Royal Decree: Gene Expression in Trans-Generationally Immune Primed Bumblebee Workers Mimics a Primary Immune Response

Abstract

Invertebrates lack the cellular and physiological machinery of the adaptive immune system, but show specificity in their immune response and immune priming. Functionally, immune priming is comparable to immune memory in vertebrates. Individuals that have survived exposure to a given parasite are better protected against subsequent exposures. Protection may be cross-reactive, but demonstrations of persistent and specific protection in invertebrates are increasing. This immune priming can cross generations ("trans-generational" immune priming), preparing offspring for the prevailing parasite environment. While these phenomena gain increasing support, the mechanistic foundations underlying such immune priming, both within and across generations, remain largely unknown. Using a transcriptomic approach, we show that exposing bumblebee queens with an injection of heat-killed bacteria, known to induce trans-generational immune priming, alters daughter (worker) gene expression. Daughters, even when unexposed themselves, constitutively express a core set of the genes induced upon direct bacterial exposure, including high expression of antimicrobial peptides, a beta-glucan receptor protein implicated in bacterial recognition and the induction of the toll signaling pathway, and slit-3 which is important in honeybee immunity. Maternal exposure results in a distinct upregulation of their daughters' immune system, with a signature overlapping with the induced individual response to a direct exposure. This will mediate mother-offspring protection, but also associated costs related to reconfiguration of constitutive immune expression. Moreover, identification of conserved immune pathways in memory-like responses has important implications for our understanding of the innate immune system, including the innate components in vertebrates, which share many of these pathways.

Fig 1. Diagram of our experimental design.

We mated full sister queens to brothers from an unrelated colony to create colony blocks with limited genetic differences between them. These sisters were then injected with heat-inactivated bacteria or left unchallenged as controls. The resulting daughters were then either left unchallenged or given an injection as above and the RNA pooled from two individuals per colony by treatment group for RNAseq. We used additional colonies to confirm our findings with qPCR.

Fig 2. The number of differentially expressed genes in naïve worker offspring of mother queens that were injected with heat killed Gram-positive bacterium (Arthrobacter globiformis) (trans-generational immunity treatment; AN), and worker offspring from naïve mother queens but themselves exposed to an immune stimulus of A. globiformis (induced immune response condition; NA).

The expression of these genes is measured relative to that of naïve worker offspring of naïve mothers (NN).

Fig 3. Log 2 fold expression based on RNAseq data (relative to naïve worker offspring from naïve mother queens) for all genes that are significantly differentially expressed in the trans-generational priming condition (naïve offspring of bacteria exposed mothers, blue).

We also show the expression of directly bacterially exposed workers from naïve mothers (red) to demonstrate the similarity of the induced response to a direct challenge to the signature of trans-generational immunity. All differentially expressed genes here are also significantly differentially expressed upon direct exposure, except for LOC100644816, which encodes for mast cell degranulating peptide. qPCR confirmation of these results can be found in S1 Fig.