Citation: Junming Shi, Weiwei Li, Yunyu Wang, Quanyan Chen, Fei Deng. Meta-Transcriptome Profiling of Novel Invasive Pest Spodoptera frugiperda in Yunnan, China .VIROLOGICA SINICA, 2020, 35(2) : 240-244.  http://dx.doi.org/10.1007/s12250-019-00188-z

Meta-Transcriptome Profiling of Novel Invasive Pest Spodoptera frugiperda in Yunnan, China

  • Corresponding author: Fei Deng, df@wh.iov.cn, ORCID: 0000-0002-5385-083X
  • Electronic supplementary material The online version of this article (https://doi.org/10.1007/s12250-019-00188-z) contains supplementary material, which is available to authorized users.
  • Received Date: 23 July 2019
    Accepted Date: 01 November 2019
    Published Date: 08 January 2020
    Available online: 01 April 2020
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    1. Bentivenha JPF, Rodrigues JG, Lima MF, Marcon P, Popham HJR, Omoto C (2019) Baseline Susceptibility of Spodoptera frugiperda (Lepidoptera: Noctuidae) to SfMNPV and evaluation of cross-resistance to major insecticides and bt proteins. J Econ Entomol 112:91–98
        doi: 10.1093/jee/toy342

    2. Buchfink B, Xie C, Huson DH (2015) Fast and sensitive protein alignment using DIAMOND. Nat Methods 12:59–60
        doi: 10.1038/nmeth.3176

    3. Carneiro TR, Fernandes OA (2012) Interspecific interaction between Telenomus remus (Hymenoptera: Platygastridae) and Trichogramma pretiosum (Hymenoptera: Trichogrammatidae) on Spodoptera frugiperda (Lepidoptera: Noctuidae) eggs. Anais Da Academia Brasileira De Ciencias 84:1127–1135
        doi: 10.1590/S0001-37652012000400027

    4. Cock MJW, Beseh PK, Buddie AG, Cafa G, Crozier J (2017) Molecular methods to detect Spodoptera frugiperda in Ghana, and implications for monitoring the spread of invasive species in developing countries. Sci Rep 7:4103
        doi: 10.1038/s41598-017-04238-y

    5. Cruz-Avalos AM, Bivián-Hernández MD, Ibarra JE, Del Rincón-Castro MC (2019) High virulence of Mexican entomopathogenic fungi againse fall armyworm, (Lepidoptera: Noctuidae). J Econ Entomol 112:99–107
        doi: 10.1093/jee/toy343

    6. Early R, González-Moreno P, Murphy ST, Day R (2018) Forecasting the global extent of invasion of the cereal pest Spodoptera frugiperda, the fall armyworm. NeoBiota 40:25–50
        doi: 10.3897/neobiota.40.28165

    7. Goergen G, Kumar PL, Sankung SB, Togola A, Tamo M (2016) First report of outbreaks of the fall armyworm Spodoptera frugiperda (J E Smith) (Lepidoptera, Noctuidae), a New Alien invasive pest in West and Central Africa. PLoS ONE 11:e0165632
        doi: 10.1371/journal.pone.0165632

    8. Guo JF, Zhao JZ, He K, Zhang F, Wang Z (2018) Watch out for the invasion of S. frugiperda to China. Plant Protect 44:5–14

    9. Harrison RD, Thierfelder C, Baudron F, Chinwada P, Midega C, Schaffner U, van den Berg J (2019) Agro-ecological options for fall armyworm (Spodoptera frugiperda JE Smith) management: providing low-cost, smallholder friendly solutions to an invasive pest. J Environ Manage 243:318–330
        doi: 10.1016/j.jenvman.2019.05.011

    10. Huson DH, Beier S, Flade I, Gorska A, El-Hadidi M, Mitra S, Ruscheweyh HJ, Tappu R (2016) MEGAN community edition—interactive exploration and analysis of large-scale microbiome sequencing data. PLoS Comput Biol 12:e1004957
        doi: 10.1371/journal.pcbi.1004957

    11. Juarez ML, Murua MG, Garcia MG, Ontivero M, Vera MT, Vilardi JC, Groot AT, Castagnaro AP, Gastaminza G, Willink E (2012) Host association of Spodoptera frugiperda (Lepidoptera: Noctuidae) corn and rice strains in Argentina, Brazil, and Paraguay. J Econ Entomol 105:573–582
        doi: 10.1603/EC11184

    12. Kakumani PK, Malhotra P, Mukherjee SK, Bhatnagar RK (2014) A draft genome assembly of the army worm, Spodoptera frugiperda. Genomics 104:134–143
        doi: 10.1016/j.ygeno.2014.06.005

    13. Kenis M, du Plessis H, Van den Berg J, Ba MN, Goergen G, Kwadjo KE, Baoua I, Tefera T, Buddie A, Cafa G, Offord L, Rwomushana I, Polaszek A (2019) Telenomus remus, a candidate parasitoid for the biological control of Spodoptera Frugiperda in Africa, is already Present on the Continent. Insects 10:92
        doi: 10.3390/insects10040092

    14. Li S-J, Huang J-P, Chang Y-Y, Quan S-Y, Yi W-T, Chen Z-S, Liu S-Q, Cheng X-W, Huang G-H (2015) Development of Microplitis similis (Hymenoptera: Braconidae) on two candidate host species, Spodoptera litura and Spodoptera exigua (Lepidoptera: Noctuidae). Fla Entomol 98:736–741
        doi: 10.1653/024.098.0250

    15. López MA, Martínez-Castillo AM, García-Gutiérrez C, Cortez-Mondaca E, Escobedo-Bonilla CM (2018) Parasitoids and entomopathogens associated with fall armyworm, Spodoptera frugiperda, in Northern Sinaloa. Southwestern Entomologist 43:867–882
        doi: 10.3958/059.043.0405

    16. Molina-Ochoa J, Carpenter JE, Heinrichs EA, Foster JE (2003) Parasitoids and parasites of Spodoptera frugiperda (Lepidoptera: Noctuidae) in the Americas and Caribbean Basin: an inventory. Fla Entomol 86:254–289
        doi: 10.1653/0015-4040(2003)086[0254:PAPOSF]2.0.CO;2

    17. Nagoshi RN (1938) The fall armyworm triose phosphate isomerase (Tpi) gene as a marker of strain identity and interstrain mating. Ann Entomol Soc Am 103:283–292
        doi: 10.1603/AN09046

    18. Ou-Yang YY, Zhao YP, Hopkins RJ, Chen XY, Huang GH, Wang X (2018) Parasitism of Two Spodoptera spp. by Microplitis prodeniae (Hymenoptera: Braconidae). J Econ Entomol 111:1131–1136
        doi: 10.1093/jee/toy085

    19. Zhang L, Jin MH, Zhang DD, Jinag YY, Liu J, Wu KM, Xiao YT (2019) Molecular identification of invasive fall armyworm Spodoptera frugiperda in Yunnan Province. Plant protection 45:19–24

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    Meta-Transcriptome Profiling of Novel Invasive Pest Spodoptera frugiperda in Yunnan, China

      Corresponding author: Fei Deng, df@wh.iov.cn
    • 1. State Key Laboratory of Virology and National Viruses Resource Center, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China
    • 2. University of Chinese Academy of Sciences, Beijing 100049, China
    • 3. Kunming Institute of Zoology, Chinese Academy Sciences, Kunming 650223, China
    • 4. Yunnan Agricultural University, Kunming 650201, China

    Abstract:  

    • Dear Editor,

      The fall armyworm, Spodoptera frugiperda (Lepidoptera: Noctuidae), a highly agricultural destructive pest of cere-als, is originally rampant in tropical and subtropical America (Kenis et al. 2019). The caterpillars of this moth feed on more than 350 plant species (Harrison et al. 2019), including several economically important cultivated grasses such as maize, rice, sorghum and sugarcane, severely threatening the food security (Cock et al. 2017). Spodoptera frugiperda is recognized to comprise two morphologically identical but genetically distinct strains with different host range. The 'rice strain'(R strain, RS) is thought to preferentially feed on rice and various pasture grasses, while the 'corn strain'(C strain, CS) on maize, cotton and sorghum (Juarez et al. 2012). Unable to tolerate freezing temperature, S. frugiperda is seldom reported outside of America. However, since its first detection in Nigeria in 2016, several congruent reports from African countries about sudden and severe upsurges of S. frugiperda raised the alarm that S. frugiperda is continuously expanding their range to become truly threat (Goergen et al. 2016). Notably, this pest is now spreading to Southeast Asia countries (including Myanmar and Thai-land) and China where the environmental requirements for this pest to establish itself presented (Early et al. 2018). Bordering with Myanmar, Yunnan Province of China is in great danger of S. frugiperda invasion while several spo-radic reports have documented the identification of S. frugiperda in Yunnan Province (https://www.ippc.int) (Guo et al. 2018; Zhang et al. 2019). Till now (May, 2019), S. frugiperda has been reported in nearly the whole south region of China where the diverse host plant sources and favorable climatic environment are provided (Kenis et al. 2019). Therefore, it's of great significance to monitor this new pest invasion and take preventive measures to mini-mize the agricultural damage of it.

      Here, we reported the meta-transcriptome profiling of S. frugiperda larva collected in Yunnan Province, China. Ten larvae of S. frugiperda were collected from four localities bordering with Myanmar in Dehong prefecture of Yunnan Province from Feb 27 to Mar 1, 2019. RNA was extracted from combined pool consisting of four whole larvae from each of the regions and subjected to RNA-seq using HiSeq 3000 sequencer. A summary of sequencing and assembly results were listed in Supplementary Table S1 and Fig. 1. Totally, ~30 G reads assembled into 531, 181 transcripts were obtained and submitted to the NCBI Sequence Read Archive (SRA) under accession number SRR9317345. As shown in Fig. 1A, most of the transcripts are less than 500 nt and the longest one reaches to 71, 803 nt. 86.3% of the reads can be mapped to the assembled transcripts with mean depth of 40 × suggesting a good quality of assembly and single base accuracy (Supple-mentary Table S1 and Fig. 1B). All the transcripts were annotated via homology-based search against the Genbank nr and trEMBL database via diamond blastx command (Buchfink et al. 2015), and taxonomy was based on the NCBI taxonomy database performed with MEGAN soft-ware (Huson et al. 2016). Nearly 38% transcripts can be annotated with a P value cutoff at 1e-5. Among the annotated sequences, bacteria is the most abundant organism with 76% reads assigned and the remaining are plant, insect, virus and fungus with 10%, 9.7%, 0.06% and 0.01% proportion respectively (Fig. 1C).

      Figure 1.  A Distribution of assembled transcripts with different length. B Distribution of sequencing depth. Bar graph representation of sequencing depth versus the percentage of transcripts having the respective depth. C A summary of transcripts annotation. The left pie shows the proportional distribution of transcripts with/without homologous reference in nr/trEMBL database. The right pie shows a detail distribution for taxon of bacteria, plant, insect, fungus, viruses and unassigned. D The top 8 taxons involving bacterias from matagenomic sequencing were presented. E Taxons involving plants from matagenomic sequencing were presented. F PCR confirmation of the detected viruses from RNA-Seq analysis in the sample. G Phylogenetic analysis of S. frugiperda. Maximum-Likelihood phylogenetic tree of S. frugiperda was constructed based on the COX I gene using Busseola spp. as outgroups. Sequences derived from RNA-seq were highlighted with a red solid circle. H Comparison of the portion of the S. frugiperda Tpi gene with Corn and Rice Strain. Strain-specific polymorphic sites were highlighted with red triangle.

      Lepidopera, as the largest order of insect including many biological and economically important species as well as some most destructive pests, genetic information of them is quite little to be known (Kakumani et al. 2014). And reference information of Lepidpopera mainly relies on Bombyx mori genome, while most of the genes of S. frugiperda remain to be annotated. So it is reasonable that 62% reads are unassigned and in 38% annotated sequences only 9.7% reads are insect-related among which 95% are S. frugiperda related. The remaining sequences mainly belong to taxons of Diptera and Hymenoptera, which contained the majority of over 150 reported parasitoids species of S. frugiperda (Molina-Ochoa et al. 2003; Loxpez et al. 2018), and their distribution and diversity varied with host geographical distribution (Molina-Ochoa et al. 2003).

      Fall armyworm is the prey of over 150 parasitoid and parasite species, and most of which belong to Hymenoptera and Diptera orders (Harrison et al. 2019). Here, to find potential effective biopesticide we investigated the basic natural parasitoids presented in S. frugiperda of China. We found the existence of sequences with 65% or so identity to Trichogramma pretiosum (Hymenoptera: Trichogram-matidae) and Cotesia marginiventris (Cresson) (Hymenoptera: Braconidae) which are commonly used in inundative release for S. frugiperda control (Carneiro and Fernandes 2012). Many other ubiquitous parasitoid wasps of Spodoptera spp. including Ceratosolen spp., Trichomalopsis spp., Copidosoma spp., Nasonia spp. in Chalcidoidea family and Glyptapanteles spp., Microplitis spp. in Braconidae family were detected in our pool (Li et al. 2015; Ou-Yang et al. 2018). Taken together, we detected possible parasitoids of S. frugiperda indicating the possible utilization of them instead of introduction of exotic parasitoids for S. frugiperda pest control. As a novel invasive pest, figuring out the parasitoids complex of S. frugiperda has important implications for the biological control in China. However, whether these species were native parasitoids of S. frugiperda or just mingled into our pool while sample collection remains further investigation.

      As for bacterium community, 2159 kinds of bacteria within 98 families were detected. The top 8 families accounting for 93% of the bacteria profile were showed in Fig. 1D. Bacteria belonging to Flavobacteriaceae and Pseudomonadaceae are significantly enriched, which are universal bacteria in plant and soil. Notably, among the bacterial sequences, 35 contigs were assigned to Bacillus thuringiensis (Bt) with 90% identity or so, and Bt is commonly used as biological insecticides to control fall armyworm (Loxpez et al. 2018). Except for bacteria, entomopathogenic fungi within the genera Beauveria and Metarhizum is also an important biopesticide (Cruz-Avalos et al. 2019). In our study, 33 kinds of fungi within 20 families were identified, among which 35 contigs were assigned to Beauveria with 60%–90% identity. This result may have implication for S. frugiperda management via Bt and Beauveria in China.

      We also detected a large quantity of sequences of plants involving 9 families (Fig. 1E). And sequences within Poaceaea, including rice, maize, sorghum and sugarcane, account for 90% of all the plant among which rice and maize were the most abundant. The diversity of the detected plants reflects the highly polyphagous character-istic of S. frugiperda. Such results provide reasonable warnings that the novel invasive pest is most likely jeop-ardize the production of maize and sugarcane in Yunnan.

      Finally, we investigate the viral community of S. frugiperda. Totally, five kinds of viruses were detected based on the homologous search involving alphabaculovirus, betabaculovirus, unclassified rhabdovirus and phage belonging to Siphoviridae (Table 1). One of the alphabaculovirus shares 99% identity with identified Autographa californica multiple nucleopolyhedrovirus (AcMNPV), which is the most-studied baculovirus. And another alphabaculovirus shared 40% identity with Clanis bilineata nucleopolyhedrovirus, which should be considered as a novel species. The betabaculovirusrelated contig shares 62% identity with strain Helicoverpa armigera granulovirus (Genbank NC_010240.1), suggesting a novel granulovirus carried by S. frugiperda. The rhabdovirus-related contig shares 92% identity with strain Spodoptera frugiperda endogenous virus rhabdovirus L-like EVE 1 also detected in S. frugiperda which remains unclassified. And a phage-related contig shares 87% identity with strain Serratia phage Eta. And the Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV), which is a valuable tool for S. frugiperda control, was absent in our viral commu-nity (Bentivenha et al. 2019). We confirmed the presence of these viral sequences in the same sample for RNA-seq analysis by PCR (Fig. 1F). Primers of each detected virus were designed based on the corresponding contigs (Sup-plementary Table S2). In conclusion, it seems no patho-genic virus to human or crops has been carried by S. frugiperda samples so far. However, whether these iden-tified viruses can serve as pathogen to fall armyworm remained to be studied.

      Contig No. Reference virus (mapped gene) Taxon Contig Length Identity with reference(aa level)
      TRINITY_DN128371_c8_g2_i1 Autographa californica nucleopolyhedrovirus strain E2 (hypothetical protein) Baculoviridae, Alphabaculovirus 428 99%
      TRINITY_DN128284_c3_g2_i1 Clanis bilineata nucleopolyhedrovirus (P87/VP80) Baculoviridae, Alphabaculovirus 824 40%
      TRINITY_DN128671_c0_g6_i1 Helicoverpa armigera granulovirus (hypothetical protein HaGV_gp053) Baculoviridae, Betabaculovirus 2345 62%
      TRINITY_DN153113_c0_g1_i1 Serratia phage Eta_0034 (hypothetical protein) Unclassified Siphoviridae 246 87%
      TRINITY_DN123344_c0_g1_i2 Spodoptera frugiperda rhabdovirus (L protein) Unclassified Rhabdoviridae 2344 92%

      Table 1.  List of BLASTX homology for contigs predicted as viruses corresponding to their closet reference.

      To clarify the species identity that invaded into China, sequence TRINITY_DN117122 (Genbank MN068212) annotated as mitochondrial cytochrome C oxidase subunit I (COI), which is commonly recognized as 'DNA barcode' for classification of S. frugiperda, was used for molecular analysis. Additionally, a 610 bp COI gene fragment was amplified by PCR (primers see Supplementary Table S2). Contig derived from RNA-Seq was identical to the PCR amplification results indicating the reliability of RNA-Seq. Combined with representative specimens of S. frugiperda and other Spodoptera spp., we performed Maximum-Likelihood phylogenetic analysis using Busseola spp. as outgroup (Fig. 1G). Obviously, two separated sub-cluster on S. frugiperda were formed, and our specimen clustered with the R strain. To futher confirm the identity of our fall armyworm sample, another nuclear marker single-nu-cleotide polymorphisms (SNPs) within the region of the fall armyworm Tpi gene between extron 3 and extron 4 were also used to distinguish strain type of S. frugiperda (Juarez et al. 2012). Contig TRINITY_DN102113 anno-tated as Tip gene and 310 bp fragments (Genbank MN066368) amplified (primers see Supplementary Table S2) were compared with referential R strain (Gen-bank GQ41 1913) and C strain (Genbank GQ41 1916). As shown in Fig. 1H, all the 17 sites of SNPs from our sample were identical to the corn-strain specific polymorphism. It seems that our sample of S. frugiperda belongs to COI-RS Tpi-C, which can be generated by matings between ricestrain females and corn-strain males (Nagoshi 1938). Because our sampling pool was a mixture of four fall armyworms from different localities, such discordance whether suggests the presence of both rice and corn strain or the only identity of COI-RS Tpi-C in China deserves further investigation.

      Summarily, this work documented the bio-community of the novel invasive pest S. frugiperda in China via meta-transcriptome, which presents a high biodiversity, espe-cially concerning bacterial community. And the presence of parasitoids and pathogens of the fall armyworm larvae in China suggests a great capacity to utilize them as biolog-ical control agents. Additionally, evidences of Corn strain and Rice strain were simultaneously detected via different molecular marker, suggesting a possible mixed species invasion or interstrain hybrids of COI-RS Tpi-C invasion.

    • This research was supported by the grant from the National Key R & D Program of China (2017YFD0200400).

    • The authors have declared no competing interests.

    • This article does not contain any studies with human or animal subjects performed by any of the authors.

    Figure (1)  Table (1) Reference (19) Relative (20)

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