Horizontally acquired antibacterial genes associated with adaptive radiation of ladybird beetles

Abstract

Background: Horizontal gene transfer (HGT) has been documented in many herbivorous insects, conferring the ability to digest plant material and promoting their remarkable ecological diversification. Previous reports suggest HGT of antibacterial enzymes may have contributed to the insect immune response and limit bacterial growth. Carnivorous insects also display many evolutionary successful lineages, but in contrast to the plant feeders, the potential role of HGTs has been less well-studied.

Results: Using genomic and transcriptomic data from 38 species of ladybird beetles, we identified a set of bacterial cell wall hydrolase (cwh) genes acquired by this group of beetles. Infection with Bacillus subtilis led to upregulated expression of these ladybird cwh genes, and their recombinantly produced proteins limited bacterial proliferation. Moreover, RNAi-mediated cwh knockdown led to downregulation of other antibacterial genes, indicating a role in antibacterial immune defense. cwh genes are rare in eukaryotes, but have been maintained in all tested Coccinellinae species, suggesting that this putative immune-related HGT event played a role in the evolution of this speciose subfamily of predominant predatory ladybirds.

Conclusion: Our work demonstrates that, in a manner analogous to HGT-facilitated plant feeding, enhanced immunity through HGT might have played a key role in the prey adaptation and niche expansion that promoted the diversification of carnivorous beetle lineages. We believe that this represents the first example of immune-related HGT in carnivorous insects with an association with a subsequent successful species radiation.

Keywords: Antibacterial activity; Cell wall hydrolase; Horizontal gene transfer; Ladybirds.

Fig. 1

Genomic features of putative eukaryotic cwh genes. Genomes of four ladybird species and high-quality genomes of four other eukaryotic species were studied

Fig. 2

Evidence of the antibacterial activity of ladybird cwh genes I: Upregulation of cwh in response to bacterial infection. The expression patterns of cwh genes in response to bacterial infection (E. coli and B. subtilis) were tested in a Cryptolaemus montrouzieri. (CM-C1a, CM-C1b and CM-C2), b Harmonia axyridis (HA-C1a, HA-C1b and HA-C2), c Propylea japonica (PJ-C1a, PJ-C1b and PJ-C2), and d Henosepilachna vigintioctopunctata (HV-C1 and HV-C2). PBS treatments were used as control. Expression levels of cwh were normalized to those of two reference genes (only one of them is shown; see details of two reference genes in Additional file 1: Table S2 and the comparative results in Additional file 1: Fig. S5). Relative expression of each cwh gene in comparison to the average level of that gene in PBS treatment was analyzed by the 2^−ΔΔCt method to calculate the fold changes. Error bars show ± standard errors with biological replicates. Bars with the same letter are not significantly different (p ≥ 0.05)

Fig. 3

Evidence of the antibacterial activity of ladybird cwh genes II: Recombinant protein activity that limited bacterial proliferation. a Western blot of the recombinant proteins of cwh1 suggested the success of cloning and recombinant expression. The bacterial killing activities of cwh1 proteins against b Escherichia coli and c Bacillus subtilis cells were measured by the increase in OD_{630 nm} within 90 min of each treatment. Error bars show ± standard errors with biological replicates. Bars with the same letter are not significantly different (p ≥ 0.05)

Fig. 4

Evidence of the antibacterial activity of ladybird cwh genes III: Downregulation of other antibacterial genes in cwh-RNAi strains. Gene expression levels of three cwh-RNAi strains of Cryptolaemus montrouzieri (RNAi of CM-C1a, CM-C1b, and CM-C2) were studied based on transcriptome sequencing data. Three biological replicates were set for each treatment, and GFP-treated individuals were used as control. Patterns of expression of immunity-related genes involving in recognition (PGRP and GNBP), signal transduction (Toll/IMD and JAK/STAT pathway), and defense execution (antimicrobial peptides (AMPs) and lysozymes) are shown. FPKM (fragments per kilobase of transcript per million fragments mapped) of PBS controls, log2(fold change) and -log10 adjust-p values (-log10(Q)) for the transcriptome comparisons of cwh-RNAi strains vs PBS control were showed. Only genes with average FPKM higher than 1 are shown

Fig. 5

Occurrence of putative cwh genes in edcysozoan genomes deposited in NCBI whole genome shotgun (WGS) assembly database. Taxon pictures are courtesy of PhyloPic (http://www.phylopic.org/)

Fig. 6

Evidence of horizontal gene transfer. The phylogenetic trees of representative cwh protein sequences were reconstructed in a maximum likelihood framework. The tree was rooted by the clade comprising mainly Firmicutes. Only node support values > 70% are shown. The NCBI protein sequence IDs and species names are color-coded according to the taxonomic group of the organisms

Fig. 7

Evolutionary history of ladybird cwh genes. The phylogenetic tree of cwh protein sequences of ladybirds was reconstructed in a maximum likelihood framework. The ladybird cwh genes are separated into two clades, cwh1 (suffix C1 or C1a or C1b) and cwh2 (suffix C2 or C2a or C2b)

Fig. 8

The consistent retention of cwh genes from the beginning of angiosperm dominance is considered relevant to the adaptive radiation of ladybirds. A phylogenetic study of ladybird Coccinellidae and Coleoptera outgroups based on 819 single-copy genes and 12 fossil calibration points revealed crown diversification of the ladybird subfamily Coccinellinae approximately 110 million years ago (MYA). Most of the Coccinellinae species contain both cwh1 (red disc) and cwh2 (blue disc). Details of the tree can be found in Additional file 1: Figs. S7 and S8