Abstract
There is growing evidence in the literature suggesting that caste differentiation in the stingless bee, Melipona scutellaris, and other bees in the genus Melipona, is triggered by environmental signals, particularly a primer pheromone. With the proper amount of food and a chemical stimulus, 25% of females emerge as queens, in agreement with a long-standing “two loci/two alleles model” proposed in the 1950s. We surmised that these larvae must be equipped with an olfactory system for reception of these chemical signals. Here we describe for the first time the diversity of antennal sensilla in adults and the morphology of larvae of M. scutellaris. Having found evidence for putative olfactory sensilla in larvae, we next asked whether olfactory proteins were expressed in larvae. Since the molecular basis of M. scutellaris is still unknown, we cloned olfactory genes encoding chemosensory proteins (CSP) and odorant-binding proteins (OBPs) using M. scutellaris cDNA template and primers designed on the basis CSPs and OBPs previously reported from the European honeybee, Apis mellifera. We cloned two CSP and two OBP genes and then attempted to express the proteins encoded by these genes. With a recombinant OBP, MscuOBP8, and a combinatorial single-chain variable fragment antibody library, we generated anti-MscuOBP8 monoclonal antibody. By immunohistochemistry we demonstrated that the anti-MscuOBP8 binds specifically to the MscuOBP8. Next, we found evidence that MscuOBP8 is expressed in M. scutellaris larvae and it is located in the mandibular region, thus further supporting the hypothesis of olfactory function in immature stages. Lastly, molecular modeling suggests that MscuOBP8 may function as a carrier of primer pheromones or other ligands.
Highlights
Stingless bees are social insects that pollinate wild and cultivated plants in tropical and subtropical regions
The geniculate antennae of adult M. scutellaris in workers (Fig 1), queens (Fig 2), and males (Fig 3) are comprised of a pedicel, scape and flagellum. As it has been described for M. quadrifasciata [18], antenna of M. scutellaris females are composed of flagellomeres (Fig 1A) while antenna of males show flagellomeres (Fig 3A)
This first scanning electron microscopy-based investigation of the morphology of M. scutellaris larvae and antennal sensilla showed that, in addition to differences among males, workers, and queens in their antennal sensilla, larval heads have structures similar to immature sensilla that possible indicate a gateway for pheromones and other chemicals from larval food
Summary
Stingless bees are social insects that pollinate wild and cultivated plants in tropical and subtropical regions. The evidence of queen development being up to 25% under ideal conditions of food led to the elegant “two loci/two alleles model” proposed by Kerr [3]. In this model, the genes of these loci would activate feminizing genes via juvenile hormone (JH), heterozygous larvae would develop into a queen by synthesizing higher titers of JH by the corpora allata glands. It was concluded that the volatile compound geraniol acts as a caste determining pheromone Environmental stimulus, such as geraniol and JH may enter into the larvae by any external structure with possible olfactory function as it occurs in adults through the antennal sensilla. CSPs and OBPs are found expressed mainly in olfactory tissues of insects, such as antennal sensilla. We expressed one putative odorant-binding protein, MscuOBP8, generated a monoclonal antibody against this protein and showed that it is expressed in larval stages
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