Invasive vectors can induce dramatic changes in disease epidemiology. While viral emergence following geographical range expansion of a vector is well known, the influence a vector can have at the level of the host's pathobiome is less well understood. Taking advantage of the formerly heterogeneous spatial distribution of the ectoparasitic mite Varroa destructor that acts as potent virus vector among honeybees Apis mellifera, we investigated the impact of its recent global spread on the viral community of honeybees in a retrospective study of historical samples. We hypothesized that the vector has had an effect on the epidemiology of several bee viruses, potentially altering their transmissibility and/or virulence, and consequently their prevalence, abundance, or both. To test this, we quantified the prevalence and loads of 14 viruses from honeybee samples collected in mite-free and mite-infested populations in four independent geographical regions. The presence of the mite dramatically increased the prevalence and load of deformed wing virus, a cause of unsustainably high colony losses. In addition, several other viruses became more prevalent or were found at higher load in mite-infested areas, including viruses not known to be actively varroa-transmitted, but which may increase opportunistically in varroa-parasitized bees.

Urbanisation is an important global driver of biodiversity change, negatively impacting some species groups whilst providing opportunities for others. Yet its impact on ecosystem services is poorly investigated. Here, using a replicated experimental design, we test how Central European cities impact flying insects and the ecosystem service of pollination. City sites have lower insect species richness, particularly of Diptera and Lepidoptera, than neighbouring rural sites. In contrast, Hymenoptera, especially bees, show higher species richness and flower visitation rates in cities, where our experimentally derived measure of pollination is correspondingly higher. As well as revealing facets of biodiversity (e.g. phylogenetic diversity) that correlate well with pollination, we also find that ecotones in insect-friendly green cover surrounding both urban and rural sites boost pollination. Appropriately managed cities could enhance the conservation of Hymenoptera and thereby act as hotspots for pollination services that bees provide to wild flowers and crops grown in urban settings.

Red Lists are very valuable tools in nature conservation at global, continental and (sub-) national scales. In an attempt to prioritise conservation actions for European butterflies, we compiled a database with species lists and Red Lists of all European countries, including the Macaronesian archipelagos (Azores, Madeira and Canary Islands). In total, we compiled national species lists for 42 countries and national Red Lists for 34 of these. The most species-rich countries in Europe are Italy, Russia and France with more than 250 species each. Endemic species are mainly found on the Macaronesian archipelagos and on the Mediterranean islands. By attributing numerical values proportionate to the threat statuses in the different national Red List categories, we calculated a mean Red List value for every country (cRLV) and a weighted Red List value for every species (wsRLV) using the square root of the country’s area as a weighting factor. Countries with the highest cRLV were industrialised (NW) European countries such as the Netherlands, Belgium, the Czech Republic and Denmark, whereas large Mediterranean countries such as Spain and Italy had the lowest cRLV. Species for which a Red List assessment was available in at least two European countries and with a relatively high wsRLV (≥ 50) are Colias myrmidone, Pseudochazara orestes, Tomares nogelii, Colias chrysotheme and Coenonympha oedippus. We compared these wsRLVs with the species statuses on the European Red List to identify possible mismatches. We discuss how this complementary method can help to prioritise butterfly conservation on the continental and/or the (sub-)national scale.

Given ongoing biodiversity decline, an important concern is that a large fraction of species diversity is not yet documented. Correct delimitation of species remains a challenge, especially for small and morphologically uniform groups such as sweat bees (Halictidae). Here, we applied an integrative taxonomic approach to study diversity within the Neotropical sweat bee subgenus Dialictus (genus Lasioglossum). We used four statistical methods to delimit species based on cytochrome oxidase subunit I gene sequences: Automatic Barcode Gap Discovery (ABGD), two variants of the General Mixed Yule Coalescent (single-threshold (stGMYC) and Bayesian (bGMYC)) and the Refined Single Linkage analysis (RESL). We detected eight principal molecular operational taxonomic units (mOTUs). Subsequently, these lineages were evaluated using ten nuclear microsatellite loci and morphological and ecological analyses. Most mOTUs could be differentiated using microsatellites and morphology (82 % identified correctly), further supporting the status of mOTUs as independent biological units. For the two most widespread mOTUs, we analysed intra-lineage geographic variation using microsatellites but did not detect additional substructuring. We further tested if the lineages showed predictable patterns of co-occurrence and habitat preferences. While we did not find any evidence of preferential association or disassociation between taxa, we detected a slight positive effect of high crop cover favouring the abundance of some lineages. We show that integrated approaches using statistical analysis of DNA barcodes jointly with additional data can provide robust and objective means of delimiting species in morphologically difficult groups.

Summary Pollination is a major ecosystem service in which insects, particularly bees, play an important role for the reproduction of most angiosperms. Currently, this service is considered under threat due to reported bee declines. Moderately urbanised areas could be important for pollinators and pollination; however, compared to agricultural and natural systems, they are poorly studied. Here, we investigated the relative effects of local habitat quality and anthropogenic land use across an agricultural to urban gradient for local plant and flying insect communities. We quantified local flower visitor networks and related network architecture to these local and landscape factors using structural equation modelling. Flower visitor network architecture is often assumed to act as a surrogate for the ecosystem service of pollination. To test this idea, we related network metrics to pollination of four experimental, insect pollinator‐dependent plant species. Overall, local land use markedly influenced plant and flying insect communities. Flower richness and bee richness were higher in urban compared to agricultural areas. Flower visitor network metrics (e.g. linkage density) increased with the proportion of urban area surrounding a site. Also, relative to agricultural areas, urban flower visitors were more generalised and foraged from a higher number of plant species, likely a consequence of higher urban flowering plant richness. However, urban bees also visited a lower proportion of the available flowering plants (higher specialisation). Surprisingly, linkage density, network specialisation and flower visitor generality were not related to pollination of our four experimental plants per se. Rather, it was the proportion of urban cover, flying insect abundance and bee richness that were positively related to pollination. Our findings show strong effects of local land use on plant and flying insect communities and flower visitor interaction networks. We observed increased overall visitation rates and pollination services to our experimental plants in urban compared to agricultural areas, despite increased urban flower visitor generality. Indeed, flower visitor network metrics were a poor proxy of provision of the ecosystem service of pollination. Nevertheless, our results point to potential facilitating effects of diverse urban floral and bee communities for pollination. A lay summary is available for this article.

Mitochondrial cytochrome oxidase I (COI) partial sequences are widely used in taxonomy for species identification. Increasingly, these sequence identities are combined with modelling approaches to delineate species. Yet the validity of species delineation based on such DNA 'barcodes' is rarely tested and may be called into question by phenomena such as ancestral polymorphisms in DNA sequences, phylogeographic divergence, mitochondrial introgression and hybridization, or distortion of mitochondrial inheritance through such factors as Wolbachia infection. The common and widespread European bumble bee Bombus lucorum s. lato contains three distinct mitochondrial DNA lineages that are assumed to represent three cryptic species, namely Bombus cryptarum, B. lucorum s. str. and B. magnus. To test whether nuclear gene pools of the three putative species were differentiated, we genotyped 304 sympatric members of the lucorum complex (54 B. cryptarum females, 168 B. lucorum s. str. females and 82 B. magnus females, as defined using mtDNA COI haplotypes) from 11 localities spread across the island of Ireland at seven nuclear microsatellite loci. Multilocus genotypes clustered into three discrete groups that largely corresponded to the three mtDNA lineages: B. cryptarum, B. lucorum s. str. and B. magnus. The good fit of mitochondrial haplotype to nuclear (microsatellite) genotypic data supports the view that these three bumble bee taxa are reproductively isolated species, as well as providing a vindication of species identity using so-called DNA barcodes.

Summary Traditional tropical agriculture often entails a form of slash‐and‐burn land management that may adversely affect ecosystem services such as pollination, which are required for successful crop yields. The Yucatán Peninsula of Mexico has a >4000 year history of traditional slash‐and‐burn agriculture, termed ‘milpa’. Hot ‘Habanero’ chilli is a major pollinator‐dependent crop that nowadays is often grown in monoculture within the milpa system. We studied 37 local farmers’ chilli fields (sites) to evaluate the effects of landscape composition on bee communities. At 11 of these sites, we undertook experimental pollination treatments to quantify the pollination of chilli. We further explored the relationships between landscape composition, bee communities and pollination service provision to chilli. Bee species richness, particularly species of the family Apidae, was positively related to the amount of forest cover. Species diversity decreased with increasing proportion of crop land surrounding each sampling site. Sweat bees of the genus Lasioglossum were the most abundant bee taxon in chilli fields and, in contrast to other bee species, increased in abundance with the proportion of fallow land, gardens and pastures which are an integral part of the milpa system. There was an average pollination shortfall of 21% for chilli across all sites; yet the shortfall was unrelated to the proportion of land covered by crops. Rather, chilli pollination was positively related to the abundance of Lasioglossum bees, probably an important pollinator of chilli, as well indirectly to the proportion of fallow land, gardens and pastures that promote Lasioglossum abundance. Synthesis and applications . Current, low‐intensity traditional slash‐and‐burn ( milpa ) agriculture provides Lasioglossum spp. pollinators for successful chilli production; fallow land, gardens and pasture therefore need to be valued as important habitats for these and related ground‐nesting bee species. However, the negative impact of agriculture on total bee species diversity highlights how agricultural intensification is likely to reduce pollination services to crops, including chilli. Indeed, natural forest cover is vital in tropical Yucatán to maintain a rich assemblage of bee species and the provision of pollination services for diverse crops and wild flowers.

Habitat fragmentation and loss are important drivers of genetic differentiation, often leading to a decrease in genetic diversity. Yet, natural populations of tropical bees often show a lack of differentiation, even in fragmented landscapes, suggesting resilience to deal with unfavourable land use. It is not clear what leads to this lack of differentiation, but large population sizes, high rates of dispersal and stable demography likely play important roles. Here, we investigate the population genetic structure and infer the present and historic demography of the eusocial stingless bee Partamona bilineata from tropical montane cloud forests in Guatemala. We used microsatellites and mitochondrial DNA to test for genetic differentiation, to infer migration rates, and to evaluate the effects of landscape. We also used demographic modelling to trace population sizes over time. We found that six populations of P. bilineata exhibited only subtle differentiation, with the exception of one site at the edge of the cloud forest, which was clearly distinct from all others. Effective population sizes (number of colonies) appeared to be rather small (18 ± 6 colonies) compared to the original sample size (N = 51 ± 9), but stable over time, and inferred rates of gene flow were low; yet, no genetic bottleneck was detected. A statistical model including elevation was the best in explaining the observed pattern of differentiation. We find that P. bilineata does not exhibit strong genetic structure, making it a resilient species for provision of pollination services. But, at the same time, our data point to the potential vulnerability of this and similar species, as effective population sizes appear to be low and hence populations may be easily affected by future environmental change. As such, P. bilineata may be representative of many other tropical stingless bees, for which lack of differentiation has been invoked.

Animal-mediated pollination is required for the reproduction of the majority of angiosperms, and pollinators are therefore essential for ecosystem functioning and the economy. Two major threats to insect pollinators are anthropogenic land-use change and the spread of pathogens, whose effects may interact to impact pollination. Here, we investigated the relative effects on the ecosystem service of pollination of (i) land-use change brought on by agriculture and urbanization as well as (ii) the prevalence of pollinator parasites, using experimental insect pollinator-dependent plant species in natural pollinator communities. We found that pollinator habitat (i.e. availability of nesting resources for ground-nesting bees and local flower richness) was strongly related to flower visitation rates at the local scale and indirectly influenced plant pollination success. At the landscape scale, pollination was positively related to urbanization, both directly and indirectly via elevated visitation rates. Bumblebees were the most abundant pollinator group visiting experimental flowers. Prevalence of trypanosomatids, such as the common bumblebee parasiteCrithidia bombi,was higher in urban compared with agricultural areas, a relationship which was mediated through higherBombusabundance. Yet, we did not find any top-down, negative effects of bumblebee parasitism on pollination. We conclude that urban areas can be places of high transmission of both pollen and pathogens.

This report presents the fifth version of the European Grassland Butterfly Indicator, one of the EU biodiversity indicators of the European Environment Agency.

The genetic structure of the earth bumblebee (Bombus terrestris L.) was examined across 22 wild populations and two commercially reared populations using eight microsatellite loci and two mitochondrial genes. Our study included wild bumblebee samples from six populations in Ireland, one from the Isle of Man, four from Britain and 11 from mainland Europe. A further sample was acquired from New Zealand. Observed levels of genetic variability and heterozygosity were low in Ireland and the Isle of Man, but relatively high in continental Europe and among commercial populations. Estimates of F-st revealed significant genetic differentiation among populations. Bayesian cluster analysis indicated that Irish populations were highly differentiated from British and continental populations, the latter two showing higher levels of admixture. The data suggest that the Irish Sea and prevailing south westerly winds act as a considerable geographical barrier to gene flow between populations in Ireland and Britain; however, some immigration from the Isle of Man to Ireland was detected. The results are discussed in the context of the recent commercialization of bumblebees for the European horticultural industry.

Riparian systems are prone to invasion by alien plant species. The spread of invasive riparian plants may be facilitated by hydrochory, the transport of seeds by water, but while ecological studies have highlighted the possible role of upstream source populations in the establishment and persistence of stands of invasive riparian plant species, population genetic studies have as yet not fully addressed the potential role of hydrochoric dispersal in such systems.A population genetics approach based on a replicated bifurcate sampling design is used to test hypotheses consistent with patterns of unidirectional, linear gene flow expected under hydrochoric dispersal of the invasive riparian plant Impatiens glandulifera in two contrasting river systems.A significant increase in levels of genetic diversity downstream was observed, consistent with the accumulation of propagules from upstream source populations, and strong evidence was found for organization of this diversity between different tributaries, reflecting the dendritic organization of the river systems studied.These findings indicate that hydrochory, rather than anthropogenic dispersal, is primarily responsible for the spread of I. glandulifera in these river systems, and this is relevant to potential approaches to the control of invasive riparian plant species.

A global decline in pollinator abundance and diversity has demanded increased research attention to the ecology and genetics of bumblebees. However, as progressively more restrictions are placed on sampling for insects, researchers are increasingly obliged to use archival specimens collected for purposes other than genetic analyses. In this study we assessed the suitability, for population genetic studies, of popular, low-cost methods for preservation and storage of bumblebee specimens. Specimens of Bombus terrestris L. were held under six storage regimes for up to two years. DNA was extracted from the samples using three extraction protocols and the quality of the DNA was examined using PCR amplification of a mitochondrial and a nuclear gene. All extraction and storage methods provided sufficient DNA for successful PCR amplification. However, samples preserved in acetone or at freezing temperatures yielded the highest DNA concentrations. DNA yields from pinned specimens at room temperature declined over time, particularly when using standard extraction techniques. DNA concentrations were significantly lower from specimens preserved in 70% ethanol compared to all other extraction techniques and declined linearly over the two years of storage. These results indicate that two of the most popular insect storage methods (pinning and storage in ethanol) should be avoided for the long-term preservation of genetic material for future studies. We suggest that optimal insect preservation methods should be incorporated into research protocols in order to best capitalise on limited collection opportunities.

The worldwide spread of diseases is considered a major threat to biodiversity and a possible driver of the decline of pollinator populations, particularly when novel species or strains of parasites emerge. Previous studies have suggested that populations of introduced European honeybee (Apis mellifera) and bumblebee species (Bombus terrestris and Bombus ruderatus) in Argentina share the neogregarine parasite Apicystis bombi with the native bumblebee (Bombus dahlbomii). In this study we investigated whether A. bombi is acting as an emergent parasite in the non-native populations. Specifically, we asked whether A. bombi, recently identified in Argentina, was introduced by European, non-native bees. Using ITS1 and ITS2 to assess the parasite's intraspecific genetic variation in bees from Argentina and Europe, we found a largely unstructured parasite population, with only 15% of the genetic variation being explained by geographic location. The most abundant haplotype in Argentina (found in all 9 specimens of non-native species) was identical to the most abundant haplotype in Europe (found in 6 out of 8 specimens). Similarly, there was no evidence of structuring by host species, with this factor explaining only 17% of the genetic variation. Interestingly, parasites in native Bombus ephippiatus from Mexico were genetically distant from the Argentine and European samples, suggesting that sufficient variability does exist in the ITS region to identify continent-level genetic structure in the parasite. Thus, the data suggest that A. bombi from Argentina and Europe share a common, relatively recent origin. Although our data did not provide information on the direction of transfer, the absence of genetic structure across space and host species suggests that A. bombi may be acting as an emergent infectious disease across bee taxa and continents.

Christian W W Pirk, Joachim R de Miranda, Matthew Kramer, Tomàs E Murray, Francesco Nazzi, Dave Shutler, Jozef J M van der Steen and Coby van Dooremalen Social Insect Research Group, Department of Zoology & Entomology, University of Pretoria, Private Bag X20 Hatfield 0028, Pretoria, South Africa. Department of Ecology, Swedish University of Agricultural Sciences, Box 7044, 750 07, Uppsala, Sweden. Biometrical Consulting Service, Agricultural Research Service/USDA, Beltsville, MD, 20705, USA. Institute of Biology, Martin Luther University Halle-Wittenberg, Hoher Weg 8, Halle (Saale) 06120, Germany. Dipartimento di Scienze Agrarie e Ambientali, Università di Udine, via delle Scienze 206, 33100 Udine, Italia. Department of Biology, Acadia University, Wolfville, Nova Scotia, B4P 2R6, Canada. Bees@wur, Bio-Interactions and Plant Health, Plant Research International, Wageningen UR, Droevendaalsesteeg 1, 6708 PB Wageningen, Netherlands.

Worldwide, wild bumble bees (Bombus spp.) are experiencing marked declines, with potentially up to 11% of species currently under threat. Recent studies from North America suggest that disease transmission from commercially reared bumble bees to wild populations has led to marked range contractions in some species. In Europe, data on the prevalence of pathogen spillover from commercial to wild bumble bee populations is lacking, despite the widespread production and transport of hives within the EU since the early 1980s. We determined the permeability of cropping systems to commercial bumble bees, and quantified the prevalence of four pathogens in commercial Bombus terrestris hives and adjacent conspecific populations at increasing distances from greenhouses in Ireland. Commercial bumble bees collected from 31% to 97% of non-crop pollen, depending on the cropping system, and hives had markedly higher frequencies of two gut parasites, Crithidia spp. and Nosema bombi, compared to adjacent populations, but were free of tracheal mites. The highest prevalence of Crithida was observed within 2 km of greenhouses and the probability of infection declined in a host sex- and pathogen-specific manner up to 10 km. We suggest implementing measures that prevent the interaction of commercially reared and wild bumble bees by integrating the enforcement of national best management practices for users of commercial pollinators with international legislation that regulates the sanitation of commercial hives in production facilities.

SummaryIn this article we provide guidelines on statistical design and analysis of data for all kinds of honey bee research. Guidelines and selection of different methods presented are, at least partly, based on experience. This article can be used: to identify the most suitable analysis for the type of data collected; to optimise one's experimental design based on the experimental factors to be investigated, samples to be analysed, and the type of data produced; to determine how, where, and when to sample bees from colonies; or just to inspire. Also included are guidelines on presentation and reporting of data, as well as where to find help and which types of software could be useful.

SummarySummaryAdult honey bees are maintained in vitro in laboratory cages for a variety of purposes. For example, researchers may wish to perform experiments on honey bees caged individually or in groups to study aspects of parasitology, toxicology, or physiology under highly controlled conditions, or they may cage whole frames to obtain newly emerged workers of known age cohorts. Regardless of purpose, researchers must manage a number of variables, ranging from selection of study subjects (e.g. honey bee subspecies) to experimental environment (e.g. temperature and relative humidity). Although decisions made by researchers may not necessarily jeopardize the scientific rigour of an experiment, they may profoundly affect results, and may make comparisons with similar, but independent, studies difficult. Focusing primarily on workers, we provide recommendations for maintaining adults under in vitro laboratory conditions, whilst acknowledging gaps in our understanding that require further attention. We specifically describe how to properly obtain honey bees, and how to choose appropriate cages, incubator conditions, and food to obtain biologically relevant and comparable experimental results. Additionally, we provide broad recommendations for experimental design and statistical analyses of data that arises from experiments using caged honey bees. The ultimate goal of this, and of all COLOSS BEEBOOK papers, is not to stifle science with restrictions, but rather to provide researchers with the appropriate tools to generate comparable data that will build upon our current understanding of honey bees.ResumenLas abejas adultas se mantienen in vitro en cajas de laboratorio para una variedad de propósitos. Por ejemplo, los investigadores pueden realizar experimentos con las abejas de miel enjauladas individualmente o en grupos para estudiar aspectos de la parasitología, toxicología y fisiología en condiciones muy controladas, o pueden meter en las cajas panales completos para obtener obreras recién emergidas de cohortes de edad conocida. Independientemente del propósito, los investigadores deben manejar una serie de variables, que van desde la selección de los sujetos a estudiar (por ejemplo, la subespecies de abeja), al ambiente experimental (por ejemplo, temperatura y humedad relativa). Aunque las decisiones tomadas por los investigadores no tienen por qué poner en peligro el rigor científico de un experimento, si que pueden afectar profundamente a los resultados, y pueden dificultar las comparaciones con estudios similares pero independientes. Centrándonos principalmente en obreras, ofrecemos recomendaciones para mantener adultos en condiciones de laboratorio in vitro, si bien reconocemos algunas lagunas en nuestro conocimiento que requieren una mayor atención. En especial, se describe cómo obtener correctamente abejas, y cómo elegir cajas adecuadas, las condiciones de incubación, y los alimentos para obtener resultados experimentales biológicamente relevantes y comparables. Además, ofrecemos recomendaciones generales para el diseño experimental y el análisis estadístico de los datos que surgen de experimentos con abejas enjauladas. El objetivo final de éste, y de todos los artículos de BEEBOOKy COLOSS, no es limitar la ciencia con restricciones, sino más bien proporcionar a los investigadores las herramientas necesarias para obtener datos comparables que se basen en el conocimiento actual de las abejas melíferas.很多研究都需要在实验室内应用蜂笼饲养成年蜜蜂，比如，研究者可能应用单个蜂笼或多个蜂笼开展严格控制条件下的寄生虫学、毒理学或生理 学研究。也可能把整个巢脾关入笼中来得到日龄明确的刚羽化出房的蜜蜂。不管目的如何，研究者必须控制多个变量：从研究对象（不同的蜜蜂 亚种）到实验环境（温度和相对湿度等。虽然研究者的选择可能不一定会损害实验的科学性，但可能会显著影响实验结果，使独立实验成为相关 实验。围绕饲养工蜂，我们推荐了在实验室条件下饲养成年蜂的方法。特别描述了如何恰当的饲养蜜蜂以及如何选择饲养笼、温箱和饲料以得到 具生物学意义并具可比性的实验结果。此外，针对实验设计和数据的统计分析还给出了大量建议。本文以及本书中所有文章所涉及的研究方法， 其最终目的是给研究者提供合适的研究工具，得到具有可比性的数据，推进我们对蜜蜂的认识，而不是设立技术障碍，限制科学发展。Keywords: Apis melliferahoney beecolony losseshoardingcagein vitrolaboratoryCOLOSSNosematoxicologyBEEBOOK