Transcriptional survey of ovarian bacteriomes in the cereal weevil,Sitophilus oryzae, shows down-regulation of immune effectors at the onset of sexual maturity

Abstract

Insects often establish long-term relationships with intracellular symbiotic bacteria, i.e. endosymbionts, that provide them with essential nutrients such as amino acids and vitamins. Endosymbionts are typically confined within specialized host cells called bacteriocytes that may form an organ, the bacteriome. Compartmentalization within host cells is paramount for protecting the endosymbionts and also avoiding chronic activation of the host immune system. In the cereal weevil Sitophilus oryzae, bacteriomes are present as a single organ at the larval foregut-midgut junction, and in adults, at the apex of midgut mesenteric caeca and at the apex of the four ovarioles. While the adult midgut endosymbionts experience a drastic proliferation during early adulthood followed by complete elimination through apoptosis and autophagy, ovarian endosymbionts are maintained throughout the weevil lifetime by unknown mechanisms. Bacteria present in ovarian bacteriomes are thought to be involved in the maternal transmission of endosymbionts through infection of the female germline, but the exact mode of transmission is not fully understood. Here, we show that endosymbionts are able to colonize the germarium in one-week-old females, pinpointing a potential infection route of oocytes. To identify potential immune regulators of ovarian endosymbionts, we have analyzed the transcriptomes of the ovarian bacteriomes through young adult development, from one-day-old adults to sexually mature ones. In contrast with midgut bacteriomes, immune effectors are downregulated in ovarian bacteriomes at the onset of sexual maturation. We hypothesize that relaxation of endosymbiont control by antimicrobial peptides might allow bacterial migration and potential oocyte infection, ensuring endosymbiont transmission.

Transcriptomic-based selection of reference genes for quantitative real-time PCR in an insect endosymbiotic model

“…RNAseq datasets were processed in our previous publication (Ferrarini et al, 2023), and raw reads are available at BioProject PRJNA918957. Briefly, quality check was performed with FastQC v0.11.8 (Andrews, 2010), and raw reads were quality trimmed using trim_galore from Cutadapt v0.6.7 (Martin, 2011), then mapped to the S. oryzae's genome (GCA_002938485.2 Soryzae_2.0) using STAR v2.7.3a (Dobin et al, 2013).…”

“…Then, uniquely mapping reads were counted with featureCounts v2.0.1 (Liao et al, 2014), and mapping quality was assessed by multiqc v1.13 (Ewels et al, 2016). Differential expression analysis was conducted with SarTOOLS, taking advantage of a global linear model from Deseq2 (Love et al, 2014, 2), and log2 fold changes and adjusted p-values were obtained for sequential pairwise comparisons, i.e., L4 vs D1, D1 vs D3 etc, and can be found in the supplementary material of our previous report (Ferrarini et al, 2023). Most graphic outputs are either performed in R, using ggplot2 (Wickham, 2009) or with GraphPad Prism software.…”

“…To find S. oryzae reference genes to be used across gut and bacteriomes development (from larval stage 4 to 9-day-old adults), we scanned a comprehensive transcriptomic dataset spanning S. oryzae life cycle (Ferrarini et al, 2023). Candidate reference genes should be stable between conditions, highly expressed, and present transcript isoforms suitable for primer design.…”

“…Analyzing transcriptomic regulations across weevil's development is required to fully understand S. oryzae and S. pierantonius relationship, and to uncover key pathways profitable for integrative pest management strategies. We have recently conducted a thorough analysis of the gut (and associated bacteriomes) transcriptome of S. oryzae across its development from larval stages up to bacterial clearance in adults (Figure 1, (Ferrarini et al, 2023)). In this work, we took advantage of the recent transcriptomic profile of S. oryzae during development (Ferrarini et al, 2023) to select a set of reliable reference genes for qRT-PCR analyses, with high stability and conservation among insects.…”

“…We have recently conducted a thorough analysis of the gut (and associated bacteriomes) transcriptome of S. oryzae across its development from larval stages up to bacterial clearance in adults (Figure 1, (Ferrarini et al, 2023)). In this work, we took advantage of the recent transcriptomic profile of S. oryzae during development (Ferrarini et al, 2023) to select a set of reliable reference genes for qRT-PCR analyses, with high stability and conservation among insects. The identified transcripts will be of use for the comparison of transcript abundance across multiple developmental stages in S. oryzae, gene silencing conditions, and comparisons with artificially-obtained aposymbiotic weevils.…”