A scaffold-level genome assembly of a minute pirate bug, Orius laevigatus (Hemiptera: Anthocoridae), and a comparative analysis of insecticide resistance-related gene families with hemipteran crop pests

Abstract

Background: Orius laevigatus, a minute pirate bug, is a highly effective beneficial predator of crop pests including aphids, spider mites and thrips in integrated pest management (IPM) programmes. No genomic information is currently available for O. laevigatus, as is the case for the majority of beneficial predators which feed on crop pests. In contrast, genomic information for crop pests is far more readily available. The lack of publicly available genomes for beneficial predators to date has limited our ability to perform comparative analyses of genes encoding potential insecticide resistance mechanisms between crop pests and their predators. These mechanisms include several gene/protein families including cytochrome P450s (P450s), ATP binding cassette transporters (ABCs), glutathione S-transferases (GSTs), UDP-glucosyltransferases (UGTs) and carboxyl/cholinesterases (CCEs).

Methods and findings: In this study, a high-quality scaffold level de novo genome assembly for O. laevigatus has been generated using a hybrid approach with PacBio long-read and Illumina short-read data. The final assembly achieved a scaffold N50 of 125,649 bp and a total genome size of 150.98 Mb. The genome assembly achieved a level of completeness of 93.6% using a set of 1658 core insect genes present as full-length genes. Genome annotation identified 15,102 protein-coding genes - 87% of which were assigned a putative function. Comparative analyses revealed gene expansions of sigma class GSTs and CYP3 P450s. Conversely the UGT gene family showed limited expansion. Differences were seen in the distributions of resistance-associated gene families at the subfamily level between O. laevigatus and some of its targeted crop pests. A target site mutation in ryanodine receptors (I4790M, PxRyR) which has strong links to diamide resistance in crop pests and had previously only been identified in lepidopteran species was found to also be present in hemipteran species, including O. laevigatus.

Conclusion and significance: This assembly is the first published genome for the Anthocoridae family and will serve as a useful resource for further research into target-site selectivity issues and potential resistance mechanisms in beneficial predators. Furthermore, the expansion of gene families often linked to insecticide resistance may be an indicator of the capacity of this predator to detoxify selective insecticides. These findings could be exploited by targeted pesticide screens and functional studies to increase effectiveness of IPM strategies, which aim to increase crop yields by sustainably, environmentally-friendly and effectively control pests without impacting beneficial predator populations.

Keywords: Beneficial predator; Comparative genomics; Crop pests; Hemiptera; Illumina; Insecticide resistance; Orius laevigatus; PacBio; Pirate bug; Whole genome sequencing.

Fig. 1

The assembly pipeline for the Orius laevigatus genome

Fig. 2

GenomeScope v2.0 profile plots of A: a transformed linear plot of k-mer frequency; and B: a transformed log plot of k-mer coverage at a k-mer length of 21 and a maximum k-mer coverage of 10,000

Fig. 3

The mitochondrial genome for Orius laevigatus, visualised using Geneious and annotation track obtained using MITOS2. The innermost graphs represent AT content shown in green, and GC content shown in blue

Fig. 4

Phylogeny and divergence of Insecta. Nodes are coloured by order, blue = Hemiptera, red = Lepidoptera, yellow = Diptera, purple = Hymenoptera, green = Coleoptera, orange = Thysanoptera, black = Chelicerata. Produced using the STAG tree inference method and full proteomes of the following species: C. lectularius: PRJNA167477, L. hesperus: PRJNA284294, R. prolixus: PRJNA13648, T. infestans: PRJNA589079, A. craccivora: PRJNA558689, A. pisum: PRJNA13657, B. tabaci: PRJNA312470, T. vaporariorum: PRJNA553773, D. citri: PRJNA2944, C. suppressalis: PRJNA506136, B. mori: PRJNA205630, D. melanogaster: PRJNA13812, A. mellifera: PRJNA471592, N. vitripennis: PRJNA575073, T. castaneum: PRJNA12540, F. occidentalis: PRJNA203209, T. palmi: PRJNA607431, T. urticae: PRJNA315122

Fig. 5

Phylogenetic tree of the Orius laevigatus glutathione S-transferases. Amino acid sequences were aligned using MAFFT and analysed using RAxML (the GAMMA LG protein model was used). The bootstrap consensus tree was inferred from 100 replicates. Coloured stars on branches indicate tandem duplications, with each colour representing a different scaffold/set of tandem duplications in the O. laevigatus assembly

Fig. 6

Phylogenetic tree of the Orius laevigatus cytochrome P450s. The Cyp397 gene family is a member of clan 3. Amino acid sequences were aligned using MAFFT and analysed using RAxML (the GAMMA LG protein model was used). The bootstrap consensus tree was inferred from 100 replicates

Fig. 7

An alignment of amino acid sequences to compare transmembrane domain 3 of the conserved ryanodine receptor (RyR) from Plutella xylostella with hemipteran species. The box indicates the I4790M RyR point mutation linked to diamide resistance (numbered according to PxRyR). A strain of P. xylostella without the point mutation was used for the alignment. RyR sequences were obtained from the UniProt database, excluding Orius laevigatus. UniProt entry names are as follows: Cimex lectularius: A0A7E4RNZ4_CIMLE, Triatoma infestans: A0A023F678_TRIIF, Lygus hesperus: A0A146M8X0_LYGHE, Dialeurodes citri: A0A141BN13_DIACT, Bemisia tabaci: A0A1U9JHP1_BEMTA, Toxoptera citricida: A0A0H3XSN1_TOXCI, Sipha flava: A0A2S2QG40_9HEMI, Plutella xylostella: I3NWV8_PLUXY, Myzus persicae: A0A0A7RS32_MYZPE, Acyrthosiphon pisum: X1WXB1_ACYPI, Aphis glycines: A0A6G0U418_APHGL