Intracellular symbiont Symbiodolus is vertically transmitted and widespread across insect orders

Abstract

Insects engage in manifold interactions with bacteria that can shift along the parasitism-mutualism continuum. However, only a small number of bacterial taxa managed to successfully colonize a wide diversity of insects, by evolving mechanisms for host-cell entry, immune evasion, germline tropism, reproductive manipulation, and/or by providing benefits to the host that stabilize the symbiotic association. Here, we report on the discovery of an Enterobacterales endosymbiont (Symbiodolus, type species Symbiodolus clandestinus) that is widespread across at least six insect orders and occurs at high prevalence within host populations. Fluorescence in situ hybridization in several Coleopteran and one Dipteran species revealed Symbiodolus' intracellular presence in all host life stages and across tissues, with a high abundance in female ovaries, indicating transovarial vertical transmission. Symbiont genome sequencing across 16 host taxa revealed a high degree of functional conservation in the eroding and transposon-rich genomes. All sequenced Symbiodolus genomes encode for multiple secretion systems, alongside effectors and toxin-antitoxin systems, which likely facilitate host-cell entry and interactions with the host. However, Symbiodolus-infected insects show no obvious signs of disease, and biosynthetic pathways for several amino acids and cofactors encoded by the bacterial genomes suggest that the symbionts may also be able to provide benefits to the hosts. A lack of host-symbiont cospeciation provides evidence for occasional horizontal transmission, so Symbiodolus' success is likely based on a mixed transmission mode. Our findings uncover a hitherto undescribed and widespread insect endosymbiont that may present valuable opportunities to unravel the molecular underpinnings of symbiosis establishment and maintenance.

Keywords: genome erosion; insect symbiont; intracellular; mixed mode transmission; parasitism-mutualism continuum; secretion systems; transovarial; vertical transmission.

Figure 1

Phylogenetic reconstruction of Symbiodolus endosymbiont strains (ES) of various hosts with representative Gammaproteobacteria and an outgroup consisting of Betaproteobacteria based on aligned 16S rRNA gene sequences. The phylogeny was reconstructed using a maximum likelihood-based method using a "TPM3 + I + R4" model, and node labels indicate branch support as estimated by 10 000 ultrafast bootstraps optimized via additional NNI based on bootstrap alignments (only values above 70 are shown); all Symbiodolus formed a monophyletic clade with three subclades as highlighted, and taxa name colors specify host order as indicated on the left.

Figure 2

Relative and absolute abundance of Symbiodolus across different insect hosts; (A) bacterial community composition of various Curculionidae, S. fagi (Silvanidae), and P. hornii (Throscidae) beetles. Each bar depicts the relative abundance of bacterial ASVs within an individual beetle, identified at family level by DADA2 analysis of the 16S rRNA gene; Symbiodolus symbiont is highlighted in magenta, all other taxa are displayed in different shades of gray; (B) violin plot of the 16S rRNA gene copy number as a proxy for symbiont titer in adults of O. gallaeciana (left, N = 23) and O. melanopus (right, N = 20); black dots represent individual data points and horizontal bars represent the mean; beetle pictures from Wikimedia commons (U. Schmidt).

Figure 3

Localization of Symbiodolus in different host species and life stages; S. clandestinus was identified in O. gallaeciana (Chrysomelidae) eggs (A), larvae (B), adult females (C), and adult males (D), confirming the presence throughout all life stages via rRNA FISH; furthermore, the Symbiodolus symbiont was found in larvae of O. melanopus (Chrysomelidae) (E), adult males of P. hornii (Throscidae) (F), adult males of N. quadrimaculatus (Curculionidae) (G), larvae of C. riparius (Diptera, Chironomidae) (H), and female adults of C. riparius (I); Symbiodolus labeled in yellow were spread in all tissues (see arrowheads that highlight a few locations with Symbiodolus), most prominently in reproductive organs, and within these organs, it was co-localized with Wolbachia, labeled in magenta, in both Ouelma species as well as in N. quadrimaculatus; eubacterial staining is shown in red, host nuclei counterstaining in cyan, and autofluorescence in gray; used abbreviations are: embryo (em), gut lumen (gl), gut epithelium (ge), ovariole (ov), oocyte (oo), seminal vesicle (sv), and accessory gland (ag); bars = 50 μm.

Figure 4

Genome characteristics and selected metabolic capabilities of different Symbiodolus strains; the phylogenetic tree on the left is taken from Fig. 1; host columns give the order, family, genus, and species of insect hosts for each analyzed symbiont strain; symbiont (draft-) genome lengths are depicted by bars, scale is in Mbp, and the largest genome was found in the ES of C. riparius with ~1.66 Mbp, the smallest was the incomplete assembly of the ES of C. marinus with ~1.35 Mbp; numbers inside bars are the respective GC contents (in %); the heatmap gives predicted functionality of amino acids pathways, vitamin and cofactors pathways, and secretion system machineries based on genomic information, and dark green fields indicate predicted functionality, light red fields indicate absence or non-functionality, and empty fields are missing data; for C. riparius, no definitive statement could be made about the presence or absence of the T4SS, as these genes were often found on plasmids and the available data did not include plasmids; for P. maculicornis, the presence of the T4SSb could not be conclusively confirmed nor disproved.