Deciphering the parasite/insecticide synergy in honeyBEE colony LOSSes using complementary approaches in Apis mellifera and the genetic model, Drosophila melanogaster – BEELOSS

To analyze the response of Apis mellifera and Drosophila melanogaster to the different stressors, we will follow physiological parameters (mortality, histopathlogical effects on tissues, parasite development), together with the dynamics of the honeybee mid-gut transcriptome (gene expression through RNAseq strategy), metabolome (identification of biomarkers of the exposition to stressors) and gut microbiota (bacterial population dynamics). Functional genomics approaches by mutagenesis and RNAi will be used for some target genes.

Intoxication by low doses of pesticides seems to alter the physiology of honeybees making them more susceptible to the infection by pathogens. Similarly, bees infected with Nosema ceranae are more susceptible to poisoning by low doses of insecticides. Preliminary results have been obtained at the molecular level.

Deciphering the molecular processes involved in toxic-pathogen interactions will be helpful in identifying both new targets for anti-parasitic drugs and biomarkers of colony health status.These data will also be useful in the context of pesticide homologation procedures.
They should also help to identify and prioritize the importance of various factors involved in honeybee colony losses.
Some perspectives will focus on the role of the gut microbiota and its modifications in response to biotic and abiotic stresses.

Preliminary data obtained before the begining of the project have been completed and will be published soon. These data concern the transcriptomic response of honeybee to the infection by Nosema ceranae and to chronic exposure to the insecticide fipronil.
These results have been recently presented to the Apimondia meeting (Kiev, september 2013).
2 posters at the ESF-EMBO meeting concerning the immunology of insects (Pultusk, Pologne, september 2013).

In the recent past, humanity has begun to realize that it depends on its environment for its own survival as brilliantly exposed by Jared Diamond in his book "Collapse". Conversely, mankind has deeply altered its environment, with sometimes devastating consequences. The honeybee illustrates these issues particularly well: it is of paramount ecological and agronomical importance; yet, bee colony losses have been recently reported worldwide at alarming rates. While the causes remain unclear, they are likely to be multifactorial and to involve honeybee pests, which include bacteria, viruses, parasites, and as of today, parasitoids such as phorid flies. The coordinator of the BEELOSS project has recently reported a synergy between an emerging microsporidian parasite, Nosema ceranae, and an insecticide, fipronil, which is still being used in some parts of the world. A dual exposure to these agents leads to the premature demise of honeybees. Microsporidia are fungal obligate intracellular pathogens. They parasitize also vertebrates, causing important losses to aquaculture for instance. Immunodeficient persons such as AIDS-infected patients are prone to microsporidial infections. Here, we propose to determine the molecular basis of the interactions between a parasite and an insecticide. Because the basal molecular mechanisms that allow the parasite to highjack the metabolism of its host are poorly known, we propose to use first a surrogate model consisting of another insect microsporidium that is somewhat phylogenetically related to N. ceranae, Tubulinosema ratisbonensis and the model genetic organism Drosophila melanogaster. Although the innate immunity of Drosophila against extracellular pathogens and viruses is relatively well known, that against intracellular eukaryotic parasites has hardly been investigated. We have developed two models to study the host-parasite interactions, one that involves permissive Drosophila cultured cells and the other that corresponds to a systemic infection in the adult fly. We propose to identify using transcriptomics, metabolomics, and a genome-wide mutagenesis screen in cell culture the host genes that are necessary for the proliferation of T. ratisbonensis, some of which are likely to be present in the honeybee genome and to also be used by N. ceranae. As the pathophysiology of N. ceranae infection in the intestinal epithelium of the honeybee remains sketchy, we propose its thorough investigation by immunohistochemistry, transcriptomics, and metabolomics. These descriptive approaches, or functional in the case of Drosophila cells, will be validated in vivo using RNAi gene knockdown technologies. The extensive studies in the fly model will guide the strategy in honeybees, which are much less easily experimentally tractable. An important aspect will be the comparison to be gained between these two systems, especially in comparing the developmental expression patterns of both parasites that should allow us to delineate common virulence factors and others that may be specific to each parasite according to its host tissue. Validation by RNAi approaches targeting parasite genes will be attempted. BEELOSS is thus characterized by extensive interactions and cross-talk between several partners that are all acknowledged specialists in their domains. Namely, Partner 1; a team of eukaryotic microbiologists, has been investigating nosemosis in honeybees and the interactions with the environment for several years. Partner 2 is a team of Drosophila geneticists that has pioneered the genetic analysis of insect innate immunity and has developed several intestinal infection models in the fly. Partner 3 runs a unique metabolomics platform in France. Finally, Partner 4 is a world expert on genome-wide RNAi screens in Drosophila cultured cells. BEELOSS will ultimately allow us to delineate molecular processes worth targeting by drugs and to identify candidate biomarkers linked to intoxicated honeybee colonies.