication and interspecific isolation in Spodoptera.     

利用mtCOI PCR-RFLP技术鉴别草地贪夜蛾与其他3种形态相近昆虫

【目的】田间调查发现草地贪夜蛾与甜菜夜蛾、斜纹夜蛾、粘虫常混合发生,传统的形态学鉴定方法不能快速鉴别出该虫,当前亟需快速鉴别该虫的方法。【方法】本研究分析了草地贪夜蛾与甜菜夜蛾、斜纹夜蛾、粘虫mtCOI基因序列的酶切位点,根据目的片段设计上游引物并进行PCR-RFLP验证。【结果】草地贪夜蛾个体在mtCOI片段的556~561 bp处均存在Sbf I内切酶酶切位点,斜纹夜蛾、甜菜夜蛾、粘虫均无Sbf I酶切位点。草地贪夜蛾PCR产物经过Sbf I内切酶酶切,可出现420 bp左右的特征带,斜纹夜蛾、甜菜夜蛾、粘虫种群均不能被Sbf I内切酶酶切。【结论】基于新设计引物扩增的mtCOI片段的PCR-RFLP方法可有效鉴别草地贪夜蛾与其他3个形态相近昆虫,研究结果为草地贪夜蛾的快速鉴别提供了方法。     

Characterization of Bacillus thuringiensis Cry toxin binding novel GPI anchored aminopeptidase from fat body of the moth Spodoptera litura

Aminopeptidase N (APN) isoforms were identified as candidate receptors for Bacillus thuringiensis Cry toxins from the midgut of several insect species. In this study a partial cDNA encoding aminopeptidase (slfbAPN) was cloned from fat body of the moth Spodoptera litura. In the deduced amino acid sequence the characteristic metallopeptidase sequences, HEXXHX₁₈E and GAMENWG were conserved but the sequence showed only 33–39% identity to other insect APNs, which were also reported to be Cry toxin receptors. The presence of a putative GPI anchor signal sequence at the C-terminus indicated that it is a membrane-anchored protein. The slfbAPN expression was restricted to the fat body as suggested by northern blot analysis of different tissues. Biochemical analyses including immunoblotting, ligand blotting and lectin blotting, demonstrated that slfbAPN is a membrane-anchored glycoprotein in the fat body and it binds to Cry toxins. The nucleotide sequence shown here has been submitted to the GenBank sequence data bank and is available under accession number EF372603.     

Noctuid migration in Texas within the nocturnal aeroecological boundary layer

Long-distance migration of adult corn earworm moths (Helicoverpazea), and several other noctuid moth species, facilitates seasonalexpansion of pest populations and consequent increased infestationsof agricultural crops on a continental scale in North America.Long-term field studies of population dynamics and migratoryflights of H. zea and fall armyworm (Spodoptera frugiperda)in the United States were evaluated using X-band radar observationsand profiles of atmospheric conditions. These studies identifiedcharacteristic patterns of migratory flight that are largelyassociated with vertical profiles of temperature and wind speed.Collective patterns of moth migrations were generally highlycorrelated with wind headings, but often at a significant angulardeviation. Preliminary analyses are presented between moth distributionsin the aerosphere estimated from discrete moth counts usingX-band radar and bulk reflectivity data from NEXRAD Dopplerradar. Identification of associations between atmospheric factorsand noctuid population dynamics and migratory flights will improvethe ability to predict infestations by pest species throughouttheir broad seasonal range expansion.     

Ectoparasitic Acugutturid Nematodes of Adult Lepidoptera

Noctuidonema guyaneme is an interesting ectoparasite of adult Lepidoptera that feeds on hosts from at least five families with its long stylet. Noctuidonema guyanense spends its entire life on the adult moth and is sustained as it is passed from moth to moth during host mating. Overlapping host generations are essential for parasite survival. This nematode occurs throughout tropical and subtropical America and is transported by at least one of its hosts, Spodoptera frugiperda, during migration to northern sites in the United States each spring. Noctuidonema guyanense debilitates its hosts. Research conducted to help determine the biological control importance of this nematode is reviewed. Two additional species, N. daptria and N. dibolia, are now known for Noctuidonema.     

Establishment and characterization of insect cell lines from 10 lepidopteran species

Summary Cell lines from selected lepidopteran species were established for the overall purpose of use in baculovirus production. A total of 36 new cell lines from 10 lepidopteran species were generated, including cell lines from a pyralid, the European corn borer,Ostrinia nubilalis, a plutellid, the diamondback moth,Plutella xylostella, as well as eight noctuids: the black cutworm,Agrotis ipsilon, the celery looper,Anagrapha falcifera, the velvetbean caterpillar,Anticarsia gemmatalis, the corn earworm,Helicoverpa zea, the tobacco budworm,Heliothis virescens, the beet armyworm,Spodoptera exigua, the fall armyworm,Spodoptera frugiperda, and the cabbage looper,Trichoplusia ni. Tissues used for cell line establishment included fat bodies, ovaries, testes, or whole embryos/larvae/pupae. All the cell lines were subcultured numerous times, characterized by isoenzyme analysis and/or deoxyribonucleic acid amplification fingerprinting using polymerase chain reaction, and stored in liquid nitrogen. Many of the cell lines were adapted to grow in serum-free medium, with cell lines fromA. ipsilon andH. virescens being adapted to suspension culture, using shaker flasks. The potential use for these cell lines in baculovirus production is discussed. All programs and services of the U.S. Department of Agriculture are offered on a nondiscriminatory basis without regard to race, color, national origin, religion sex, age, marital status, or handicap.     

Starvation‐induced autophagy in Spodoptera frugiperda Sf9 ovarian cells

Autophagy controls insect development and can be targeted for pest control in agriculture. In the present study, starvation‐induced autophagy is investigated in the insect species Spodoptera frugiperda. Bioinformatics analysis and a search of the EST database (http://bioweb.ensam.inra.fr/spodobase) identifies a putative ATG8 gene of S. frugiperda. To generate a biomarker of autophagosome, the DNA sequence encoding the open reading frame of this gene is amplified and cloned into a pIEX‐4‐mCherry‐EGFP‐SfATG8 recombinant vector. Sf9 cells are then transfected with this expression vector and starved in phosphate‐buffered saline solution for 4 h to induce autophagy, which is examined by LysoTracker staining (Life Technologies, Grand Island, New York), western blotting and fluorescence microscopy. The results obtained show that starvation stimulates lipidation of SfATG8‐PE and the formation of autophagosomes, providing a foundation for further research with respect to autophagy in insects.     

草地贪夜蛾综合防控技术研究进展

草地贪夜蛾是世界重大入侵性害虫,我国于2019年初发现草地贪夜蛾大面积危害西南地区玉米、高粱、甘蔗等多种作物,随后迅速扩散至主要玉米产区,严重威胁和影响我国粮食安全。国内研究人员已开展了大量关于草地贪夜蛾监测预警与防控技术的研究,在其种群发生规律监测、迁飞路径研判、化学防治、生物防治、物理防治等方面取得了系列进展,基本明确了草地贪夜蛾在我国西南、江南江淮、北方各地区的发生与危害规律,建立了一套草地贪夜蛾综合防控技术体系,有效阻断了其大面积扩散危害。本文综述了2019—2022年间国内草地贪夜蛾防控技术的研究进展,以期为开发草地贪夜蛾综合防控技术提供参考。     

Development and encapsulation of the endoparasitoid,Microplitis croceipes (Hym.: Braconidae), in six candidate host species (Lep.)

Encapsulation and development of the endoparasitoid,Microplitis croceipes (Cresson), were studied in six atypical lepidopteran host species whose usual host isHelicoverpa zea (Boddie). The candidate hosts examined were: the fall armywormSpodoptera frugiperda (J. E. Smith); the beet armyworm,Spodoptera exigua (Hübner); the cabbage looper,Trichoplusia ni (Hübner); the greater wax moth,Galleria mellonella (L.); the Indian meal moth,Plodia interpunctella (Hübner); and the diamondback moth,Plutella xylostella (L.). BothS. exigua andT. ni were completely unsuitable forM. croceipes development due to the high rate of eggs that were encapsulated within three days after parasitism. Encapsulation inS. frugiperda included mainly parasitoid eggs and was first detected six days after parasitization at 25°C and two days at 30°C. Encapsulation inG. mellonella occurred only in the larval stage of the parasitoid. InP. interpunctella, parasitoid larvae reached the 3rd stadium, but none of them pupated. OnlyS. frugiperda andG. mellonella supported successful development ofM. croceipes from egg to adult. The percentage of parasitoids reaching the adult stage in these hosts was higher at 30°C than at 25°C (13% vs. 4% inS. frugiperda, and 21% vs. 3% inG. mellonella, respectively). However, these percentages were too low to substitute them as a more economical host for rearingM. croceipes. This biological information will be useful in additional laboratory studies directed toward reducing the rate of encapsulation (e.g., manipulation of host rearing temperature) to increase production ofM. croceipes on these hosts.     

Detecting host–parasitoid interactions in an invasive Lepidopteran using nested tagging DNA metabarcoding

Determining the host–parasitoid interactions and parasitism rates for invasive species entering novel environments is an important first step in assessing potential routes for biocontrol and integrated pest management. Conventional insect rearing techniques followed by taxonomic identification are widely used to obtain such data, but this can be time‐consuming and prone to biases. Here, we present a next‐generation sequencing approach for use in ecological studies which allows for individual‐level metadata tracking of large numbers of invertebrate samples through the use of hierarchically organised molecular identification tags. We demonstrate its utility using a sample data set examining both species identity and levels of parasitism in late larval stages of the oak processionary moth (Thaumetopoea processionea—Linn. 1758), an invasive species recently established in the United Kingdom. Overall, we find that there are two main species exploiting the late larval stages of oak processionary moth in the United Kingdom with the main parasitoid (Carcelia iliaca—Ratzeburg, 1840) parasitising 45.7% of caterpillars, while a rare secondary parasitoid (Compsilura concinnata—Meigen, 1824) was also detected in 0.4% of caterpillars. Using this approach on all life stages of the oak processionary moth may demonstrate additional parasitoid diversity. We discuss the wider potential of nested tagging DNA metabarcoding for constructing large, highly resolved species interaction networks.     

Fungal endophytes of native grasses decrease insect herbivore preference and performance

Endophytic fungal symbionts of grasses are well known for their protective benefit of herbivory reduction. However, the majority of studies on endophyte–grass symbioses have been conducted on economically important, agricultural species—particularly tall fescue (Lolium arundinaceum) and perennial ryegrass (Lolium perenne)—raising the hypothesis that strong benefits are the product of artificial selection. We examined whether fungal endophytes found in natural populations of native grass species deterred insect herbivores. By testing several native grass–endophyte symbiota, we examined phylogenetic signals in the effects of endophytes on insects and compared the relative importance of herbivore and symbiotum identity in the outcome of the interactions. Preference was assessed using three herbivore species [Spodoptera frugiperda (Lepidoptera), Schistocerca americana (Orthoptera), Rhopalosiphum padi (Hemiptera)] and ten native symbiota, which spanned seven grass genera. We also assessed herbivore performance in a no choice experiment for five native symbiota against S. frugiperda. We compared greenhouse and laboratory trials with natural levels of herbivory measured in experimental field populations. In all cases, we included the agronomic grass species, L. arundinaceum, to compare with results from the native grasses. Both in the field and in experimental trials, herbivores showed a significant preference for endophyte-free plant material for the majority of native grasses, with up to three times lower herbivory for endophyte-symbiotic plants; however, the degree of response depended on the identity of the herbivore species. Endophyte presence also significantly reduced performance of S. frugiperda for the majority of grass species. In contrast, the endophyte in L. arundinaceum had few significant anti-herbivore effects, except for a reduction in herbivory at one of two field sites. Our results demonstrate that the mechanisms by which native symbionts deter herbivores are at least as potent as those in model agricultural systems, despite the absence of artificial selection.     

Human Pancreatic Procolipase Expressed in Insect Cells: Purification and Characterization

A cDNA clone encoding human pancreatic procolipase was incorporated into a recombinant baculovirus. Spodoptera frugiperda insect cells infected with the recombinant baculovirus secreted procolipase into the medium, which could be isolated in a single step by immunoaffinity chromatography. The highly purified protein reactivated human pancreatic lipase in a concentration-dependent fashion and was efficiently converted to colipase by limited trypsin digestion. This expression system is suitable for producing amounts of procolipase adequate for biophysical studies.     

High risk of the fall armyworm invading Japan and the Korean Peninsula via overseas migration

The fall armyworm, Spodoptera frugiperda (J.E. Smith), is an invasive pest species in the Old World. This species is originally native to the Americas. Since 2016, S. frugiperda has spread widely and rapidly throughout Africa, the Middle East, India, Southeast Asia and, most recently, to southern China. By May 2019, S. frugiperda had appeared in 13 provinces in southern China and will spread further into northern China. It is highly likely that S. frugiperda will enter Japan and Korea via overseas migrations as have many other migratory pests. To evaluate the risk of S. frugiperda invasion in Japan and the Korean Peninsula, we modelled the rate of expansion and future potential migratory range of the insect by a trajectory analytical approach using the flight behaviour of S. frugiperda and meteorological data of the past five years (2014–2018). If S. frugiperda can fly for up to 36 continuous hours over water, then our results predict migration from southern and eastern China into Japan and Korea. Most likely, Japan would be invaded from Fujian and Zhejiang in 1 June–15 July, and Kyushu, Shikoku and southwestern Honshu could face the highest risk of S. frugiperda invasion. Korea would most likely be reached by S. frugiperda from northern Zhejiang, Jiangsu, Anhui and Shandong in 1 June–15 July and later. Our results indicated a very high risk that S. frugiperda would annually invade Japan and the Korean Peninsula and cause a possible significant decrease in agricultural productivity.     

Characterization of two groups of Spodoptera exigua Hübner (Lepidoptera: Noctuidae) C‐type lectins and insights into their role in defense against the densovirus JcDV

Insect innate immunity relies on numerous soluble and membrane‐bound receptors, named pattern recognition proteins (PRPs), which enable the insect to recognize pathogen‐associated molecular patterns. C‐type lectins are among the best‐studied PRPs and constitute the most diverse family of animal lectins. Here we have characterized two groups of Spodoptera exigua C‐type lectins that differ in their phylogeny, domain architecture, and expression pattern. One group includes C‐type lectins with similar characteristics to other lepidopteran lectins, and a second group includes bracoviral‐related lectins (bracovirus‐like lectins, Se‐BLLs) recently acquired by horizontal gene transfer. Subsequently, we have investigated the potential role of some selected lectins in the susceptibility to Junonia coenia densovirus (JcDV). For this purpose, three of the bracoviral‐related lectins were expressed, purified, and their effect on the densovirus infection to two different Spodoptera species was assessed. The results showed that Se‐BLL3 specifically reduce the mortality of Spodoptera frugiperda larvae caused by JcDV. In contrast, no such effect was observed with S. exigua larvae. In a previous work, we have also shown that Se‐BLL2 increased the tolerance of S. exigua larvae to baculovirus infection. Taken together, these results confirm the implication of two different C‐type lectins in antiviral response and reflect the biological relevance of the acquisition of bracoviral genes in Spodoptera spp.     

Inga laurina trypsin inhibitor (ILTI) obstructs Spodoptera frugiperda trypsins expressed during adaptive mechanisms against plant protease inhibitors

Plant protease inhibitors (PIs) are elements of a common plant defense mechanism induced in response to herbivores. The fall armyworm, Spodoptera frugiperda, a highly polyphagous lepidopteran pest, responds to various PIs in its diet by expressing genes encoding trypsins. This raises the question of whether the PI‐induced trypsins are also inhibited by other PIs, which we posed as the hypothesis that Inga laurina trypsin inhibitor (ILTI) inhibits PI‐induced trypsins in S. frugiperda. In the process of testing our hypothesis, we compared its properties with those of selected PIs, soybean Kunitz trypsin inhibitor (SKTI), Inga vera trypsin inhibitor (IVTI), Adenanthera pavonina trypsin inhibitor (ApTI), and Entada acaciifolia trypsin inhibitor (EATI). We report that ILTI is more effective in inhibiting the induced S. frugiperda trypsins than SKTI and the other PIs, which supports our hypothesis. ILTI may be more appropriate than SKTI for studies regarding adaptive mechanisms to dietary PIs.     

Nucleotide sequence and molecular characterization of the structural glycoprotein gp41 gene homologue of <Emphasis Type="Italic">Spodoptera litura</Emphasis> nucleopolyhedrosis virus (SpltNPV-I)

The structural glycoprotein gene gp41 homologue of Spodoptera litura nucleopolyhedrosis virus (SpltNPV-I *) was identified in the 4.0 kb EcoRI-L fragment of the viral genome. The nucleotide sequence of 2063 bp of this fragment revealed an open reading frame of 1014 nucleotides to encode a polypeptide of 337 amino acids. Analysis of nucleotide and deduced amino acid sequences of the putative ORF indicated its identity with gp41 protein of other baculoviruses sharing maximum homology with that of Spodoptera frugiperda nucleopolyhedrosis virus (SfNPV). The coding sequence was preceded by an AT-rich region containing the consensus baculoviral late promoter motif RTAAG. The putative SpltNPV gp41 ORF was abundantly expressed as a 37 kDa apoprotein in E. coli and as a 50 kDa glycoprotein in Sf9 cells. The recombinant protein expressed in insect cells was glycosylated (20%) and has GlcNAc as the terminal sugar. The gene is conserved among baculoviruses and places SpltNPV-I close to Spodoptera frugiperda and Spodoptera exigua NPVs in phylogenetic tree.Assigned GenBank accession no. for the nucleotide sequence data is AF445192.abbreviated as SlNPV in earlier publications and GenBank     

Toxicity of a <Emphasis Type="Italic">Bacillus thuringiensis israelensis</Emphasis>-like strain against <Emphasis Type="Italic">Spodoptera frugiperda</Emphasis>

Bacillus thuringiensis (Bt) Berliner is a promising agent for microbial control of agriculturally and medically important insects. This study aimed at searching for Bt strains encoding Cry proteins that act more efficiently against fall armyworm. Thirty Bt strains were isolated from soil samples in Pernambuco State and evaluated through bioassays. Among these, strain I4A7 was the most efficient against the fall armyworm, Spodoptera frugiperda (J. E. Smith, 1797) (Lepidoptera: Noctuidae), and thus it was characterized by biochemical sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and molecular (polymerase chain reaction (PCR) and sequencing reaction) methods. The protein pattern of this strain on a SDS–PAGE was similar to that of B. thuringiensis israelensis (Bti). Moreover, I4A7 cry DNA sequence showed high identity (99–100%) to genes cry4Aa, 4Ba, 10Aa, 11Aa, cyt1Aa and cyt2B from Bti. The toxicity of the newly isolated Bti-like strain upon S. frugiperda should be considered as this strain might be used in combination with other Bt strains, such as B. thuringiensis var. kurstaki (Btk). Handling Editor: Helen Roy.