Transcriptomic Immune Response of the Cotton Stainer Dysdercus fasciatus to Experimental Elimination of Vitamin-Supplementing Intestinal Symbionts

Abstract

The acquisition and vertical transmission of bacterial symbionts plays an important role in insect evolution and ecology. However, the molecular mechanisms underlying the stable maintenance and control of mutualistic bacteria remain poorly understood. The cotton stainer Dysdercus fasciatus harbours the actinobacterial symbionts Coriobacterium glomerans and Gordonibacter sp. in its midgut. The symbionts supplement limiting B vitamins and thereby significantly contribute to the host’s fitness. In this study, we experimentally disrupted the symbionts’ vertical transmission route and performed comparative transcriptomic analyses of genes expressed in the gut of aposymbiotic (symbiont-free) and control individuals to study the host immune response in presence and absence of the mutualists. Annotation of assembled cDNA reads identified a considerable number of genes involved in the innate immune system, including different protein isoforms of several immune effector proteins (specifically i-type lysozyme, defensin, hemiptericin, and pyrrhocoricin), suggesting the possibility for a highly differentiated response towards the complex resident microbial community. Gene expression analyses revealed a constitutive expression of transcripts involved in signal transduction of the main insect immune pathways, but differential expression of certain antimicrobial peptide genes. Specifically, qPCRs confirmed the significant down-regulation of c-type lysozyme and up-regulation of hemiptericin in aposymbiotic individuals. The high expression of c-type lysozyme in symbiont-containing bugs may serve to lyse symbiont cells and thereby harvest B-vitamins that are necessary for subsistence on the deficient diet of Malvales seeds. Our findings suggest a sophisticated host response to perturbation of the symbiotic gut microbiota, indicating that the innate immune system not only plays an important role in combating pathogens, but also serves as a communication interface between host and symbionts.

Quantitative PCR primer design affects quantification of dsRNA‐mediated gene knockdown

“…This cDNA was used as a template for the synthesis of dsRNA for D. fasciatus ' antimicrobial peptide genes defensin 1 and defensin 2, which have low expression levels (<10 RPKM), and c‐type lysozyme gene with a higher expression level (>100 RPKM) using MEGAscript® RNAi kit (Thermo Fisher Scientific) following the manufacturer's protocol. The expression levels of the three genes are based on a previously published transcriptomic data which reported the normalized gene transcripts of the c‐type lysozyme to be at least 200 times higher than those of the different isoforms of the defensin genes (Bauer, Salem, Marz, Vogel, & Kaltenpoth, ). The synthesized dsRNA was used for in vivo RNAi gene knockdown that was performed by feeding 15 replicate individuals of second instar D. fasciatus nymphs that had been deprived of water for 24 hr with dsRNA for defensins (i.e., defensin 1 and defensin 2 in combination) or c‐type lysozyme, or nonsense dsRNA for GFP for the control group, respectively.…”

“…This cDNA was used as a template for the synthesis of dsRNA for D. fasciatus' antimicrobial peptide genes defensin 1 and defensin 2, which have low expression levels (<10 RPKM), and c-type lysozyme gene with a higher expression level (>100 RPKM) using MEGAscript® RNAi kit (Thermo Fisher Scientific) following the manufacturer's protocol. The expression levels of the three genes are based on a previously published transcriptomic data which reported the normalized gene transcripts of the c-type lysozyme to be at least 200 times higher than those of the different isoforms of the defensin genes (Bauer, Salem, Marz, Vogel, & Kaltenpoth, 2014).…”

Symbiont transmission entails the risk of parasite infection

“…Transcriptome sequencing of poly-A enriched mRNAs, assembly, annotation and analysis were described previously [9,20]. The sequence data were deposited in the European Nucleotide Archive, accession number PRJEB6171 (http://www.ebi.ac.uk/ena/data/view/PRJEB6171).…”

Nutrition meets the microbiome: micronutrients and the microbiota

“…For example, insects' host mutualistic bacteria that supply them with B vitamins can act as a defense barrier against pathogens. In a recent study examining the mechanism underlying the stable maintenance and control of mutualistic bacteria, elimination of the gut symbionts resulted in up‐ and downregulation of antimicrobial defensins, which may also be involved in lysis of symbiont bacteria (downregulation of c‐type lysozyme) to harvest the symbiont B vitamins. B vitamins (vitamin B 2 and folate) can serve as precursors to activate special immune T cells, called mucosa‐associated invariant T (MAIT) cells .…”