Nutritional, foraging, and nesting preferences of three species of solitary bees, Megachile rotundata, Megachile pugnata, and Megachile sculpturalis

Flupyradifurone is a novel butenolide insecticide, first approved as an active substance for use in plant protection products by Commission Implementing Regulation (EU) 2015/2084. Following concerns that this substance may pose high risks to humans and the environment, the French authorities, in November 2020, asked the Commission to restrict its uses under Article 69 of Regulation (EC) No 1107/2009. To support this request, competent Authorities from France cited a series of literature papers investigating its hazards and/or exposure to humans and the environment. In addition, in June 2020, the Dutch Authorities notified the Commission, under Article 56 of Regulation (EC) No 1107/2009, of new information on flupyradifurone on the wild bee species Megachile rotundata. This notification is also referred to in the French notification on flupyradifurone. Consequently, the EFSA PPR Panel was mandated to quantify the likelihood of this body of evidence constituting proof of serious risks to humans or the environment. Therefore, the EFSA PPR Panel evaluated the likelihood of these studies indicating new or higher hazards and exposure to humans and the environment compared to previous EU assessments. A stepwise methodology was designed, including: (i) the initial screening; (ii) data extraction and critical appraisal based on the principles of OHAT/NTP; (iii) weight of evidence, including consideration of the previous EU assessments; (iv) uncertainty analysis, followed, whenever relevant, by an expert knowledge elicitation process. For the human health, only one study was considered relevant for the genotoxic potential of flupyradifurone in vitro. These data did not provide sufficient information to overrule the EU assessment, as in vivo studies already addressed the genotoxic potential of flupyradifurone. Environment: All available data investigated hazards in bee species. For honey bees, the likelihood of the new data indicating higher hazards than the previous EU assessment was considered low or moderate, with some uncertainties. However, among solitary bee species – which were not addressed in the previous EU assessment – there was evidence that Megachile rotundata may be disproportionately sensitive to flupyradifurone. This sensitivity, which may partially be explained by the low bodyweight of this species, was mechanistically linked to inadequate bodily metabolisation processes.

Social bees have been extensively studied for their gut microbial functions, but the significance of the gut microbiota in solitary bees remains less explored. Solitary bee, Megachile rotundata females provision their offspring with pollen from various plant species, harboring a diverse microbial community that colonizes larvae guts. The Apilactobacillus is the most abundant microbe, but evidence concerning the effects of Apilactobacillus and other provision microbes on growth and survival are lacking. We hypothesized that the presence of Apilactobacillus in abundance would enhance larval and prepupal development, weight, and survival, while the absence of intact microbial communities was expected to have a negative impact on bee fitness. We reared larvae on pollen provisions with naturally collected microbial communities (Natural pollen) or devoid of microbial communities (Sterile pollen). We also assessed the impact of introducing Apilactobacillus micheneri by adding it to both types of pollen provisions. Feeding larvae with sterile pollen + A. micheneri led to the highest mortality rate, followed by natural pollen + A. micheneri, and sterile pollen. Larval development was significantly delayed in groups fed with sterile pollen. Interestingly, larval and prepupal weights did not significantly differ across treatments compared to natural pollen-fed larvae. 16S rRNA gene sequencing found a dominance of Sodalis, when A. micheneri was introduced to natural pollen. The presence of Sodalis with abundant A. micheneri suggests potential crosstalk between both, shaping bee nutrition and health. Hence, this study highlights that the reliance on nonhost-specific environmental bacteria may not impact fitness of M. rotundata.IMPORTANCEThis study investigates the impact of environmentally acquired gut microbes of solitary bee fitness with insights into the microbial ecology of bee and their health. While the symbiotic microbiome is well-studied in social bees, the role of environmental acquired microbiota in solitary bees remains unclear. Assessing this relationship in a solitary pollinator, the leaf-cutting bee, Megachile rotundata, we discovered that this bee species does not depend on the diverse environmental bacteria found in pollen for either its larval growth or survival. Surprisingly, high concentrations of the most abundant pollen bacteria, Apilactobacillus micheneri did not consistently benefit bee fitness, but caused larval mortality. Our findings also suggest an interaction between Apilactobacillus and the Sodalis and perhaps their role in bee nutrition. Hence, this study provides significant insights that contribute to understanding the fitness, conservation, and pollination ecology of other solitary bee species in the future.

Pollinator-plant interactions represent a core mutualism that underpins biodiversity in terrestrial ecosystems, and the loss of flowering plants is a major driver of pollinator declines. Bee attraction to flowers is mediated by both quantity of resources (the number of available flowers for exploration) and quality of resources (pollen nutritional value), but whether and how bees prioritize these factors is not well understood. Here, we leveraged a unique plant system to investigate the floral factors influencing bee foraging decisions. Recombinant inbred plant lines were generated by crossing the self-fertilizing Capsella rubella and the pollinator-dependent outcrosser C. grandiflora, to produce plants that varied across floral traits. Using enclosed arenas, we evaluated the foraging behavior of two solitary bee species, Osmia cornifrons and Megachile rotundata, to the isolated inflorescences from these lines. Visits from O. cornifrons were significantly positively correlated with the number of flowers, while M. rotundata visits were significantly positively associated with pollen nutrition, with a preference for plants with higher pollen protein-to-lipid content. Further experiments using artificial flowers confirmed that M. rotundata preferred flowers with higher protein:lipid ratios, while O. cornifrons visits were unaffected by nutrition. These studies demonstrate that, although both bee species collect pollen as their sole source of protein and lipids for themselves and/or their offspring, they differentially prioritize resource quantity (number of flowers) and quality (pollen nutritional content). These studies lay the groundwork for understanding how different foraging strategies evolved, and influence, plant-pollinator ecological networks.

Managed and wild bees contribute to alfalfa (Medicago sativa) pollination

During community assembly, early arriving exotic species might suppress other species to a greater extent than do native species. Because most exotics were intentionally introduced, we hypothesize there was human selection on regeneration traits during introduction. This could have occurred at the across- or within-species level (e.g. during cultivar development). We tested these predictions by seeding a single species that was either native, exotic 'wild-type' (from their native range), or exotic 'cultivated' using 28 grassland species in a glasshouse experiment. Priority effects were assessed by measuring species' effect on establishment of species from a seed mix added 21 d later. Exotic species had higher germination and earlier emergence dates than native species, and differences were found in both 'wild' and 'cultivated' exotics. Exotic species reduced biomass and species diversity of later arriving species much more than native species, regardless of seed source. Results indicate that in situations in which priority effects are likely to be strong, effects will be greater when an exotic species arrives first than when a native species arrives first; and this difference is not merely a result of exotic species cultivation, but might be a general native-exotic difference that deserves further study.

The mechanisms that underlie senescence are not well understood in insects. Telomeres are conserved repetitive sequences at chromosome ends that protect DNA during replication. In many vertebrates, telomeres shorten during cell division and in response to stress and are often used as a cellular marker of senescence. However, little is known about telomere dynamics across the lifespan in invertebrates. We measured telomere length in larvae, prepupae, pupae, and adults of two species of solitary bees, Osmia lignaria and Megachile rotundata. Contrary to our predictions, telomere length was longer in later developmental stages in both O. lignaria and M. rotundata. Longer telomeres occurred after emergence from diapause, which is a physiological state with increased tolerance to stress. In O. lignaria, telomeres were longer in adults when they emerged following diapause. In M. rotundata, telomeres were longer in the pupal stage and subsequent adult stage, which occurs after prepupal diapause. In both species, telomere length did not change during the 8 months of diapause. Telomere length did not differ by mass similarly across species or sex. We also did not see a difference in telomere length after adult O. lignaria were exposed to a nutritional stress, nor did length change during their adult lifespan. Taken together, these results suggest that telomere dynamics in solitary bees differ from what is commonly reported in vertebrates and suggest that insect diapause may influence telomere dynamics.

Pollinators face many stressors, including reduced floral diversity. A low-diversity diet can impair organisms' ability to cope with additional stressors, such as pathogens, by altering the gut microbiome and/or immune function, but these effects are understudied for most pollinators. We investigated the impact of pollen diet diversity on two ecologically and economically important generalist pollinators, the social bumble bee (Bombus impatiens) and the solitary alfalfa leafcutter bee (Megachile rotundata). We experimentally tested the effect of one-, two-, or three-species pollen diets on gut bacterial communities in both species, and the melanization immune response in B. impatiens. Pollen diets included dandelion (Taraxacum officinale), staghorn sumac (Rhus typhina), and hawthorn (Crataegus sp.) alone, each pair-wise combination, or a mix of all three species. We fed bees their diet for 7 days and then dissected out guts and sequenced 16S rRNA gene amplicons to characterize gut bacterial communities. To assess melanization in B. impatiens, we inserted microfilament implants into the bee abdomen and measured melanin deposition on the implant. We found that pollen diet did not influence gut bacterial communities in M. rotundata. In B. impatiens, pollen diet composition, but not diversity, affected gut bacterial richness in older, but not newly-emerged bees. Pollen diet did not affect the melanization response in B. impatiens. Our results suggest that even a monofloral, low-quality pollen diet such as dandelion can support diverse gut bacterial communities in captive-reared adults of these bee species. These findings shed light on the effects of reduced diet diversity on bee health.

Cultivation of pollinator-dependent crops has expanded globally, increasing our reliance on insect pollination. This essential ecosystem service is provided by a wide range of managed and wild pollinators whose abundance and diversity are thought to be in decline, threatening sustainable food production. The Western honey bee (Apis mellifera) is amongst the best-monitored insects but the state of other managed pollinators is less well known. Here, we review the status and trends of all managed pollinators based on publicly accessible databases and the published literature. We found that, on a global scale, the number of managed A. mellifera colonies has increased by 85% since 1961, driven mainly by Asia. This contrasts with high reported colony overwinter mortality, especially in North America (average 26% since 2007) and Europe (average 16% since 2007). Increasing agricultural dependency on pollinators as well as threats associated with managing non-native pollinators have likely spurred interest in the management of alternative species for pollination, including bumble bees, stingless bees, solitary bees, and flies that have higher efficiency in pollinating specific crops. We identify 66 insect species that have been, or are considered to have the potential to be, managed for crop pollination, including seven bumble bee species and subspecies currently commercially produced mainly for the pollination of greenhouse-grown tomatoes and two species that are trap-nested in New Zealand. Other managed pollinators currently in use include eight solitary bee species (mainly for pollination services in orchards or alfalfa fields) and three fly species (mainly used in enclosures and for seed production). Additional species in each taxonomic category are under consideration for pollinator management. Examples include 15 stingless bee species that are able to buzz-pollinate, will fly in enclosures, and some of which have a history of management for honey production; their use for pollination is not yet established. To ensure sustainable, integrated pollination management in agricultural landscapes, the risks, as well as the benefits of novel managed pollinator species must be considered. We, therefore, urge the prioritization of biodiversity-friendly measures maintaining native pollinator species diversity to provide ecosystem resilience to future environmental changes.

Pathogens and lack of floral resources interactively impair global pollinator health. However, epidemiological and nutritional studies aimed at understanding bee declines have historically focused on social species, with limited evaluations of solitary bees. Here, we asked whether Crithidia bombi, a trypanosomatid gut pathogen known to infect bumble bees, could infect the solitary bees Osmia lignaria (females) and Megachile rotundata (males), and whether nutritional stress influenced infection patterns and bee survival. We found that C. bombi was able to infect both solitary bee species, with 59% of O. lignaria and 29% of M. rotundata bees experiencing pathogen replication 5–11 days following inoculation. Moreover, access to pollen resulted in O. lignaria living longer, although it did not influence M. rotundata survival. Access to pollen did not affect infection probability or resulting pathogen load in either species. Similarly, inoculating with the pathogen did not drive survival patterns in either species during the 5–11-day laboratory assays. Our results demonstrate that solitary bees can be hosts of a known bumble bee pathogen, and that access to pollen is an important contributing factor for bee survival, thus expanding our understanding of factors contributing to solitary bee health.

Wild bees are important pollinators for agricultural crops and solitary species such as Osmia bicornis are particularly suitable for pollination management. Wild bees share floral resources with managed honey bees and may be exposed to emerging infectious diseases. Although studies have explored the prevalence of pathogens in solitary wild bee species, data regarding the impact of pathogens on solitary bee health are lacking. We carried out experiments examining whether the solitary bee species O. bicornis is susceptible to infection with the emerging pathogen Nosema ceranae and recorded the impact of exposure on survival. The results obtained indicate that N. ceranae may be able to infect O. bicornis but its impact on host fitness is negligible: survival rates did not differ between control and inoculated bees, although male survival was marginally lower after infection. To explore the possible field‐relevance of our findings, we collected wild bees near an infected and a non‐infected hive and showed that N. ceranae was shared between managed and wild bees, although only the in presence of infected honey bees. The findings of the present study show that O. bicornis is susceptible to pathogen spillover and could act as a potential reservoir host for N. ceranae in pollinator networks. Additional studies on this species incorporating sublethal effects, multiple infections and other interacting stressors are warranted.

Recent work has demonstrated that many bee species have specific cytochrome P450 enzymes (P450s) that can efficiently detoxify certain insecticides. The presence of these P450s, belonging or closely related to the CYP9Q subfamily (CYP9Q-related), is generally well conserved across the diversity of bees. However, the alfalfa leafcutter bee, Megachile rotundata, lacks CYP9Q-related P450s and is 170-2500 times more sensitive to certain insecticides than bee pollinators with these P450s. The extent to which these findings apply to other Megachilidae bee species remains uncertain. To address this knowledge gap, we sequenced the transcriptomes of four Megachile species and leveraged the data obtained, in combination with publicly available genomic data, to investigate the evolution and function of P450s in the Megachilidae. Our analyses reveal that several Megachilidae species, belonging to the Lithurgini, Megachilini and Anthidini tribes, including all species of the Megachile genus investigated, lack CYP9Q-related genes. In place of these genes Megachile species have evolved phylogenetically distinct CYP9 genes, the CYP9DM lineage. Functional expression of these P450s from M. rotundata reveal they lack the capacity to metabolize the neonicotinoid insecticides thiacloprid and imidacloprid. In contrast, species from the Osmiini and Dioxyini tribes of Megachilidae have CYP9Q-related P450s belonging to the CYP9BU subfamily that are able to detoxify thiacloprid. These findings provide new insight into the evolution of P450s that act as key determinants of insecticide sensitivity in bees and have important applied implications for pesticide risk assessment.

Pollinator habitat can be planted on farms to enhance floral and nesting resources, and subsequently, pollinator populations. There is ample evidence linking such plantings to greater pollinator abundance on farms, but less is known about their effects on pollinator reproduction. We placed Bombus impatiens Cresson (Hymenoptera: Apidae) and Megachile rotundata (F.) (Hymenoptera: Megachilidae) nests out on 19 Mid-Atlantic farms in 2018, where half (n = 10) the farms had established wildflower plantings and half (n = 9) did not. Bombus impatiens nests were placed at each farm in spring and mid-summer and repeatedly weighed to capture colony growth. We quantified the relative production of reproductive castes and assessed parasitism rates by screening for conopid fly parasitism and Nosema spores within female workers. We also released M. rotundata cocoons at each farm in spring and collected new nests and emergent adult offspring over the next year, recording female weight as an indicator of reproductive potential and quantifying Nosema parasitism and parasitoid infection rates. Bombus impatiens nests gained less weight and contained female workers with Nosema spore loads over 150 times greater on farms with wildflower plantings. In contrast, M. rotundata female offspring weighed more on farms with wildflower plantings and marginally less on farms with honey bee hives. We conclude that wildflower plantings likely enhance reproduction in some species, but that they could also enhance microsporidian parasitism rates in susceptible bee species. It will be important to determine how wildflower planting benefits can be harnessed while minimizing parasitism in wild and managed bee species.

Trait divergence and the ecosystem impacts of invading species

ABSTRACTThe study of nesting biology is an important biological aspect for both conservation and management, but despite this, only a few of the over 20,000 described bee species have had their nesting biology studied. The aim of this study was to describe and analyse the nesting biology and dynamic of an aggregation of the cavity-nesting bee species Centris analis during one generation. Twenty-eight females were marked and had their nesting activities documented, 64 nests were sampled, and 113 individuals emerged from these nests. An asymmetry in the sex allocation was observed for this species, and the sex ratio was male-biased in the first nests built. Nonetheless, considering an individual-level perspective, the females showed different strategies, with some of them producing more daughters, other females producing more sons, and still others producing an equal proportion. The productivity per female was low compared to that of other solitary bee species, and the females displayed an aggressive usurpation behaviour against conspecific females and performed an intraspecific parasitism. As the substrate used to nest is a limiting factor for this species, the usurpation behaviour may be an important strategy that could increase the chance of nesting. A few males used the nesting site as a resting place and the fidelity observed to this locale was high. This study provides new information about the natural history of this bee species, which is an important pollinator of both crops and native plant species.

Landscapes with high amounts of mass-flowering fruit crops reduce the reproduction of two solitary bees