Project: Optimizing a novel, safe and efficient RNA Interference protection strategy for honeybee Nosema disease.

Honeybees produce honey, but are more important for human welfare because they directly contribute to pollination of many crops. Recent honeybee colony declines have been primarily attributed to pathogenic organisms causing disease. Nosema disease (Nosemosis) is one of the most prevalent adult honey bee maladies and is caused by two described species of microsporidia (Nosema apis and Nosema ceranae). Microsporidia are obligate intracellular parasites of other eukaryotes, and are characterized by extreme genomic and cellular reduction. Nosema ceranae causes digestive disorders, shortened life span, decrease of hive population size, and reduction of honey production and crop products that rely on bees for pollination (Fries 2010 Nosema ceranae in European honey bees (Apis mellifera) J. Inv. Path 103:73-79). Bees infected with Nosema ceranae have a higher hunger level that leads to a lower survival and display precocious and risky foraging. _x000D_Nosemosis is widespread across the world. Diana Cox-Foster and co-workers found that there was extensive coverage of Nosema in almost all samples taken from hives that were afflicted by colony collapse disorder (CCD) (Cox Foster et al. 2007 A metagenomic survey of microbes in honey bee colony collapse disorder Science 318:283-7). Moreover, subsequent work published recently, indicates that nosema pathogenesis, together with increased viral load, are the best predictors of weak and collapsing colonies (VanEngelsdorp et al. 2009 Colony Collapse Disorder: A Descriptive Study. PLoS One Aug 4(8):e6481)._x000D_In Europe, disappearing colony syndrome has been directly attributed to Nosema ceranae. It is very pathogenic when experimentally inoculated into Apis mellifera and natural infection has been associated with a syndrome of gradual depopulation, copious colony death in autumn or winter, and poor honey production. In fact, the risk of colony depopulation is six times higher in colonies infected with N. ceranae than in uninfected ones. Recently, Spanish researchers showed that natural Nosema ceranae infection can cause the sudden collapse of bee colonies, establishing a direct correlation between Nosema ceranae infection and the death of honey bee colonies under field conditions. (Higes et al. 2008. How natural infection by Nosema ceranae causes honeybee colony collapse. Environmental Microbiology 10:2659–2669). Signs of colony weakness were not evident until the queen could no longer replace the loss of the infected bees. Furthermore, their results demonstrate that healthy colonies nearby an infected hive can also become infected._x000D_The objective of this project is to provide safe and efficacious control on Nosema Ceranae facilitated by RNA interference technology. This will be facilitated by the recent demonstration that Nosema Ceranae gene transcripts can be reduced by feeding honeybees with dsRNA (Paldi et al. 2010. Effective gene silencing in a microsporidian parasite associated with honeybee (Apis mellifera) colony declines. Appl Environ Microbiol. 76:5960-4). The rationale of the project is to optimize a RNAi approach to silencing genes of Nosema, based on correlating epidemiological data with the manifestation of the interactions between Nosema and honeybee at the transcriptomic level._x000D_The project was envisioned, initiated and developed by Beeologics, a company with expertise in honeybee RNAi product development. The consortium is composed of the two leading honeybee veterinary centers in Italy, where Nosema Ceranae has been found to be highly prevalent and where significant colony losses have been reported in recent years. In addition, a leading reputable Pan-European genomics center will provide the transcriptomics analysis and targets for gene silencing, and future market dissemination will be facilitated by the presence of a large global ag-chemical company.

Acronym	NOLESSBEES (Reference Number: 5928)
Duration	01/01/2011 - 31/12/2013
Project Topic	RNA interference approach will provide European beekeepers with an optimized, safe and efficacious application for combating honeybee colony declines that are caused by the pathogen Nosema ceranae. This product will be realized using a novel approach of phenotype correlated transcriptomics.
Project Results (after finalisation)	_x000D_Goals of Nolessbee project was to investigate if the RNAi techology could be used as a control method to reduce impact of Nosema ceranae on bees and therefore to imrove beehealth in commercial colonies . Role of Syngenta France was , with help of a start up company : RNAgro to:_x000D_1-sample bee populations form different part of France and assess level of Nosema infection _x000D_2-Test in mini hives populations _x000D_3-Identify pathogenic strains of Nosema cerenae and/or different susceptibility level of bee populations _x000D_4-Transfert these strains to Parco Technologico Padano (PTP) to study the transcriptome profile of Nosema ceranae strains in order to design a double strand RNA interference molecule (Ds RNAi )_x000D_5-Test in minihives the effects of these Ds RNAi on silencing Nosema and bee health _x000D__x000D_On this basis the CO results were :_x000D_Monitoring carried on bees from commercial apiaries showed either large variability in pathogenicity of Nosema ceranae or important differences in susceptibility of bees populations. _x000D_Successful nosema infection mini box assays were performed in France. Focusing on bee populations with high pathogenic effects of Nosema ceranae, samples were collected and sent to deep sequenced using Illumina technology in Parco Tecnologico Padano where the sequencing has been done. The results of the deep sequencing were analyzed using bioinformatic tools and 5 potential genes were identified as potential targets to be used in RNA silencing. and the design of ds RNA was performed ._x000D_Then ds RNA manufacturing was performed by Beeologics _x000D_The sequences were produced and dsRNA was sent to France for further testing._x000D_Test in minihives the effects of these Ds RNAi on silencing Nosema and bee health could not be performed on time due to bankrupt of RNAgro .
Network	Eurostars
Call	Eurostars Cut-Off 5

Project partner

Number	Name	Role	Country
5	BEEOLOGICS IL Ltd.	Coordinator	Israel
5	Istituto Zooprofilattico Sperimentale delle Regioni Lazio e Toscana	Partner	Italy
5	ISTITUTO ZOOPROFILATTICO SPERIMENTALE DELLE VENEZIE	Partner	Italy
5	Parco Tecnologico Padano S.r.l.	Partner	Italy
5	Syngenta Agro S.A.S.	Partner	France