Thiamethoxam exposure deregulates short ORF gene expression in the honey bee and compromises immune response to bacteria

Abstract

Running title: Long-term low dose Thiamethoxam exposure induces short ORFs AbstractMaximizing crop yields heavily relies on the use of agrochemicals to control insect pests. One of the most widely used insecticides are neonicotinoids. Here, we analysed the impact of sub-lethal chronic long-term exposure to the neonicotinoid Thiamethoxam on gene expression and alternative splicing in brains of Africanized honey bees Apis mellifera. Our results reveal a small number of differentially regulated genes showing a concentration dependent response to two low doses of chronic, 10-day Thiamethoxam exposure. Unexpectedly, most of these genes have no annotated function, but encode short Open Reading Frames (sORFs), a characteristic feature of anti-microbial peptides. Likewise, we find that Thiamethoxam exposure sensitizes bees to infection by non-pathogenic bacteria Bacillus badius and Ochrobactrum anthropi. Moreover, infection with estimated single Serratia marcescens kills bees arguing that Varroa mites may essentially contribute to colony collapse by penetrating the cuticle to spread this pathogen. Our results implicate an altered immune response to Thiamethoxam exposure compromising the immune response leading to a decline in bee populations.Understanding the risk for honey bees requires detailed knowledge of cellular and molecular effects that result from the exposure to an insecticide in order to mitigate negative effects or refine the target specificity towards pest species.Changes in gene expression and processing of RNAs, including alternative splicing, are one of the options available to an organism to respond to environmental perturbations 15,16 . Sub-lethal exposure of xenobiotics can induce modulation of splicing reactions [17][18][19] .Neonicotinoid exposure has been linked to a decline in bee health including a reduction of immune competence 20 ;; 21-23 . Insects do not have antibodies and rely on the innate immune system to fight microbial infections. The best insight on insect immunity stems from studies in the fruit fly Drosophila melanogaster, where cellular and humoral immune responses have been identified 24,25 . The cellular response is mediated by three types of hematopoetic cell lineages 24,26,27 , while the humoral immune system can be split into Toll and Imd pathways [reviewed in 28 ]. The Tollpathway is triggered following an immune challenge by Gram-positive bacteria and fungi, ultimately leading to expression of antimicrobial peptides (AMPs) that are then secreted from the fat body into the hemolymph 24,29,30 . In contrast, the Immune deficiency (Imd) pathway leads to expression of a different set of AMPs after a Gramnegative bacterial infection activates pattern recognition receptors and a complex intracellular signaling cascade 24,25,29 . AMPs are short peptides of 10-100 amino acids and are characterised by an evolutionary dynamism among insect species 31 .We have previously shown that worker-bee larvae in colonies contaminated with the neonicotinoid imidacloprid, have altered expression o...

Dynamically expressed single ELAV/Hu orthologue elavl2 of bees is required for learning and memory

“…Alternative splicing could serve as an adaptive mechanism to changes in perception, but also to environmental conditions such as toxic insult 77 . Although learning and memory is affected by neonicotinoids in insects, we did not find any changes in elavl2 alternative splicing 44,78,79 . Since we also could not detect any alternative splicing changes after learning in mRNA from central brains, drastic changes in alternative splicing relevant to learning and memory might occur only in few cells.…”

“…RNA extraction from whole bees or dissected bee brains and RT-PCR was done as described 87 . Expression of elavl2 at different timepoints was compared to Appl expression using primers AM elav qF3 and AM elav qR3 to amplify the constant part of elav and normalized to unpaired control animals using qPCR as described 33,79 .…”

CMTr mediated 2′-O-ribose methylation status of cap-adjacent nucleotides across animals

“…After 48 hours, larvae were washed off and collected on a fine mesh and the temperature shifted to 29º C. Groups of 30 larvae were transferred to food vials and flies were reared at designated temperatures. Honey bees were obtained and maintained as described (Decio et al 2019;Decio et al 2021).…”

Gene expression in bumble bee larvae differs qualitatively between high and low concentration imidacloprid exposure levels

“…Gene expression (RNA-seq) studies have shown that exposure to sublethal neonicotinoid concentrations can affect gene expression in bees [40][41][42][43][44][45] . Molecular responses to exposure may vary depending on the context: for instance, research on B. terrestris found that clothianidin had a greater impact than imidacloprid on gene expression in head tissue, and the impact was greater in workers than in queens 42 .…”