Sex, offspring and carcass determine antimicrobial peptide expression in the burying beetle

Abstract

The burying beetle Nicrophorus vespilloides has emerged as a model system for the investigation of adaptations that allow the utilization of carrion as a diet and as a resource for reproduction. The survival of beetles and their offspring given their exposure to soil-dwelling and cadaver-borne microbes requires mechanisms that reduce bacterial contamination in the diet and that achieve sanitation of the microhabitat. To explore the role of antimicrobial peptides (AMPs) in this context, we analyzed burying beetle males and females at different stages of their breeding cycle using the RNA-Seq and proteomics approaches. To address variation in immune functions, we investigated the impact of adult sex, the presence or absence of offspring (social context), and the presence of carrion (environmental context) on the expression of the identified immune effector genes. We found that particular AMPs are sex-specific and tightly regulated by the presence of a carcass or offspring and identified the two most context-dependent antimicrobial proteins in anal secretions. The context-specific expression dynamics of particular AMPs and lysozymes reveals a complex regulatory system, reflecting adaptations to specific ecological niches. This study highlights how burying beetles cope with microorganisms found on carrion and identifies candidates for both internal and external immunity.

Figure 1. Overview of the experimental setup.

Total RNA and anal secretions were collected from (1) males and females that were only allowed to mate (Mm, MF); (2) Mated males and females with carrion and larvae (CLm, CLf), and (3) mated males and females with carrion but without larvae (Cm, Cf).

Figure 2. Heat map showing the relative expression levels of AMPs and lysozymes in males and females and in relation to social and environmental context.

Significant differences for females are shown with an asterisk (* < 0.05, ** < 0.01, *** < 0.001). RPL7 and RPS4e are used as housekeeping genes and are shown to confirm the uniform expression of these control genes across tissues. The map is based on log2-transformed RPKM values (blue represents weakly-expressed genes, and red represents strongly-expressed genes).

Figure 3. The protein composition of anal secretions from male (m) and female (f) beetles in the three experimental groups (M, C and CL) was investigated by SDS-PAGE.

Lanes 1 and 8 show molecular size markers (PM) with molecular weights in kDa. Immunity-related protein bands identified by LC-MS^E are indicated by white boxes and arrows.

Figure 4. Proposed effects of anal secretions produced by burying beetles on social and personal (internal) immune function in adults and larvae.

Shown are both potential direct and indirect effects of antimicrobials applied to carrion (external immunity).