Bee Trypanosomatids: First Steps in the Analysis of the Genetic Variation and Population Structure of Lotmaria passim, Crithidia bombi and Crithidia mellificae.

Trypanosomatids are among the most prevalent parasites in bees but, despite the fact that their impact on the colonies can be quite important and that their infectivity may potentially depend on their genotypes, little is known about the population diversity of these pathogens. Here we cloned and sequenced three non-repetitive single copy loci (DNA topoisomerase II, glyceraldehyde-3-phosphate dehydrogenase and RNA polymerase II large subunit, RPB1) to produce new genetic data from Crithidia bombi, C. mellificae and Lotmaria passim isolated from honeybees and bumblebees. These were analysed by applying population genetic tools in order to quantify and compare their variability within and between species, and to obtain information on their demography and population structure. The general pattern for the three species was that (1) they were subject to the action of purifying selection on nonsynonymous variants, (2) the levels of within species diversity were similar irrespective of the host, (3) there was evidence of recombination among haplotypes and (4) they showed no haplotype structuring according to the host. C. bombi exhibited the lowest levels of synonymous variation (πS= 0.06 ± 0.04 %) - and a mutation frequency distribution compatible with a population expansion after a bottleneck - that contrasted with the extensive polymorphism displayed by C. mellificae (πS= 2.24 ± 1.00 %), which likely has a more ancient origin. L. passim showed intermediate values (πS= 0.40 ± 0.28 %) and an excess of variants a low frequencies probably linked to the spread of this species to new geographical areas.

Detection of Microsporidia in Pollinator Communities of a Mediterranean Biodiversity Hotspot for Wild Bees.

Insect pollination is crucial for the maintenance of natural and managed ecosystems but the functioning of this ecosystem service is threatened by a worldwide decline of pollinators. Key factors in this situation include the spread and interspecific transmission of pathogens worldwide through the movement of managed pollinators. Research on this field has been mainly conducted in some particular species, while studies assessing the interspecific transmission of pathogens at a community level are scarce. However, this information is pivotal to design strategies to protect pollinators. Herein, we analysed the prevalence of two common microsporidia pathogens of managed honey bees (Nosema ceranae and N. apis) in bee communities of semiarid Mediterranean areas from the Southeast of the Iberian Peninsula. Our results confirm the ability of N. ceranae to disperse across wild bee communities in semiarid Mediterranean ecosystems since it was detected in 36 Apoidea species (39% of the sampling; for the first time in nine genera). The prevalence of the pathogen did not show any phylogenetic signal which suggests a superfamily host range of the pathogen or that wild bees may be acting only as vectors of N. ceranae. In addition, N. apis was detected in an Eucera species, which is the second time it has been detected by molecular techniques in a host other than the honey bee. Our study represents the primary assessment of the prevalence of microsporidia at community level in Mediterranean areas and provides outstanding results on the ability of Nosema pathogens to spread across the landscape.

Spatial and temporal patterns of genetic diversity in Bombus terrestris populations of the Iberian Peninsula and their conservation implications.

The bumblebee Bombus terrestris is used worldwide for crop pollination. Despite its positive impact on crop yield, it has become a widespread threat to biodiversity due to its interactions with local bumblebee populations. Commercial subspecies introduced to the Iberian Peninsula since the 1990s without any regulation have colonized the environment, with evidence of naturalization and introgression with the endemic subspecies Bombus terrestris lusitanicus. We have used mitochondrial and nuclear genetic data to describe the current genetic diversity of the Iberian population and to estimate the expansion of commercial bumblebees. Samples from the natural distribution range of the commercial subspecies, the natural intergradation area between the two subspecies and from a period prior to the use of commercial colonies (i.e., before the 1990s) have been used for comparison. Our results show that the mitochondrial haplotype of the commercial breeds has spread throughout the territory, which, together with subtle changes observed in the nuclear genetic diversity of the populations, indicates that hybridization and consequent introgression are occurring in most of the peninsula. It is, therefore, necessary to improve the existing legislation concerning the management and exportation of commercial bumblebees to conserve locally adapted populations.

Wide diversity of parasites in Bombus terrestris (Linnaeus, 1758) revealed by a high-throughput sequencing approach.

Assessing the extent of parasite diversity requires the application of appropriate molecular tools, especially given the growing evidence of multiple parasite co-occurrence. Here, we compared the performance of a next-generation sequencing technology (Ion PGM ™ System) in 12 Bombus terrestris specimens that were PCR-identified as positive for trypanosomatids (Leishmaniinae) in a previous study. These bumblebees were also screened for the occurrence of Nosematidae and Neogregarinorida parasites using both classical protocols (either specific PCR amplification or amplification with broad-range primers plus Sanger sequencing) and Ion PGM sequencing. The latter revealed higher parasite diversity within individuals, especially among Leishmaniinae (which were present as a combination of Lotmaria passim, Crithidia mellificae and Crithidia bombi), and the occurrence of taxa never reported in these hosts: Crithidia acanthocephali and a novel neogregarinorida species. Furthermore, the complementary results produced by the different sets of primers highlighted the convenience of using multiple markers to minimize the chance of some target organisms going unnoticed. Altogether, the deep sequencing methodology offered a more comprehensive way to investigate parasite diversity than the usual identification methods and provided new insights whose importance for bumblebee health should be further analysed.

Antennal morphology and sensillar equipment vary with pollen diet specialization in Andrena bees.

Several studies recently reported that specialized (oligolectic) bees, which collect pollen from few host plants, use, besides visual cues, specific volatiles to find their hosts. Generalist (polylectic) bees, on the other hand, likely have to recognize a wider range of volatiles because they forage on many plant species. Bee antennal sensory equipment may thus be under selection to optimize plant host recognition. This selection may have led to variation in sensory equipment morphology with diet specialization (lecty). We tested if lecty correlates with antennal morphology and abundance of the main olfactory/gustatory sensilla (sensilla trichoidea (ST), placoidea (SP), sensilla basiconica (SB)) in the genus Andrena (Hymenoptera: Andrenidae). Across 24 species, and after having controlled for body size, we found polylectic species to have a longer and narrower flagellomer F9 (the one with highest abundance of sensilla), and to have a greater ST density on F9, compared with oligolectic species. Neither SP density nor SB number varied with lecty. A cluster analysis furthermore depicted groups of species that reasonably reflect diet specialization. Our results are in line with the previously observed lower number of glomeruli in the brain of oligolectic, compared with polylectic, bees. A formal correction for phylogeny is necessary to confirm our preliminary conclusion that pollen diet specialization has driven the morphology of the peripheral sensory system in this bee genus.

Phenology drives species interactions and modularity in a plant - flower visitor network.

Phenology is often identified as one of the main structural driving forces of plant - flower visitor networks. Nevertheless, we do not yet have a full understanding of the effects of phenology in basic network build up mechanisms such as ecological modularity. In this study, we aimed to identify the effect of within-season temporal variation of plant and flower visitor activity on the network structural conformation. Thus, we analysed the temporal dynamics of a plant - flower visitor network in two Mediterranean alpine communities during one complete flowering season. In our approach, we built quantitative interaction networks and studied the dynamics through temporal beta diversity of species, interaction changes and modularity analysis. Within-season dissimilarity in the identity of interactions was mainly caused by species replacement through time (species turnover). Temporal replacement of species and interactions clearly impacted modularity, to the extent that species phenology emerged as a strong determinant of modularity in our networks. From an applied perspective, our results highlight the importance of considering the temporal variation of species interactions throughout the flowering season and the requirement of making comprehensive temporal sampling when aiming to build functionally consistent interaction networks.

A new multiplex PCR protocol to detect mixed trypanosomatid infections in species of Apis and Bombus.

Trypanosomatids are highly prevalent pathogens of Hymenoptera; however, most molecular methods used to detect them in Apis and Bombus spp. do not allow the identification of the infecting species, which then becomes expensive and time consuming. To overcome this drawback, we developed a multiplex PCR protocol to readily identify in a single reaction the main trypanosomatids present in these hymenopterans (Lotmaria passim, Crithidia mellificae and Crithidia bombi), which will facilitate the study of their epidemiology and transmission dynamics. A battery of primers, designed to simultaneously amplify fragments of the RNA polymerase II large subunit (RPB1) of L. passim, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of C. mellificae and the DNA topoisomerase II (TOPII) of C. bombi, was tested for target specificity under single and mixed template conditions using DNA extracted from cell cultures (L. passim ATCC PRA403; C. mellificae ATCC 30254) and from a bumblebee specimen infected with C. bombi only (14_349). Once validated, the performance of the method was assessed using DNA extractions from seven Apis mellifera (Linnaeus, 1758) and five Bombus terrestris (Linnaeus, 1758) field samples infected with trypanosomatids whose identity had been previously determined by PCR-cloning and sequencing (P-C-S). The new method confirmed the results obtained by P-C-S: two of the honeybee samples were parasitized by L. passim, C. mellificae and C. bombi at the same time, whereas the other five were infected with L. passim only. The method confirmed the simultaneous presence of L. passim and C. mellificae in two B. terrestris, where these parasites had not previously been reported.

Eumelanin and pheomelanin are predominant pigments in bumblebee (Apidae: Bombus) pubescence.

BACKGROUND: Bumblebees (Hymenoptera: Apidae: Bombus) are well known for their important inter- and intra-specific variation in hair (or pubescence) color patterns, but the chemical nature of the pigments associated with these patterns is not fully understood. For example, though melanization is believed to provide darker colors, it still unknown which types of melanin are responsible for each color, and no conclusive data are available for the lighter colors, including white. METHODS: By using dispersive Raman spectroscopy analysis on 12 species/subspecies of bumblebees from seven subgenera, we tested the hypothesis that eumelanin and pheomelanin, the two main melanin types occurring in animals, are largely responsible for bumblebee pubescence coloration. RESULTS: Eumelanin and pheomelanin occur in bumblebee pubescence. Black pigmentation is due to prevalent eumelanin, with visible signals of additional pheomelanin, while the yellow, orange, red and brown hairs clearly include pheomelanin. On the other hand, white hairs reward very weak Raman signals, suggesting that they are depigmented. Additional non-melanic pigments in yellow hair cannot be excluded but need other techniques to be detected. Raman spectra were more similar across similarly colored hairs, with no apparent effect of phylogeny and both melanin types appeared to be already used at the beginning of bumblebee radiation. DISCUSSION: We suggest that the two main melanin forms, at variable amounts and/or vibrational states, are sufficient in giving almost the whole color range of bumblebee pubescence, allowing these insects to use a single precursor instead of synthesizing a variety of chemically different pigments. This would agree with commonly seen color interchanges between body segments across Bombus species.

Updated list of bumblebees (Hymenoptera: Apidae) from the Spanish Pyrenees with notes on their decline and conservation status.

The Pyrenees, where Euro-Siberian, Mediterranean and alpine faunas join together, have a large biodiversity of bumblebees. We compiled historical literature records of bumblebee species from the Spanish Pyrenees, and then compared these to contemporary surveys to assess trends in elevational distribution. Twenty-eight species (including thirty-five subspecies) were found in the contemporary survey. Nine species and two subspecies previously present were not detected, some included on the Spanish Red List. With the exception of a few species, a reduction of the altitudinal range and an orophilous tendency was observed at both upper and lower elevational levels, suggesting an upward trend towards better-preserved high areas. Our results reinforce the need to develop new protection programs and more restrictive conservation measures for bumblebee populations, species and their habitats.

The subgenus Andrena (Micrandrena) (Hymenoptera: Andrenidae) in the Iberian Peninsula.

This work presents the results obtained after a revision of the current species of the subgenus Andrena (Micrandrena) Ashmead, 1899 that appear in the Iberian Peninsula. It provides a key to the species, diagnosis, biological data, and comments about the 19 species and 5 subspecies. Additionally, it includes a redescription of five species because their original descriptions were inadequate. Newly reported for the Iberian Peninsula are Andrena floricola Eversmann, 1852 and Andrena strohmella Stöckhert, 1928. This study is the first contribution to resolve certain taxonomic problems in the subgenus Andrena (Micrandrena); however, the use of additional tools like morphometric and molecular characters are recommended in the future to confirm the taxonomic status of Andrena spreta Pérez, 1895.