The Insecticide Imidacloprid Decreases Nannotrigona Stingless Bee Survival and Food Consumption and Modulates the Expression of Detoxification and Immune-Related Genes.

Stingless bees are ecologically and economically important species in the tropics and subtropics, but there has been little research on the characterization of detoxification systems and immune responses within them. This is critical for understanding their responses to, and defenses against, a variety of environmental stresses, including agrochemicals. Therefore, we studied the detoxification and immune responses of a stingless bee, Nanotrigona perilampoides, which is an important stingless bee that is widely distributed throughout Mexico, including urban areas, and has the potential to be used in commercial pollination. We first determined the LC50 of the neonicotinoid insecticide imidacloprid for foragers of N. perilampoides, then chronically exposed bees for 10 days to imidacloprid at two field-realistic concentrations, LC10 (0.45 ng/µL) or LC20 (0.74 ng/µL), which are respectively 2.7 and 1.3-fold lower than the residues of imidacloprid that have been found in honey (6 ng/g) in central Mexico. We found that exposing N. perilampoides stingless bees to imidacloprid at these concentrations markedly reduced bee survival and food consumption, revealing the great sensitivity of this stingless bee to the insecticide in comparison to honey bees. The expression of detoxification (GSTD1) and immune-related genes (abaecin, defensin1, and hymenopteacin) in N. perilampoides also changed over time in response to imidacloprid. Gene expression was always lower in bees after 8 days of exposure to imidacloprid (LC10 or LC20) than it was after 4 days. Our results demonstrate that N. perilampoides stingless bees are extremely sensitive to imidacloprid, even at low concentrations, and provide greater insight into how stingless bees respond to pesticide toxicity. This is the first study of its kind to look at detoxification systems and immune responses in Mexican stingless bees, an ecologically and economically important taxon.

Rapid evolution of chemosensory receptor genes in a pair of sibling species of orchid bees (Apidae: Euglossini).

BACKGROUND: Insects rely more on chemical signals (semiochemicals) than on any other sensory modality to find, identify, and choose mates. In most insects, pheromone production is typically regulated through biosynthetic pathways, whereas pheromone sensory detection is controlled by the olfactory system. Orchid bees are exceptional in that their semiochemicals are not produced metabolically, but instead male bees collect odoriferous compounds (perfumes) from the environment and store them in specialized hind-leg pockets to subsequently expose during courtship display. Thus, the olfactory sensory system of orchid bees simultaneously controls male perfume traits (sender components) and female preferences (receiver components). This functional linkage increases the opportunities for parallel evolution of male traits and female preferences, particularly in response to genetic changes of chemosensory detection (e.g. Odorant Receptor genes). To identify whether shifts in pheromone composition among related lineages of orchid bees are associated with divergence in chemosensory genes of the olfactory periphery, we searched for patterns of divergent selection across the antennal transcriptomes of two recently diverged sibling species Euglossa dilemma and E. viridissima. RESULTS: We identified 3185 orthologous genes including 94 chemosensory loci from five different gene families (Odorant Receptors, Ionotropic Receptors, Gustatory Receptors, Odorant Binding Proteins, and Chemosensory Proteins). Our results revealed that orthologs with signatures of divergent selection between E. dilemma and E. viridissima were significantly enriched for chemosensory genes. Notably, elevated signals of divergent selection were almost exclusively observed among chemosensory receptors (i.e. Odorant Receptors). CONCLUSIONS: Our results suggest that rapid changes in the chemosensory gene family occurred among closely related species of orchid bees. These findings are consistent with the hypothesis that strong divergent selection acting on chemosensory receptor genes plays an important role in the evolution and diversification of insect pheromone systems.

Nectar minerals as regulators of flower visitation in stingless bees and nectar hoarding wasps.

Various nectar components have a repellent effect on flower visitors, and their adaptive advantages for the plant are not well understood. Persea americana (avocado) is an example of a plant that secretes nectar with repellent components. It was demonstrated that the mineral constituents of this nectar, mainly potassium and phosphate, are concentrated enough to repel honey bees, Apis mellifera, a pollinator often used for commercial avocado pollination. Honey bees, however, are not the natural pollinator of P. americana, a plant native to Central America. In order to understand the role of nectar minerals in plant-pollinator relationships, it is important to focus on the plant's interactions with its natural pollinators. Two species of stingless bees and one species of social wasp, all native to the Yucatan Peninsula, Mexico, part of the natural range of P. americana, were tested for their sensitivity to sugar solutions enriched with potassium and phosphate, and compared with the sensitivity of honey bees. In choice tests between control and mineral-enriched solutions, all three native species were indifferent for mineral concentrations lower than those naturally occurring in P. americana nectar. Repellence was expressed at concentrations near or exceeding natural concentrations. The threshold point at which native pollinators showed repellence to increasing levels of minerals was higher than that detected for honey bees. The results do not support the hypothesis that high mineral content is attractive for native Hymenopteran pollinators; nevertheless, nectar mineral composition may still have a role in regulating flower visitors through different levels of repellency.

Conservation genetics of neotropical pollinators revisited: microsatellite analysis suggests that diploid males are rare in orchid bees.

Allozyme analyses have suggested that Neotropical orchid bee (Euglossini) pollinators are vulnerable because of putative high frequencies of diploid males, a result of loss of sex allele diversity in small hymenopteran populations with single locus complementary sex determination. Our analysis of 1010 males from 27 species of euglossine bees sampled across the Neotropics at 2­11 polymorphic microsatellite loci revealed only five diploid males at an overall frequency of 0.005 (95% CIs 0.002­0.010); errors through genetic nondetection of diploid males were likely small. In contrast to allozyme-based studies, we detected very weak or insignificant population genetic structure, even for a pair of populations >500 km apart, possibly accounting for low diploid male frequencies. Technical flaws in previous allozyme-based analyses have probably led to considerable overestimation of diploid male production in orchid bees. Other factors may have a more immediate impact on population persistence than the genetic load imposed by diploid males on these important Neotropical pollinators.

An olfactory shift is associated with male perfume differentiation and species divergence in orchid bees.

Saltational changes may underlie the diversification of pheromone communication systems in insects, which are normally under stabilizing selection favoring high specificity in signals and signal perception. In orchid bees (Euglossini), the production of male signals depends on the sense of smell: males collect complex blends of volatiles (perfumes) from their environment, which are later emitted as pheromone analogs at mating sites. We analyzed the behavioral and antennal response to perfume components in two male morphotypes of Euglossa cf. viridissima from Mexico, which differ in the number of mandibular teeth. Tridentate males collected 2-hydroxy-6-nona-1,3-dienyl-benzaldehyde (HNDB) as the dominant component of their perfume. In bidentate males, blends were broadly similar but lacked HNDB. Population genetic analysis revealed that tri- and bidentate males belong to two reproductively isolated lineages. Electroantennogram tests (EAG and GC-EAD) showed substantially lower antennal responses to HNDB in bidentate versus tridentate males, revealing for the first time a mechanism by which closely related species acquire different chemical compounds from their habitat. The component-specific differences in perfume perception and collection in males of two sibling species are in agreement with a saltational, olfaction-driven mode of signal perfume evolution. However, the response of females to the diverged signals remains unknown.

Enfleurage, lipid recycling and the origin of perfume collection in orchid bees.

Enfleurage, the extraction of elusive floral scents with the help of a lipophilic carrier (grease), is widely used in the perfume industry. Male neotropical orchid bees (Euglossini), which accumulate exogenous fragrances as pheromone analogues, use a similar technique. To collect fragrances, the bees apply large amounts of straight-chain lipids to odoriferous surfaces from their cephalic labial glands, which dissolve the volatiles, and the mixture is then transferred to voluminous hind-leg pockets. Here, we show that males do in fact operate a lipid conveyor belt to accumulate and concentrate their perfume. From the hind-leg pockets of caged male Euglossa viridissima, deuterated derivatives of carrier lipids were consecutively sequestered, shuttled back to the labial glands and reused on consecutive bouts of fragrance collection. Such lipid cycling is instrumental in creating complex perfume bouquets. Furthermore, we found that labial glands of male orchid bees are strikingly similar to those of scent-marking male bumblebees in terms of size, form and structure. This, and a prominent overlap in secretory products, led us to propose that perfume collection evolved from scent-marking in ancestral corbiculate bees.