The crucial role of the pollination activity of domestic honeybees is at risk because of an alarming increase in numbers of colony losses in the last years. Among the multiple factors that impact colony fitness, the sensitivity of honeybees to sublethal doses of insecticides, affecting crucial functions involved in foraging, including sensory processing, learning, memory and motor functions have been revealed. Acute toxicity of pyrethroids, the most commonly used insecticides, mainly occurs via binding to voltage-gated sodium (Na+) channels. However, it is clear now that calcium (Ca2+) channels are affected at similar doses. Because Na+ and Ca2+ channels are key actors of neuronal and muscular excitability, they are involved in many neural processes such as locomotion, sensory processing, learning and memory formation in vertebrates and invertebrates. They are thus both suspected of participating in sublethal toxicity pyrethroids. Oddly, the biophysical properties, regulation and pharmacology of Na+ and Ca2+ channels are still poorly studied in honeybees. Moreover, their subunit composition and molecular identity in the tissues potentially affected by these insecticides (muscle, central and peripheral nervous system) is largely unknown. We have identified in the genomic honeybee database the pore-forming subunit genes of the voltage–gated Na+ channels (2 genes? and Ca2+ channels (3 genes) as well as respectively 5 and 4 genes for regulatory subunits. Our preliminary cloning (for all Na+ and Ca2+ channels subunits) and expression studies (for one regulatory subunit) have revealed unique amino-acids sequences and specific biophysical and regulatory properties in honeybees. In this integrative project, we propose to analyse the role of these genes in honeybee physiology and behaviour (locomotion, olfactory perception, orientation, learning, memory) and in the sensitivity of this insect to sublethal doses of pyrethroids. • Task 1 will characterize the effects of sublethal doses of type I and type II pyrethroids on honeybee physiology. We will determine sublethal doses of pyrethroids on honeybees. We will then characterize their effects on Na+ and Ca2+ currents in honey bee muscle cells and central and peripheral neurons in vitro, as well as their effects and dependence on calcium homeostasis. We will also analyze the effect of pyrethroids on gene expression. • Task 2 will address the molecular characterization of Na+ and Ca2+ channel subunits identified in honeybees. We will define their expression profiles, biophysical properties and sensitivity to pyrethroids using heterologous expression. Their roles in Na+ and Ca2+ influxes in muscle cells and neurons will be challenged using siRNA and specific antibodies. Cell lines expressing actual combinations of subunits found in honeybees will be produced as screening tools for in vitro toxicological tests. • Task 3 will unravel the role of Na+ and Ca2+ channel subunits in sensory, cognitive and behavioural tasks, so that the sublethal toxicity of pyrethroids can be understood. The impact of siRNA and of sublethal doses of pyrethroids will be evaluated on olfactory processing, learning and memory, locomotor activity and free-flying foraging behaviour. This integrative approach brings together 4 partners with combined expertise in apidology, behavioural neurophysiology, toxicology, molecular cloning, electrophysiology and heterologous expression. It will improve our understanding of the neurobiology of sensory and cognitive functions in honeybees and clarify acute and chronic toxicity of pyrethroids and their effects on these processes. This project will also provide screening tools for evaluating the toxicity of phytopharmaceutical products toward these key channels.

Understanding the link between individual behavior and population-level phenomena has long been central in ecology and evolutionary biology. Behavior is a response to intrinsic and extrinsic factors including individual state, ecological factors or social interactions. Within a group each individual can be seen as part of a network of social interactions varying in strength, type and dynamic. The structure of this network can deeply impact the ecology and evolution of individuals, populations and species. Within a group social transmission of behavior can take many forms and may deeply affect individual’s behavior. Social learning has been studied mostly in fish, birds and mammals including humans. In insects, social learning has been unambiguously demonstrated in social Hymenoptera but this probably reflects limited research effort and recent evidence show that even non-eusocial insects such as Drosophila can copy the behavior of others. Compared to individual learning, which requires a trial and error period in every generation, social learning can potentially result in stable tradition transmission across generations. Despite the potential importance of social transmission on animal behavior relatively little is known about the processes which may facilitates or prevent this transmission and the relationship between social network structure and efficiency of social transmission. The goal of this project is to study the genetic and socio-environmental factors affecting social transmission with the integration of experimental approach, social network analysis and modeling. More specifically DrosoNet focuses on the mechanisms of information transfers that generate social learning. The originality of the program is to integrate complementary approaches (behavioral and social) devoted traditionally to very distinct biological models characterized by strongly divergent group organizations. This approach can help us identify patterns of social interaction (including how they change with time) that can lay the foundation for understanding key components of social transmission, and for making comparison of different populations, context or species, in order to understand at a global scale the emergence of cultures. Using Drosophila as an experimental model system and social network analysis the project will investigate (1) how the structure of a group affects social transmission (2) how individuals treat different source of information (in particular personal vs. social information) (3) the genetic bases of social learning ability. Importantly we aim at combining different approaches in order to understand whether a relationship between social network structures and dynamic can reflect the efficiency of social transmission i.e. can we use social network analysis in order to predict social transmission of information and ultimately the evolutionary trajectory of a group?

In a context of resource scarcity, population growth, environment destruction and food supply dependency, new solutions to produce food resources need to be explored. These solutions notably need to be environmentally safe, socially acceptable, healthy, relevantly nutritive, and economically loyal and realistic. An insect bioreactor seems a credible solution for human food production, as well as cosmetics, biomaterials, pharmaceutics and animal feed. Thus, insect was recently identified as a strategic development axis by several international institutions. The Food and Agriculture Organization of the United Nations (FAO) promotes the development of the insect industry as a new resource for animal feed and human food. Since 2010, two large-scale European research projects have already been launched to anticipate and help the development of the emerging insect industry. France is represented in neither of them. Following the second thematic axis of the ANR ALID 2012 call, Project DESIRABLE will notably explore a solution for underexploited waste streams, and for local supply of protein-rich materials, as the industry highly relies on importations of products that contribute to natural resources depletion. Our project aims to study and quantify the input of insect bioreactors in the valorization of underused industrial by-products by insect bioconversion upstream, and the relevance and sustainability of insects as a source of animal feed downstream. Even though insects also seem a pertinent source of food for humans, this thematic is partially explored in other European projects. Thus, Project DESIRABLE will focus on the potential incorporation of insects in fish & poultry diets, as a substitute for fish-based and soybean-based meals. Project DESIRABLE is an ambitious and completely integrated research project, which aims to cover the new insect industry as a whole, from organic waste collection to the final consumption of fish and poultry. Focus will be made on the innovative bioconversion and separation unit, which will be called biorefinery in reference to similar projects using a vegetal reactor. Lab-scale and pilot-scale experiments will be undertaken for every biorefinery component: insect rearing, insect transformation and meals formulation. The global vision will be brought by the realization of a deep Life Cycle Assessment of the system. Project DESIRABLE is based on the following fundamental hypothesis: (1) the sustainability of the insect alternative would notably rely on organic by-products utilization, in order to improve global waste management and limit competition with other treatment streams (2) the mealworm (Tenebrio molitor) and the black soldier fly (Hermetia illucens) will be the only insects studied in this project, as they are the object of many publications and seem to be pertinent species according to the literature. Project DESIRABLE gathers excellent competencies on a very wide scientific range, either academic and SME. In-depth studies and analysis will be performed on every biorefinery component and will be globally consolidated at industry scale. Skills brought by public laboratories in the fields of entomology, food technology, biochemistry, animal nutrition, sensory analysis, consumer acceptance, environmental LCA, socio-economic LCA, as well as SME competencies in insect rearing and transformation technologies will ensure the optimization of the insect biorefinery blueprints and the evaluation of industrial spinoffs. Project DESIRABLE constitutes a major milestone in the development of the innovative, local, sustainable and strategic insect industry.

The current rate of species extinction in the biosphere would be comparable with those of the last massive extinctions. The reduction in the richness of species and genetic diversity is accompanied by deterioration of a great number of ecosystemic services like pollination by animals (i.e. zoogamy). Several biotic (ex.: pathogens, alien species) and abiotic (ex.: habitat loss and fragmentation, agrochemicals, climate change) factors are probably involved in this disturbance of pollination and on the decline of pollinating species leading to a loss of genetic diversity. Genetic diversity of a species is a key factor to counter infection by native and invasive pathogens and to respond to abiotic factor pressures (ex. pollution) in any habitat. Species with an important distribution range, like the honeybee, Apis mellifera, have acquired a great adaptive potential to diverse environmental conditions. Covering Europe, Middle East and Africa, A. mellifera is subdivided into at least 26 physiologically, behaviourally and morphologically distinct subspecies. These subspecies have been grouped into 5 evolutionary branches according to the genetic structure. As an agronomical species of interest, the natural distribution of A. mellifera subspecies has been disturbed for many decades by beekeeping activities, particularly because of international trade of honeybees (ex.: colonies, queens, drones). These movements, which tend to homogenize the diversity, were particularly amplified this last decade due to livestock rebuilding to counter the effects of colony losses. An interesting assumption is that current honeybee declines observed in European apiaries can be caused by commercial and European trades of honeybees by (i) the introduction (for their apicultural traits) of non- adapted and artificially maintained colonies, and (ii) the spread of allochtone and invasive pathogens carried by allochtones bees. Genetic surveys have demonstrated that some populations of honeybee subspecies are adapted to local climate and flora. Those populations thus constitute particularly interesting populations to study and preserve in a context of sustainable beekeeping. The aim of our proposal is to set up, according to a North/South gradient, genetic conservatories of original naturally distributed honeybee populations. These honeybee conservation areas will have as missions: (i) to characterize the genetic and eco-ethologic diversity of honeybees from the West-Mediterranean lineage, (ii) to preserve the genetic diversity of those populations, (iii) to constitute a reserve of diversity usable by the honeybee industry and by beekeepers, (iv) to study the impact of the domesticated honeybee in the maintenance of local floristic diversity, and (v) to be able to use the honeybee as a bio-collector and as a biological indicator of environmental quality. Our Proposal thus comprises several parts which join to form a unit based on research on genetic and behavioral diversity of local honeybee populations and more applied aiming at answering a societal problem which is the conservation of a key species for the environment and human being. We will perform an impact study (using morphological and molecular tools) to determine if each area is appropriate or not for the conservation of honeybee populations, and we will monitor the spatio-temporal dynamics of key parasites involved in the arms race in each studied area (i.e. Varroa, virus, microsporodia, and bacteria).

BLINDTest is a basic research project involving three collaborative partners: two research teams and a bioinformatics platform. Through a comparative transcriptomics approach, we would like to identify mutations involved in the physiological, morphological and behavioral adaptive evolution of cavefish populations of the species Astyanax mexicanus. Indeed, the long term occupancy by surface river-dwelling fish of an open and empty ecological niche such as the cave system implies two major adaptive challenges: finding food and finding mates in the total and permanent darkness. The main reason why we choose to study this species is that it presents two very different types of morphs. The first corresponds to normal, river-dwelling fish of Central and South America. The second corresponds to a few dozens of populations (29 known today) of blind and de-pigmented fish which inhabit caves of the Sierra de El Abra region in Mexico. Cave colonization occurred several times and in an independent manner, since about 1 million years. Some cavefish population may be relatively recent, while others are more ancient, but they all correspond to the parallel evolution of several phenotypic traits in relation to the life in caves. In this project, we propose to compare the transcriptomes of surface fish and three independently-evolved cavefish populations and analyze them, in order to answer the three following questions: 1) What is the extent and what is the nature of structural variations in the transcriptomes of cave-adapted versus surface populations? 2) Can we find some examples of parallel evolution at transcriptome level in independently-evolved natural populations? 3) Are the structural transcriptome changes responsible for the physiological, morphological and behavioral adaptation to caves observed in cave populations? BLINDTest will take advantage of the possibilities offered by second generation sequencing technologies to describe transcriptome-wide the genetic polymorphism that arose within and between natural populations of a vertebrate species presenting populations living in radically different environments. We will then further test functionally the adaptive nature of observed genetic changes through a functional approach including crosses between populations of fish, transgenesis experiments, as well as developmental and behavioral analyses. This work, which will also include the identification of thousands of selectively neutral SNPs, will constitute a solid basis for future population genetics studies and for testing hypotheses of selection at a few loci (for instance selective sweep) and searching QTLs.