Paratransgenesis feasibility in the honeybee (Apis mellifera) using Fructobacillus fructosus commensal.

AbstractAims: To establish the molecular tools for honeybee paratransgenesis.Methods and Results: Commensal bacteria were isolated from two honeybees.Based on 16S ribosomal RNA sequence analysis, some isolates were identiﬁedas Fructobacillus fructosus ,Lactobacillus kunkeei Gilliamella apicolaAcinetobacter spp, Arthrobacter spp and Pseudomonas spp. Rolling circle andtheta replicons were successfully introduced into F. fructosus and Lact. kunkeei.Green ﬂuorescent protein was expressed into both species. The 7 3KbLactococcus lactis subsp. cremoris MG1363 operon encoding a cluster of ﬁvegenes involved in the metabolism of galactose via the Leloir pathway wasfunctionally expressed into a non-galactose-fermenting strain of F. fructosusenabling it to grow on galactose as a sole carbon source.Conclusions: Fructophilic lactic acid bacteria, F. fructosus and Lact. kunkeei,are amenable to extensive genetic manipulations.Signiﬁcance and Impact of the Study: This is the ﬁrst study demonstratingthe feasibility of genetically engineering honeybee commensals, thusestablishing the tools necessary for honeybee paratransgenesis.IntroductionHoneybees (Apis mellifera) are, by far, the most relevantpollinators for a vast array of crops. The added value ofpollination is in the tens of billions of dollars worldwide,thus exceeding by two logs the value of honey, wax, propo-lis and bee venom combined (Gallai et al. 2009; van En-gelsdorp and Meixner 2010; Calderone 2012). Honeybeesare highly social insects living in colonies of 10 000–50 000 sterile females (workers, who perform differenttasks, mostly according to their age), few hundred males(called drones) and, typically, one fertile female commonlyreferred to as the queen. The queen is responsible for lay-ing eggs and for directing the life of the entire colony byproducing pheromones having prominent effects upon thecolony (Bloch 2010 and references therein). More recently,the concept of the hive, intended as a ‘superorganism’composed by the different castes of bees as well as themacro and microbiota, has been gaining interest, fosteredby the ﬁndings of the interactions between mammals andtheir microbiomes. A closer look to the role of the hivemicrobiome reveals that it is not much different fromhumans. Namely, it seems to be involved in the modula-tion of insect health according to in vitro and in vivo data(Alippi and Reynaldi 2006; Evans and Armstrong 2006;Forsgren et al. 2010; Vasquez et al. 2012); it providesnutrients in forms of micro- and macro-nutrients (Yoderet al. 2011; Engel et al. 2012); and it stabilizes the mixtureof pollen and nectar by converting this into bee bread(Vasquez and Olofsson 2009; DeGrandi-Hoffman et al.2011). Similar to mammals, variations in the microbiomecomposition have been associated to pathological statesalthough it is unclear whether such changes are to be con-sidered a cause or an effect. (Cox-Foster et al. 2007). Theimportance of the bee microbiome is relevant in maintain-ing adequate hive health, and now has become a focus ofintense research. This is in large part due to the steadydecline of honeybee colonies in the past 15 years resultingin the dramatic decline both in commercial and inamateur operations. A survey carried out in the winter2006–2007 revealed that 23% of commercial beekeeperslost an average of 35% of their colonies (van Engelsdorp