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.

Optimising the production of insects for food and feed and ensuring their health are growing concerns for producers. Insects suffer from a range of insect pathogenic microorganisms, and the management of such diseases is essential. One solution is the introduction of beneficial probiotic bacteria into the diet of the insects. Here, we show that a lactic acid bacterial strain, Pediococcus pentosaceus, isolated from the gut of the mealworm, Tenebrio molitor, was able to inhibit the growth of selected insect pathogens in vitro. Using in vivo assessments of the host's fitness benefits conferred by the lactic bacterium we show a significant effect of P. pentosaceus on larval growth rate and survival into adulthood. Gut microbiota analysis focusing on bacterial composition based on 16S rRNA gene amplicon sequencing suggests that P. pentosaceus could have successfully colonised the guts, or altered their bacteria, of the larvae that received it. Finally, we discuss our results in the context of mass insect production systems and outline the remaining work needed to explore and secure the role of beneficial bacterial additives in the field.

ABSTRACTFungi in the genusMetarhiziumare soil-borne plant-root endophytes and rhizosphere colonisers, but also potent insect pathogens with highly variable host ranges. These ascomycete fungi are predominantly asexually reproducing and ancestrally haploid, but two independent origins of persistent diploidy within the Coleoptera-infectingM. majusspecies complex are known and has been attributed to incomplete chromosomal segregation following meiosis during the sexual cycle. There is also evidence for infrequent sexual cycles in the locust-specific pathogenic fungusMetarhizium acridum(Hypocreales: Clavicipitaceae), which is an important entomopathogenic biocontrol agent used for the control of grasshoppers in agricultural systems as an alternative to chemical control. Here, we show that the genome of theM. acridumisolate ARSEF 324, which is formulated and commercially utilised under the name ‘Green Guard’, is functionally diploid. We used single-molecule real-time (SMRT) sequencing technology to complete a high-quality assembly of ARSEF 324. Kmer frequencies, intragenomic collinearity between contigs and single nucleotide variant read depths across the genome revealed the first incidence of diploidy described within the speciesM. acridum. The haploid assembly of 44.7 Mb consisting of 20.8% repetitive elements, which is the highest proportion described of anyMetarhiziumspecies. The genome assembly and the inferred diploid state, can shed light on past research on this strain and could fuel future investigation into the fitness landscape of aberrant ploidy levels, not least in the contest of biocontrol agents.

Entomopathogenic fungi are widespread in terrestrial ecosystems and can be used as models for studying host-pathogen interactions. However, the associations between fungal virulence and ecosystem abundance, and the importance of virulence for ecological fitness are poorly understood. To study these associations, we investigated five selected strains from a natural community of the entomopathogenic genus Beauveria spp. representing different phylogenetic clades with distinct abundance and distribution patterns within an agricultural field and its neighboring hedgerow. We measured pathogen fitness traits related to virulence in a naive host, the beetle Tenebrio molitor. We found that the two strains belonging to the most abundant Beauveria bassiana clade, which was the only clade represented in the agricultural field, exhibited the highest relative virulence in terms of host mortality, transmission probability, in vitro growth rate and germination speed. In addition, gene expression of specific virulence factors was higher compared to the strain belonging to the second most abundant B. bassiana clade. The study highlights the relevance of fungal virulence traits and adaptations to explain abundance patterns within natural communities of entomopathogenic fungi. (C) 2020 Elsevier Ltd and British Mycological Society. All rights reserved.

Physiological constraints restrict specialist pathogens from infecting new hosts. From an applied perspective, a narrow host range makes specialist pathogens interesting for targeting specific pest insects since they have minimal direct effects on non-target species. Entomopathogenic fungi of the genus Entomophthora are dipteran-specific but have not been investigated for their ability to infect the spotted wing drosophila (SWD; Drosophila suzukii) a fruit-damaging pest invasive to Europe and America. Our main goal was to study whether SWD is in the physiological host range of the entomophthoralean species E. muscae. We investigated pathogenicity and virulence of E. muscae towards its main natural host, the housefly Musca domestica, and towards SWD. We found that E. muscae readily infected and significantly reduced survival of SWD by 27.3% with the majority of flies dying 4-8 days post-exposure. In comparison with SWD, infection of the natural host M. domestica resulted in an even higher mortality of 62.9% and larger conidial spores of E. muscae, reflecting the physiological constraints of the pathogen in the atypical host. We demonstrated that pathogens of the E. muscae species complex that typically have a narrow natural host range of one or few dipteran species are able to infect SWD, and we described a new method for in vivo transmission and infection of an entomophthoralean fungus to SWD.

Bees are considered to be threatened globally, with severe overwinter losses of the most important commercial pollinator, the Western honeybee, a major concern in the Northern Hemisphere. Emerging infectious diseases have risen to prominence due to their temporal correlation with colony losses. Among these is Deformed wing virus (DWV), which has been frequently linked to colony mortality. We now provide evidence of a strong statistical association between overwintering colony decline in the field and the presence of DWV genotype-B (DWV-B), a genetic variant of DWV that has recently been shown to be more virulent than the original DWV genotype-A. We link the prevalence of DWV-B directly to a quantitative measure of overwinter decline (workforce mortality) of honeybee colonies in the field. We demonstrate that increased prevalence of virus infection in individual bees is associated with higher overwinter mortality. We also observed a substantial reduction of infected colonies in the spring, suggesting that virus-infected individuals had died during the winter. Our findings demonstrate that DWV-B, plus possible A/B recombinants exhibiting DWV-B at PCR primer binding sites, may be a major cause of elevated overwinter honeybee loss. Its potential emergence in naïve populations of bees may have far-reaching ecological and economic impacts.

Task allocation in social insect colonies is generally organised into an age-related division of labour, termed the temporal polyethism schedule, which may in part have evolved to reduce infection of the colony’s brood by pests and pathogens. The temporal polyethism schedule is sensitive to colony perturbations that may lead to adaptive changes in task allocation, maintaining colony homeostasis. Though social insects can be infected by a range of parasites, little is known of how these parasites impact within-colony behaviour and the temporal polyethism schedule. We use honey bees ( Apis mellifera ) experimentally infected by two of their emerging pathogens, Deformed wing virus (DWV), which is relatively understudied concerning its behavioural impact on its host, and the exotic microsporidian Nosema ceranae . We examined parasite effects on host temporal polyethism and patterns of activity within the colony. We found that pathogens accelerated the temporal polyethism schedule, but without reducing host behavioural repertoire. Infected hosts exhibited increased hyperactivity, allocating more time to self-grooming and foraging-related tasks. The strength of behavioural alterations we observed was found to be pathogen specific; behavioural modifications were more pronounced in virus-treated hosts versus N. ceranae -treated hosts, with potential benefits for the colony in terms of reducing within-colony transmission. Investigating the effects of multiple pathogens on behavioural patterns of social insects could play a crucial role in understanding pathogen spread within a colony and their effects on colony social organisation.

Emerging infectious diseases (EIDs) have contributed significantly to the current biodiversity crisis, leading to widespread epidemics and population loss. Owing to genetic variation in pathogen virulence, a complete understanding of species decline requires the accurate identification and characterization of EIDs. We explore this issue in the Western honeybee, where increasing mortality of populations in the Northern Hemisphere has caused major concern. Specifically, we investigate the importance of genetic identity of the main suspect in mortality, deformed wing virus (DWV), in driving honeybee loss. Using laboratory experiments and a systematic field survey, we demonstrate that an emerging DWV genotype (DWV-B) is more virulent than the established DWV genotype (DWV-A) and is widespread in the landscape. Furthermore, we show in a simple model that colonies infected with DWV-B collapse sooner than colonies infected with DWV-A. We also identify potential for rapid DWV evolution by revealing extensive genome-wide recombination in vivo . The emergence of DWV-B in naive honeybee populations, including via recombination with DWV-A, could be of significant ecological and economic importance. Our findings emphasize that knowledge of pathogen genetic identity and diversity is critical to understanding drivers of species decline.

Transmission among colonies is a central feature for the epidemiology of honey bee pathogens. High colony abundance may promote transmission among colonies independently of apiary layout, making colony abundance a potentially important parameter determining pathogen prevalence in populations of honey bees. To test this idea, we sampled male honey bees (drones) from seven distinct drone congregation areas (DCA), and used their genotypes to estimate colony abundance at each site. A multiplex ligation dependent probe amplification assay (MLPA) was used to assess the prevalence of ten viruses, using five common viral targets, in individual drones. There was a significant positive association between colony abundance and number of viral infections. This result highlights the potential importance of high colony abundance for pathogen prevalence, possibly because high population density facilitates pathogen transmission. Pathogen prevalence in drones collected from DCAs may be a useful means of estimating the disease status of a population of honey bees during the mating season, especially for localities with a large number of wild or feral colonies.

Nosema apis and Nosema ceranae are gut parasites that infect western honey bees (Apis mellifera) worldwide. N. ceranae is an exotic infectious disease agent of A. mellifera, having been originally described in the Asian honey bee (Apis cerana), while N. apis is native to the western honey bee. To better understand the dynamics and epidemiology of the two pathogens, we examined the impact of European isolates of both Microsporidia on the longevity of European A. mellifera in a controlled laboratory experiment. N. ceranae caused slightly higher host mortality compared to N. apis, but differences in virulence were subtle and non-significant. Variation across published studies may reflect geographic differences in the coadaptation of hosts and parasites and seasonal differences in host susceptibility.

Nosema ceranae, an emerging pathogen of the western honeybee (Apis mellifera), is implicated in recent pollinator losses and causes severe energetic stress. However, whether precocious foraging and accelerated behavioural maturation in infected bees are caused by the infection itself or via indirect energetic stress remains unknown. Using a combination of nutritional and infection treatments, we investigated how starvation and infection alters the regulation of adipokinetic hormone (AKH) and octopamine, two highly conserved physiological pathways that respond to energetic stress by mobilizing fat stores and increasing search activity for food. Although there was no response from AKH when bees were experimentally infected with N. ceranae or starved, supporting the notion that honeybees have lost this pathway, there were significant regulatory changes in the octopamine pathway. Significantly, we found no evidence of acute energetic stress being the only cause of symptoms associated with N.ceranae infection. Therefore, the parasite itself appears to alter regulatory components along a highly conserved physiological pathway in an infection-specific manner. This indicates that pathogen-induced behavioural alteration of chronically infected bees should not just be viewed as a coincidental short-term by-product of pathogenesis (acute energetic stress) and may be a result of a generalist manipulation strategy to obtain energy for reproduction.

Pathogens and parasites may facilitate their transmission by manipulating host behavior. Honeybee pathogens and pests need to be transferred from one colony to another if they are to maintain themselves in a host population. Inter-colony transmission occurs typically through honeybee workers not returning to their home colony but entering a foreign colony ("drifting"). Pathogens might enhance drifting to enhance transmission to new colonies. We here report on the effects infection by ten honeybee viruses and Nosema spp., and Varroa mite infestation on honeybee drifting. Genotyping of workers collected from colonies allowed us to identify genuine drifted workers as well as source colonies sending out drifters in addition to sink colonies accepting them. We then used network analysis to determine patterns of drifting. Distance between colonies in the apiary was the major factor explaining 79% of drifting. None of the tested viruses or Nosema spp. were associated with the frequency of drifting. Only colony infestation with Varroa was associated with significantly enhanced drifting. More specifically, colonies with high Varroa infestation had a significantly enhanced acceptance of drifters, although they did not send out more drifting workers. Since Varroa-infested colonies show an enhanced attraction of drifting workers, and not only those infected with Varroa and its associated pathogens, infestation by Varroa may also facilitate the uptake of other pests and parasites.

Two pathogens co-infecting a common host can either interact positively (facilitation), negatively (competition) or act independently. A correlative study has suggested that two pathogens of the honey bee, Nosema ceranae and Deformed wing virus (DWV), interact negatively within a host (Costa et al., 2011). To test this hypothesis, we sequentially co-infected honey bees with these pathogens in a reciprocally crossed experimental design. Prior establishment in the host ventriculus by N. ceranae inhibited DWV while prior infection by DWV did not impact N. ceranae, highlighting an asymmetry in the competitive interaction between these emerging pathogens.

There is increasing appreciation that hosts in natural populations are subject to infection by multiple parasite species. Yet the epidemiological and ecological processes determining the outcome of mixed infections are poorly understood. Here, we use two intracellular gut parasites (Microsporidia), one exotic and one co-evolved in the western honeybee (Apis mellifera), in an experiment in which either one or both parasites were administered either simultaneously or sequentially. We provide clear evidence of within-host competition; order of infection was an important determinant of the competitive outcome between parasites, with the first parasite significantly inhibiting the growth of the second, regardless of species. However, the strength of this ‘priority effect’ was highly asymmetric, with the exotic Nosema ceranae exhibiting stronger inhibition of Nosema apis than vice versa. Our results reveal an unusual asymmetry in parasite competition that is dependent on order of infection. When incorporated into a mathematical model of disease prevalence, we find asymmetric competition to be an important predictor of the patterns of parasite prevalence found in nature. Our findings demonstrate the wider significance of complex multi-host–multi-parasite interactions as drivers of host–pathogen community structure.

SummarySummaryMethods are described for working with Nosema apis and Nosema ceranae in the field and in the laboratory. For fieldwork, different sampling methods are described to determine colony level infections at a given point in time, but also for following the temporal infection dynamics. Suggestions are made for how to standardise field trials for evaluating treatments and disease impact. The laboratory methods described include different means for determining colony level and individual bee infection levels and methods for species determination, including light microscopy, electron microscopy, and molecular methods (PCR). Suggestions are made for how to standardise cage trials, and different inoculation methods for infecting bees are described, including control methods for spore viability. A cell culture system for in vitro rearing of Nosema spp. is described. Finally, how to conduct different types of experiments are described, including infectious dose, dose effects, course of infection and longevity tests.ResumenSe describen procedimientos para trabajar con Nosema apis y Nosema ceranae en el campo y en el laboratorio. Para el trabajo de campo, se describen diferentes métodos de muestreo para determinar infecciones al nivel de colonia en un momento determinado, y también para el seguimiento de la dinámica temporal de infección. Se hacen sugerencias para la forma de estandarizar los ensayos de campo para evaluar los tratamientos y el impacto de la enfermedad. Los métodos de laboratorio descritos incluyen diferentes formas de determinar los niveles de infección al nivel de colonia y de abeja individual, y los métodos para la determinación de las especies, incluyendo microscopía óptica, microscopía electrónica y métodos moleculares (PCR). Se hacen sugerencias para estandarizar los ensayos con cajas, y se describen diferentes métodos de inoculación para infectar abejas, incluyendo métodos de control para la viabilidad de las esporas. Se describe un sistema de cultivo celular para la cría in vitro de Nosema spp. Finalmente, se describe cómo llevar a cabo diferentes tipos de experimentos, incluyendo la dosis infecciosa, efectos de la dosis, curso de la infección y las pruebas de longevidad.本文描述了实验室及野外实验中关于孢子虫Nosema apis 和 Nosema ceranae 的研究方法。对于野外实验，本文列举了多种取样方法，用于研究特定时间段内，蜂群群体感染水平，以及随后开展长期感染规律的研究。同时也对如何标准化评估蜂场治疗效果、感病程度及选用哪些指标用于标准化评估提出了建议。实验室方法包括，确定蜂群感染水平及个体蜜蜂感染水平的方法以及测定孢子虫种类的方法，如光学显微镜法，电子显微镜法以及分子方法（PCR）。对于如何标准化蜂笼实验的各项指标提出了建议，并描述了感染蜜蜂的不同接种方法，包括孢子生存能力的对比法。描述了孢子虫的一种体外细胞培养体系。最后描述了如何进行不同类型的实验，包括感染剂量、剂量效能、感染过程以及寿命试验。Keywords: Nosema apisNosema ceranaefield methodslaboratory methodssampling methodsinfection dynamicsinfection levelmicroscopyspecies identificationstandardised cage trialsinoculation methodsspore viabilitycell cultureinfectious dosedose effectscourse of infectionlongevity testshoney beeBEEBOOKCOLOSS

Click to increase image sizeClick to decrease image sizeKeywords: Nosema ceranaehoney beesThailandApis ceranaApis dorsataApis floreaApis mellifera

Methods are described for working with Nosema apis and Nosema ceranae in the field and in the laboratory. For fieldwork, different sampling methods are described to determine colony level infections at a given point in time, but also for following the temporal infection dynamics. Suggestions are made for how to standardise field trials for evaluating treatments and disease impact. The laboratory methods described include different means for determining colony level and individual bee infection levels and methods for species determination, including light microscopy, electron microscopy, and molecular methods (PCR). Suggestions are made for how to standardise cage trials, and different inoculation methods for infecting bees are described, including control methods for spore viability. A cell culture system for in vitro rearing of Nosema spp. is described. Finally, how to conduct different types of experiments are described, including infectious dose, dose effects, course of infection and longevity tests.

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

Two pairs of sympatric three-spined stickleback Gasterosteus aculeatus morphs and two single morph populations inhabiting mud and lava or rocky benthic habitats in four Icelandic lakes were screened for parasites and genotyped for MHC class IIB diversity. Parasitic infection differed consistently between G. aculeatus from different benthic habitats. Gasterosteus aculeatus from the lava or rocky habitats were more heavily infected in all lakes. A parallel pattern was also found in individual MHC allelic variation with lava G. aculeatus morphs exhibiting lower levels of variation than the mud morphs. Evidence for selective divergence in MHC allele number is ambiguous but supported by two findings in addition to the parallel pattern observed. MHC allele diversity was not consistent with diversity reported at neutral markers (microsatellites) and in Þingvallavatn the most common number of alleles in each morph was associated with lower infection levels. In the Þingvallavatn lava morph, lower infection levels by the two most common parasites, Schistocephalus solidus and Diplostomum baeri, were associated with different MHC allele numbers.