Weevil endosymbiont dynamics is associated with a clamping of immunity

Abstract

Background: Insects subsisting on nutritionally unbalanced diets have evolved long-term mutualistic relationships with intracellular symbiotic bacteria (endosymbionts). The endosymbiont population load undergoes changes along with insect development. In the cereal weevil Sitophilus oryzae, the midgut endosymbionts Sodalis pierantonius drastically multiply following adult metamorphosis and rapidly decline until total elimination when the insect achieves its cuticle synthesis. Whilst symbiont load was shown to timely meet insect metabolic needs, little is known about the host molecular and immune processes underlying this dynamics.

Methods: We performed RNA sequencing analysis on weevil midguts at three representative phases of the endosymbiont dynamics (i.e. increase, climax and decrease). To screen genes which transcriptional changes are specifically related to symbiont dynamics and not to the intrinsic development of the midgut, we further have monitored by RT-qPCR sixteen gene transcript levels in symbiotic and artificially non-symbiotic (aposymbiotic) weevils. We also localized the endosymbionts during the elimination process by fluorescence microscopy.

Results: Functional analysis of the host differentially expressed genes by RNA sequencing showed that the main transcriptional changes occur during endosymbiont growth phase and affect cell proliferation, apoptosis, autophagy, phagocytosis, and metabolism of fatty acids and nucleic acids. We also showed that symbiont dynamics alters the expression of several genes involved in insect development. Our results strengthened the implication of apoptosis and autophagy processes in symbiont elimination and recycling. Remarkably, apart from the coleoptericin A that is known to target endosymbionts and controls their division and location, no gene coding antimicrobial peptide was upregulated during the symbiont growth and elimination phases.

Conclusion: We show that endosymbiont dynamics parallels numerous transcriptional changes in weevil developing adults and affects several biological processes, including metabolism and development. It also triggers cell apoptosis, autophagy and gut epithelial cell swelling and delamination. Strikingly, immunity is repressed during the whole process, presumably avoiding tissue inflammation and allowing insects to optimize nutrient recovery from recycled endosymbiont.

Fig. 1

Analysis of Differentially Expressed Genes (DEGs) between Day 1, Day 6 and Day 9 after emergence. a qPCR quantification of endosymbiont DNA from 4th instar larvae (L4), late pupae (Ny), and adult males (Dx). Red arrows represent the UIM (Ultimate Insect Molting) and the emergence of the young adult from the wheat grain. Green arrows highlight the time points chosen for the RNAseq analysis. Adapted from Vigneron et al., [13]. b DEGs between D1 and D6 and between D6 and D9. The analysis shows a large proportion of transcriptional changes (up- and down-regulations) for the D1-D6 comparisonRed: significantly down-regulated genes; green: significantly up-regulated genes; black: non-differentially expressed genes. c Number and proportion of genes that are significantly differentially expressed between D1 and D6 and between D6 and D9

Fig. 2

Main KEGG terms regrouping DEGs from D1 to D6. Are described each KEGG pathway from which at least five sequences are differentially expressed from D1 to D6. Terms were manually grouped into the five functional categories. The arrows indicate whether the pathway is globally overactive or underactive in D6 when compared to D1 (green up arrow: most of the term-attached sequences are up in D6; red down arrow: most of the term-attached sequences are down in D6; no arrow: a similar number of term-attached sequences are up or down in D6, allowing no reliable interpretation about the term activity changes)

Fig. 3

Kinetics of expression of developmental, apoptosis- and autophagy-related genes identified through the RNAseq analysis. Transcript level quantification has been performed both on symbiotic (red, plain line) and aposymbiotic (blue, dotted line) individuals. Each point represents the mean of five replicates, and the error bars represent the standard errors. The central dotted line on each plot symbolizes D6, when the endosymbiont dynamics switches from a bursting increase to a decrease due to their host-controlled recycling

Fig. 4

Expression of immune-related genes in symbiotic and aposymbiotic individuals. Transcript level quantifications have been performed both on symbiotic (red, plain line) and aposymbiotic (blue, dotted line) midguts. Each point represents the mean of five replicates, and the error bars represent the standard errors. The central dotted line on each plot symbolizes D6, when the endosymbiont dynamics switches from a bursting increase to a decrease due to their host-controlled recycling

Fig. 5

a Larval bacteriome showing S. pierantonius (red) secluded inside bacteriocytes. b Overview of an adult midgut showing S. pierantonius in bacteriocytes, epithelia and delaminating cells. c Delaminating epithelial cells in an adult weevil midgut, showing S. pierantonius in their cytosol