Integrative analysis reveals cryptic speciation linked to habitat differentiation within Albanian populations of the anomalous blues (Lepidoptera, Lycaenidae, Polyommatus Latreille, 1804).

The Balkan Peninsula is one of the greatest hotspots for biodiversity in Europe. While the region has been investigated thoroughly, some parts remain understudied and may still harbour undiscovered diversity, even in well-studied organisms such as Lepidoptera. Here we investigated the group of the so-called anomalous blue butterflies, also known as 'brown complex' of the subgenus Agrodiaetus Hübner, 1822 including the taxa of the entire Polyommatusaroaniensis (Brown, 1976) species complex. This species complex is distributed in the southern part of the Balkan Peninsula and known to be represented by three closely related allopatric species, differentiated by their chromosome numbers (n) and mitochondrial (mt) DNA. These are P.aroaniensis sensu stricto (Southern Greece, Peloponnese, n=47-48; mt haplogroup aroa1), P.timfristos Lukhtanov, Vishnevskaya et Shapoval, 2016 (Central Greece, Attika, n=38, aroa2) and P.orphicus Kolev, 2005 (North-Eastern Greece, Southern Bulgaria, n=41-42, orph1). Based on an analysis of chromosomal, molecular and morphological markers, we demonstrate that a fourth taxon of this species complex exists in Albania. This taxon possesses the mt haplogroup aroa3, which is the most differentiated within the entire P.aroaniensis species complex, and the karyotype (n=42-43), which differs by one fixed chromosome fission from P.orphicus. The Albanian taxon seems to be ecologically specialised (habitat on dark-coloured, ophiolitic substrate soils) and differs in colouration (wing reflectance) from the others taxa of the P.aroaniensis species group. Based on the evidence here presented and following the current view of the taxonomy of the group, we propose considering the Albanian taxon as a new species, here described as Polyommatuslurae sp. nov. At the contact zone between the new species and P.orphicus, in addition to typical ones, we detected specimens with haplogroup orph2, karyotype n=43 and intermediate morphology, which seem to represent P.lurae × P.orphicus hybrids.

Managed bumble bees acquire parasites from their foraging environment: A case study on parasite spillback.

The use of commercially reared bumble bees in agricultural environments has been recognized as a potential threat to wild pollinators due to competition, genetic contamination, and most notably, disease transmission. Higher parasite prevalence near greenhouses where managed bumble bees are used has been linked to parasite spillover from managed to wild bees. However, pathogen transmission is not unidirectional, and can also flow from wild to managed bees. These newly infected managed bees can subsequently re-infect (other) wild bees, in a process known as spillback, which is an alternative explanation for the increased parasite prevalence near greenhouses. Reducing parasite prevalence in managed bees is key to controlling host-parasite dynamics in cases of spillover; in spillback, producing managed bees that are resilient to infection is important. Here we establish that the managed bumble bee Bombus terrestris can acquire parasites from their foraging environment, which is the major infection route for Apicystis spp. and Crithidia spp., but not for Nosema spp.. Managed B. terrestris were found to have a higher prevalence of Crithdia and a higher load of Apicystis than local wild conspecifics, showing that for these parasites, spillback is a possible risk scenario.

Reduced nest development of reared Bombus terrestris within apiary dense human-modified landscapes.

Wild bees are in decline on a local to global scale. The presence of managed honey bees can lead to competition for resources with wild bee species, which has not been investigated so far for human-modified landscapes. In this study we assess if managed honey bee hive density influence nest development (biomass) of bumble bees, an important trait affecting fitness. We hypothesize that domesticated honey bees can negatively affect Bombus terrestris nest development in human-modified landscapes. In Flanders, Belgium, where such landscapes are dominantly present, we selected 11 locations with landscape metrics ranging from urban to agricultural. The bee hive locations were mapped and each location contained one apiary dense (AD) and one apiary sparse (AS) study site (mean density of 7.6 ± 5.7 managed honey bee hives per km2 in AD sites). We assessed the effect of apiary density on the reproduction of reared B. terrestris nests. Reared B. terrestris nests had more biomass increase over 8 weeks in apiary sparse (AS) sites compared to nests located in apiary dense (AD) sites. This effect was mainly visible in urban locations, where nest in AS sites have 99.25 ± 60.99 g more biomass increase compared to nest in urban AD sites. Additionally, we found that managed bumble bee nests had higher biomass increase in urban locations. We conclude that the density of bee hives is a factor to consider in regard to interspecific competition between domesticated honey bees and bumble bees.

Varroa destructor Macula-like virus, Lake Sinai virus and other new RNA viruses in wild bumblebee hosts (Bombus pascuorum, Bombus lapidarius and Bombus pratorum).

Pollinators such as bumblebees (Bombus spp.) are in decline worldwide which poses a threat not only for ecosystem biodiversity but also to human crop production services. One main cause of pollinator decline may be the infection and transmission of diseases including RNA viruses. Recently, new viruses have been discovered in honeybees, but information on the presence of these in wild bumblebees is largely not available. In this study, we investigated the prevalence of new RNA viruses in Bombus species, and can report for the first time Varroa destructor Macula-like virus (VdMLV) and Lake Sinai virus (LSV) infection in multiple wild bumblebee hosts of Bombus pascuorum, Bombus lapidarius and Bombus pratorum. We sampled in 4 locations in Flanders, Belgium. Besides, we confirmed Slow bee paralysis virus (SBPV) in wild bumblebees, but no positive samples were obtained for Big Sioux river virus (BSRV). Secondly, we screened for the influence of apiaries on the prevalence of these viruses. Our results indicated a location effect for the prevalence of VdMLV in Bombus species, with a higher prevalence in the proximity of honeybee apiaries mainly observed in one location. For LSV, the prevalence was not different in the proximity or at a 1.5 km-distance of apiaries, but we reported a different isolate with similarities to LSV-2 and "LSV-clade A" as described by Ravoet et al. (2015), which was detected both in Apis mellifera and Bombus species. In general, our results indicate the existence of a disease pool of new viruses that seems to be associated to a broad range of Apoidae hosts, including multiple Bombus species.

16S rRNA Amplicon Sequencing Demonstrates that Indoor-Reared Bumblebees (Bombus terrestris) Harbor a Core Subset of Bacteria Normally Associated with the Wild Host.

A MiSeq multiplexed 16S rRNA amplicon sequencing of the gut microbiota of wild and indoor-reared Bombus terrestris (bumblebees) confirmed the presence of a core set of bacteria, which consisted of Neisseriaceae (Snodgrassella), Orbaceae (Gilliamella), Lactobacillaceae (Lactobacillus), and Bifidobacteriaceae (Bifidobacterium). In wild B. terrestris we detected several non-core bacteria having a more variable prevalence. Although Enterobacteriaceae are unreported by non next-generation sequencing studies, it can become a dominant gut resident. Furthermore the presence of some non-core lactobacilli were associated with the relative abundance of bifidobacteria. This association was not observed in indoor-reared bumblebees lacking the non-core bacteria, but having a more standardized microbiota compared to their wild counterparts. The impact of the bottleneck microbiota of indoor-reared bumblebees when they are used in the field for pollination purpose is discussed.

Commercial bumblebee hives to assess an anthropogenic environment for pollinator support: a case study in the region of Ghent (Belgium).

Anthropogenic changes of the environment influence the distribution and abundance of pollinators such as bumblebees and have been proposed as one of the main causes in their worldwide decline. In order to evaluate the impact of expanding anthropogenic landscapes on supporting pollinator potential, reliable tools are needed. Bombus terrestris is one of the most abundant bumblebee species in Europe, and these bumblebees are known as generalist pollinators of not only wild flowers in nature but also of crops in agriculture. For more than two decades, these bumblebees have been commercially mass reared for biological pollination in greenhouses. In this project, we placed commercial hives of the bumblebee B. terrestris containing one queen and 40 workers, in three different locations in the region of Ghent (Belgium), and the performance of these hives was followed during a 4-week period in spring 2012. In parallel, we determined the floral richness and diversity index in the chosen study sites. The sites consisted of a rich urban environment with patchy green areas opposed to an urban environment with poor landscape metrics; a third rural study site showed average positive landscape metrics. The results demonstrated that the hive biomass and numbers of workers increased significantly in the rich compared to the poor environment, providing a mechanism to discriminate between study sites. In addition, the bumblebee-collected pollen showed that the flowering plants Salix spp. and Rosaceae/Prunus spp. are dominant food sources in all anthropogenic environments during early spring. Finally, the results are discussed in relation to the optimization of the experimental setup and to the use of commercial bumblebee hives in assessing local pollinator support within any given environment.