From eggs to guts: Symbiotic association of Sodalis nezarae sp. nov. with the Southern green shield bug Nezara viridula

Abstract

Phytophagous insects engage in symbiotic relationships with bacteria that contribute to digestion, nutrient supplementation, and development of the host. The analysis of shield bug microbiomes has been mainly focused on the gut intestinal tract predominantly colonized by Pantoea symbionts and other microbial community members in the gut or other organs have hardly been investigated. In this study, we reveal that the Southern green shield bug Nezara viridula harbours a Sodalis symbiont in several organs, with a notable prevalence in salivary glands, and anterior regions of the midgut. Removing external egg microbiota via sterilization profoundly impacted insect viability but did not disrupt the vertical transmission of Sodalis and Pantoea symbionts. Based on the dominance of Sodalis in testes, we deduce that N. viridula males could be involved in symbiont vertical transmission. Genomic analyses comparing Sodalis species revealed that Sodalis sp. Nvir shares characteristics with both free-living and obligate insect-associated Sodalis spp. Sodalis sp. Nvir also displays genome instability typical of endosymbiont lineages, which suggests ongoing speciation to an obligate endosymbiont. Together, our study reveals that shield bugs harbour unrecognized symbionts that might be paternally transmitted.

Keywords: Pantoea; Sodalis; egg microbiome; gut microbiota; insect-microbe symbiosis; symbionts.

Figure 1.

The effect of egg-surface sterilization on survival and gut microbial community of N. viridula. (a) Survival rate was monitored from hatching until reaching adulthood in nonsterilized (control) and egg-surface sterilized populations of N. viridula. Two egg clusters in a sterilized population (n = 95) were treated with a 1 min wash in 70% ethanol, while two nonsterilized egg clusters (n = 89) were subjected to 1 min wash in demineralized autoclaved water. (b) External appearance of the control and treated adult females. (c) Gut microbial community composition in adult N. viridula individuals based on 16S rRNA gene amplicon sequencing subjected to symbiont removal via egg-surface sterilization or nonsterilized. The taxonomy is displayed at the genus level. ‘Others’ represents the amplicon sequence variants (ASVs) that average below 0.5% of all reads. Individual bar graphs represent the sequencing of one adult insect per bar, with three biological replicates (n = 3) per treatment group.

Figure 2.

Sodalis distribution in N. viridula. (a) Hatching of nymphs from the egg of N. viridula. (b) Organization of organs dissected from adult N. viridula. M1, midgut first section; M2, midgut second section; M3, midgut third section; and M4, midgut fourth section with crypts. (c) Diagnostic PCR detection of obligate Pantoea and Sodalis symbionts in nonsterilized (n = 47) and surface-sterilized eggs (n = 48) and Sodalis in organs of female and male adult N. viridula. Negative control was nuclease-free water, while positive control was the DNA extracted from the isolated Sodalis strain from the N. viridula salivary glands. DNA extracted from eggs, organs, and tissue was normalized to an equal concentration of 2 ng µl⁻¹.

Figure 3.

Colonization of N. viridula salivary glands by Gammaproteobacteria and Sodalis. Confocal laser scanning micrographs show the adult N. viridula salivary gland where bacteria are visualized with fluorescence in situ hybridization. (a) Overview micrographs of a principal salivary gland. A square in the left panel marks the localization of the magnified panel in (b) scale bar = 100 µm. Left panel, detection of all bacteria with probe EUB-mix in cyan (Fluos); middle panel, γ-proteobacteria detected with probe GAM42A in yellow (Cy5); and right panel, Sodalis detected with probe Sod1238R in magenta (Cy3). (b) Magnified confocal micrographs indicated in panel (a) with a square of the principal salivary gland. Scale bar = 5 µm. The images shown here are representative of FISH micrographs collected from three individual insects (n = 3).

Figure 4.

Colonization of the N. viridula gut by Gammaproteobacteria and Sodalis. Confocal micrographs show the adult N. viridula intestinal tract. Left panel bacteria shows FISH-probe Eub-mix in cyan (Fluos). Middle panel γ-Proteobacteria shows FISH-probe GAM42A in yellow (Cy5). Right panel Sodalis shows FISH-probe Sod1238R in magenta (Cy3). Open arrowhead points at FISH-stained Sodalis. (a) M1 section of the gut. Scale bar = 10 µm. (b) M2 section of the gut. Scale bar = 10 µm. (c) M3 section of the gut. Scale bar = 10 µm. (d) M4 section of the gut with crypts. Scale bar = 25 µm. (e) Hindgut. Scale bar = 5 µm. The images shown here are representative of multiple FISH micrographs collected from three individual insects (n = 3).

Figure 5.

Colonization of N. viridula testes and Malpighian tubules by Gammaproteobacteria and Sodalis. Confocal micrographs show the adult N. viridula testes (a) and (b) Malpighian tubules. Left panel bacteria shows FISH-probe Eub-mix in cyan (Fluos). Middle panel γ-Proteobacteria shows FISH-probe GAM42A in yellow (Cy5). Right panel Sodalis shows FISH-probe Sod1238R in magenta (Cy3). (a) Testes. Scale bar = 10 µm. (b) Malpighian tubules. Scale bar = 5 µm. The images shown here are representative of multiple FISH micrographs collected from three individual insects (n = 3).

Figure 6.

Phylogenetic placement and genomic features of Sodalis sp. Nvir. (a) Excerpt of maximum-likelihood phylogenetic placement of the novel Sodalis sp. Nvir within the Sodalis clade. Names on leaves specify the bacterial genera, species, and strains (in grey). Red squared brackets denote a two-way comparison of 16S rRNA gene identity values. Values at nodes indicate the UltraFast bootstrap support values in percentages. An asterisk (*) denotes a support of 100%. Sodalis sp. SPI-1 was excluded for the visual grouping representing 16S rRNA gene identity value scores. (b) Circular diagrams of selected Sodalis genomes. Numbers at inner ticks denote the location within the genome in mega base pairs. The outer ring represents contigs/molecules of the genome assembly, with alternate grey and black colours separating them. Phage regions are coloured as specified in the colour key at the top of the diagrams. Squared brackets indicate the two-way ANI values.