Genome-enabled insights into the biology of thrips as crop pests

Abstract

Background: The western flower thrips, Frankliniella occidentalis (Pergande), is a globally invasive pest and plant virus vector on a wide array of food, fiber, and ornamental crops. The underlying genetic mechanisms of the processes governing thrips pest and vector biology, feeding behaviors, ecology, and insecticide resistance are largely unknown. To address this gap, we present the F. occidentalis draft genome assembly and official gene set.

Results: We report on the first genome sequence for any member of the insect order Thysanoptera. Benchmarking Universal Single-Copy Ortholog (BUSCO) assessments of the genome assembly (size = 415.8 Mb, scaffold N50 = 948.9 kb) revealed a relatively complete and well-annotated assembly in comparison to other insect genomes. The genome is unusually GC-rich (50%) compared to other insect genomes to date. The official gene set (OGS v1.0) contains 16,859 genes, of which ~ 10% were manually verified and corrected by our consortium. We focused on manual annotation, phylogenetic, and expression evidence analyses for gene sets centered on primary themes in the life histories and activities of plant-colonizing insects. Highlights include the following: (1) divergent clades and large expansions in genes associated with environmental sensing (chemosensory receptors) and detoxification (CYP4, CYP6, and CCE enzymes) of substances encountered in agricultural environments; (2) a comprehensive set of salivary gland genes supported by enriched expression; (3) apparent absence of members of the IMD innate immune defense pathway; and (4) developmental- and sex-specific expression analyses of genes associated with progression from larvae to adulthood through neometaboly, a distinct form of maturation differing from either incomplete or complete metamorphosis in the Insecta.

Conclusions: Analysis of the F. occidentalis genome offers insights into the polyphagous behavior of this insect pest that finds, colonizes, and survives on a widely diverse array of plants. The genomic resources presented here enable a more complete analysis of insect evolution and biology, providing a missing taxon for contemporary insect genomics-based analyses. Our study also offers a genomic benchmark for molecular and evolutionary investigations of other Thysanoptera species.

Keywords: Chemosensory receptors; Detoxification; Hemipteroid assemblage; Innate immunity; Insect genomics; Opsins; Salivary glands; Thysanoptera; Tospovirus; Western flower thrips.

Fig. 1

Illustration of how curated gene sets intertwine with understanding of biological processes of Frankliniella occidentalis. a Developmental stages. Vertical bars: (left) embryonic and postembryonic stages associated with developmental and sex-specific expression analyses of genes underlying molting and metamorphosis through neometaboly; (right) larval and adult stages feed and are associated with divergent clades and expansions in gene families related to host selection and feeding (vision, chemosensation) and detoxification of xenobiotics; propupal and pupal stages do not feed; adults reproduce by arrhenotokous parthenogenesis. Modified from [2], permission of CAB International through PLSclear. b Cartoon depicting principal and tubular salivary glands (PSG, TSG) associated with enriched expression of specific genes, and the midgut (MG), hindgut (HG), Malpighian tubules (MPT), and fat body (FB), important sites for detoxification and innate immunity gene sets along with the hemolymph and cuticle. Modified from [3], permission by Elsevier. c Scanning electron micrograph (SEM) of adult pre-probing behavior highlights compound eyes used in visual aspects of host finding (associated with opsin genes); external antennal and mouthcone sensory structures essential to host finding, choice, and feeding; likely associated with expanded gene families underlying chemosensation. Internal leaf anatomy shows cells most commonly fed on. Modified from [4], permission from Springer-Verlag. d SEM showing the tips of the single mandible (Md) and paired maxillae (Mx) forming the feeding tube. Modified from [5], permission of Elsevier. e Mouthcone paraglossal sensory pegs (numbered, left paraglossa)—pegs 1–5, 7–10, are dual function (mechano- and chemosensory), peg 6 is mechanosensory; their location suggests importance in detecting plant surface microtopography and chemistry during host and feeding-site selection and association with divergent and expanded gene families related to environmental sensing. Modified from [5], permission of Elsevier

Fig. 2

Phylogeny and orthology of Frankliniella occidentalis with other arthropods, with genome and gene set completeness assessments. a The phylogenomic analysis was based on the aligned amino acid sequences of 1604 single-copy orthologs and placed F. occidentalis (shown in red) as basal to the hemipteran species Acyrthosiphon pisum and Cimex lectularius (shown in purple). All nodes have bootstrap support of 100% and the scale bar corresponds to substitutions per site. OrthoDB orthology delineation with the protein-coding genes from the F. occidentalis official gene set identify genes with orthologs in all or most of the representative insects and the outgroup species, Daphnia pulex, as well as those with more limited distributions or with no confidently identifiable arthropod orthologs. b Assessments using the 1066 arthropod Benchmarking Universal Single-Copy Orthologs (BUSCOs) show few missing genes (5 for the assembly, 4 for the OGS) from F. occidentalis, with better OGS completeness than A. pisum, C. lectularius, and P. humanus. The F. occidentalis official gene set (OGS) scores better than its genome assembly, indicating that the gene annotation strategy has successfully managed to capture even difficult to annotate genes. The left bars for each species, also outlined with a dashed line, show the results based on the genome, whereas the right bars show the results for the OGSs. Species names abbreviations: Dmela—Drosophila melanogaster, Dplex—Danaus plexippus, Tcast—Tribolium castaneum, Amell—Apis mellifera, Phuma—Pediculus humanus, Apisu—Acyrthosiphon pisum, Clect—Cimex lectularius, Focci—Frankliniella occidentalis, Dpule—Daphnia pulex

Fig. 3

Distribution of transcription factor families across insect genomes and stage-specific expression in Frankliniella occidentalis. a Heatmap depicting the abundance of transcription factor (TF) families across a collection of insect genomes. Each entry indicates the number of TF genes for the given family in the given genome, based on presence of DNA-binding domains (DBD). Color key is depicted at the top (light blue means the TF family is completely absent)—note log (base 2) scale. Species were hierarchically clustered using average linkage clustering. F. occidentalis is boxed. See Additional file 2: Table S5 for TF genes with predicted DBDs. b Expression of specific TFs enriched within each developmental stage (larvae, propupae, and adult) based on data presented in Additional file 3. Sample designations: L1 = first-instar larvae, P1 = propupae, and A1 = adults (mixed males and females) of healthy cohorts (H) from three biological replicates (0, 1, 2)

Fig. 4

Genes/contigs with enriched expression in the salivary glands of Frankliniella occidentalis. RNA-seq reads generated from male and female principal and tubular salivary glands collectively [151] and whole bodies (this study) were used for the enrichment analysis. a Venn diagram depicting the overlap in transcript sequences enriched in the salivary glands of males, females, and combined sexes compared respectively to whole bodies. b Percent reads from salivary glands and whole-body RNA-seq datasets mapped to the putative 123 salivary gland-associated sequences. c Number of reads from the female salivary gland RNA-seq dataset mapping to each of the 123 salivary gland-associated sequences. d Reads mapped by fold change for 14 sequences with the highest number of mapped reads denoted in panel c. “Thrips-specific unknown protein” signifies hypothetical proteins with no match to proteins in other organisms and “unknown” indicates uncharacterized proteins in other arthropods. Details of expression and potential functions are denoted in Additional file 2: Table S11 (Excerpt D). e Specific sequences with functional assignments suggesting they are enzymatic, and based on comparison with other insects systems, could be involved in plant feeding and digestion. Details of expression and potential functions are denoted in Additional file 2: Table S11 (Excerpt E)

Fig. 5

Unique and shared innate immunity-associated transcripts in three thrips vector species of orthotospoviruses. Whole-body, assembled transcriptomes obtained from published orthotospovirus-thrips RNA-seq studies [–26] were mined for putative innate immune transcripts using an innate immune-associated protein database derived from ImmunoDb (http://cegg.unige.ch/Insecta/immunodb). a Venn diagram depicting overlap in orthologous clusters (bold) and transcripts (in parentheses) of innate immune-associated protein sequences in Frankliniella occidentalis (tomato spotted with virus), F. fusca (tomato spotted wilt virus), and Thrips palmi (capsicum chlorosis virus) using Orthovenn.v2. b Number of transcripts classified into innate immune categories (roles) and shared across all three vector species. Sequences may fall into more than one category. See Additional file 2: Table S17 and S18, respectively, for innate immune genes and transcript sets; Additional file 8 for Orthovenn outputs

Fig. 6

Identification of co-expressed genes (modules) and gene ontologies associated with three developmental stages of Frankliniella occidentalis. a Association between modules of co-expressed genes (colored boxes stacked on left of figure) and developmental stage, depicting the gene correlation network. Weighted gene co-expression network analysis [43] was performed on a matrix of normalized read counts (FPKM values) obtained from a published F. occidentalis RNA-seq study involving three biological replicates of healthy first-instar larvae, propupae, and adults (mixed males and females) [24]. Modules of co-expressed genes were determined by the dynamic tree cutting algorithm with a minimum of 20 genes per module. Modules that exhibited the highest correlation (red color) with a developmental stage are indicated by an asterisk (*). Transcript IDs of co-expressed genes within these significant stage-associated modules are presented in Additional file 3. b–d REVIGO (REduce and Visualize Gene Ontologies, [255]) was used to visualize specific GO terms comprised of non-redundant sequences enriched in each developmental stage; sizes of delineated blocks indicate the number of genes within each GO category. Refer to Additional file 3 for more detailed REVIGO maps with identities of each GO term (block) indicated

Fig. 7

Conserved sex-specific gene expression in thrips. Genome-assembled transcripts derived from RNA-seq reads for females, males, and pre-adults (larval and pupal combined) of Frankliniella occidentalis (this study, PRJNA203209) were compared to transcripts generated de novo from publicly available RNA-seq data sets for Frankliniella cephalica (PRJNA219559), Gynaikothrips ficorum (PRJNA219563), and Thrips palmi (PRJNA219609). Venn diagrams depict the number of transcript sequences associated with a females, b males, and c pre-adult stages of thrips. Highly enriched sequences (> 1000 unique reads and > 4-fold difference) conserved in d female and e male thrips. Open circles in d and e represent highly enriched gene ontology (GO) terms; especially notable genes are labeled. See methods for enrichment criteria and Additional file 9 for sex-specific genes sets and associated normalized (TPM = transcripts per million) fold change values (relative to the other sex)