Trisiloxane Surfactants Negatively Affect Reproductive Behaviors and Enhance Viral Replication in Honey Bees.

Trisiloxane surfactants are often applied in formulated adjuvant products to blooming crops, including almonds, exposing the managed honey bees (Apis mellifera) used for pollination of these crops and persisting in colony matrices, such as bee bread. Despite this, little is known regarding the effects of trisiloxane surfactants on important aspects of colony health, such as reproduction. In the present study, we use laboratory assays to examine how exposure to field-relevant concentrations of three trisiloxane surfactants found in commonly used adjuvant formulations affect queen oviposition rates, worker interactions with the queen, and worker susceptibility to endogenous viral pathogens. Trisiloxane surfactants were administered at 5 mg/kg in pollen supplement diet for 14 days. No effects on worker behavior or physiology could be detected, but our results demonstrate that hydroxy-capped trisiloxane surfactants can negatively affect queen oviposition and methyl-capped trisiloxane surfactants cause increased replication of Deformed Wing Virus in workers, suggesting that trisiloxane surfactant use while honey bees are foraging may negatively impact colony longevity and growth. Environ Toxicol Chem 2024;43:222-233. © 2023 SETAC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Examination of Hivetop Incubator Efficacy for Emerging Osmia lignaria (Hymenoptera: Megachilidae) and the Impact on Apis mellifera (Hymenoptera: Apidae) Colonies.

Osmia lignaria Say is used in combination with Apis mellifera L. to pollinate U.S. orchard crops. The deployment of O. lignaria requires artificial warming to synchronize adult bee emergence with crop bloom. However, current methods for emerging bees are time consuming and inefficient; the Hivetop Incubator (HTI) invention creates a space atop an A. mellifera hive whose heat flows through the screened bottom of the HTI to incubate cocooned O. lignaria adults therein. In response to HTI heat, O. lignaria adults chew out of cocoons, find an exit hole, and fly away to nest in provided nesting sites and, thereby, pollinate a crop. Objectives for studies performed in Utah and Washington were to: 1) determine whether HTIs inhibit A. mellifera thermoregulation or colony growth, 2) compare O. lignaria emergence duration from an HTI with and without hive heat, and 3) assess whether O. lignaria females leave HTIs located at the orchard edge to nest throughout the orchard. We found no significant differences between the internal temperatures of A. mellifera colonies with and without HTIs and no impact on A. mellifera food storage or brood production. Osmia lignaria in hive-heated HTIs emerged 3× faster than bees in unheated HTIs. Heated HTIs were significantly cooler than hive temperatures but significantly warmer than HTIs atop empty hive boxes. Osmia lignaria nest distribution was not correlated to the location of HTIs at the orchard edge. Overall, HTIs were effective for timely, on-site emergence of O. lignaria for orchard pollination without negatively impacting A. mellifera colonies.

Novel Microsatellite Markers for Osmia lignaria (Hymenoptera: Megachilidae): A North American Pollinator of Agricultural Crops and Wildland Plants.

Comprehensive decisions on the management of commercially produced bees, depend largely on associated knowledge of genetic diversity. In this study, we present novel microsatellite markers to support the breeding, management, and conservation of the blue orchard bee, Osmia lignaria Say (Hymenoptera: Megachilidae). Native to North America, O. lignaria has been trapped from wildlands and propagated on-crop and used to pollinate certain fruit, nut, and berry crops. Harnessing the O. lignaria genome assembly, we identified 59,632 candidate microsatellite loci in silico, of which 22 were tested using molecular techniques. Of the 22 loci, 12 loci were in Hardy-Weinberg equilibrium (HWE), demonstrated no linkage disequilibrium (LD), and achieved low genotyping error in two Intermountain North American wild populations in Idaho and Utah, USA. We found no difference in population genetic diversity between the two populations, but there was evidence for low but significant population differentiation. Also, to determine if these markers amplify in other Osmia, we assessed 23 species across the clades apicata, bicornis, emarginata, and ribifloris. Nine loci amplified in three species/subspecies of apicata, 22 loci amplified in 11 species/subspecies of bicornis, 11 loci amplified in seven species/subspecies of emarginata, and 22 loci amplified in two species/subspecies of ribifloris. Further testing is necessary to determine the capacity of these microsatellite loci to characterize genetic diversity and structure under the assumption of HWE and LD for species beyond O. lignaria. These markers will inform the conservation and commercial use of trapped and managed O. lignaria and other Osmia species for both agricultural and nonagricultural systems.

Effects of Provision Type and Pesticide Exposure on the Larval Development of Osmia lignaria (Hymenoptera: Megachilidae).

Wild and managed bee populations are in decline, and one of many environmental causes is the impact of pesticides on developing bees. For solitary bees, delayed larval development could lead to asynchronous adult emergence, unhealthy and inefficient adult pollinators, and decreased brood production and survival. We examined a methodology for testing Osmia lignaria Say (Hymenoptera: Megachilidae) larval responses to pesticide exposure using a laboratory bioassay. We created two provision types: a homogenized blend of O. lignaria provisions from an apple orchard and homogenized almond pollen pellets collected by honey bees plus sugar water. Pesticides were administered to the provisions to compare toxic effects. We recorded larval developmental durations for second-fifth instar and for fifth instar to cocoon initiation for larvae fed provisions treated with water (control) or doses of three pesticides and a representative spray-tank mixture (acetamiprid, boscalid/pyraclostrobin, dimethoate, and acetamiprid plus boscalid/pyraclostrobin). All larvae survived to cocoon initiation when only water was added to provisions. Impacts of pesticide treatments significantly differed between the apple and almond homogenates. The greatest treatment effects occurred when the homogenized almond provision was mixed with acetamiprid alone and when combined with boscalid/pyraclostrobin. Optimizing bioassays through the use of appropriate larval food for exposing solitary bee larvae to agrochemicals is crucial for assessing risks for pollinators.

Genetic and Usurpation Data Support High Incidence of Bumble Bee Nest Invasion by Socially Parasitic Bumble Bee, Bombus insularis.

Cuckoo bumble bees (Psithyrus) (Lepeletier, 1832) (Hymenoptera: Apidae) are a unique lineage of bees that depend exclusively on a host bumble bee species to provide nesting material, nutritional resources, and labor to rear offspring. In this study, we document usurpation incidence and population genetic data of Bombus insularis (Smith, 1861) (Hymenoptera: Apidae), a bumble bee species in the Psithyrus subgenus, on field-deployed B. huntii colonies in northern Utah, United States. Within 12 d of deploying B. huntii Greene, 1860 (Hymenoptera: Apidae) colonies at two field sites, 13 of the 16 colonies contained at least one established B. insularis female. Although our results demonstrate that field-deployed bumble bee colonies are highly susceptible to B. insularis usurpation, applying a fabricated excluder to prevent the inquiline from invading a colony was 100% effective. Sibship analysis using microsatellite genotype data of 59 B. insularis females estimates that they originated from at least 49 unique colonies. Furthermore, sibship analysis found siblings distributed between the field sites that were 7.04 km apart. Our result suggests that B. insularis females have the capacity to disperse across the landscape in search of host colonies at distances of at least 3.52 km and up to 7.04 km. Our study underscores the detrimental impact B. insularis usurpation has on the host bumble bee colony. As B. insularis significantly impacts the success of bumble bee colonies, we briefly discuss how the utilization of excluders may be useful for commercial bumble bee colonies that are used to pollinate open field crops.

Diet Breadth Affects Bacterial Identity but Not Diversity in the Pollen Provisions of Closely Related Polylectic and Oligolectic Bees.

Mounting evidence suggests that microbes found in the pollen provisions of wild and solitary bees are important drivers of larval development. As these microbes are also known to be transmitted via the environment, most likely from flowers, the diet breadth of a bee may affect the diversity and identity of the microbes that occur in its pollen provisions. Here, we tested the hypothesis that, due to the importance of floral transmission of microbes, diet breadth affects pollen provision microbial community composition. We collected pollen provisions at four sites from the polylectic bee Osmia lignaria and the oligolectic bee Osmia ribifloris. We used high-throughput sequencing of the bacterial 16S rRNA gene to characterize the bacteria found in these provisions. We found minimal overlap in the specific bacterial variants in pollen provisions across the host species, even when the bees were constrained to foraging from the same flowers in cages at one site. Similarly, there was minimal overlap in the specific bacterial variants across sites, even within the same host species. Together, these findings highlight the importance of environmental transmission and host specific sorting influenced by diet breadth for microbes found in pollen provisions. Future studies addressing the functional consequences of this filtering, along with tests for differences between more species of oligoletic and polylectic bees will provide rich insights into the microbial ecology of solitary bees.

Bombus (Hymenoptera: Apidae) Microcolonies as a Tool for Biological Understanding and Pesticide Risk Assessment.

Bumble bees provide valuable pollination services to many wild and agricultural plants. Populations of some bumble bee species are in decline, prompting the need to better understand bumble bee biology and to develop methodologies for assessing the effects of environmental stressors on these bees. Use of bumble bee microcolonies as an experimental tool is steadily increasing. This review closely examines the microcolony model using peer-reviewed published literature identified by searching three databases through November 2018. Microcolonies have been successfully used for investigating a range of endpoints including behavior, the gut microbiome, nutrition, development, pathogens, chemical biology, and pesticides/xenobiotics. Methods for the initiation and monitoring of microcolonies, as well as the recorded variables were catalogued and described. From this information, we identified a series of recommendations for standardizing core elements of microcolony studies. Standardization is critical to establishing the foundation needed to support use of this model for biological response investigations and particularly for supporting use in pesticide risk assessment.

Floral and Foliar Source Affect the Bee Nest Microbial Community.

Managed pollinators such as the alfalfa leafcutting bee, Megachile rotundata, are essential to the production of a wide variety of agricultural crops. These pollinators encounter a diverse array of microbes when foraging for food and nest-building materials on various plants. To test the hypothesis that food and nest-building source affects the composition of the bee-nest microbiome, we exposed M. rotundata adults to treatments that varied both floral and foliar source in a 2 × 2 factorial design. We used 16S rRNA gene and internal transcribed spacer (ITS) sequencing to capture the bacterial and fungal diversity of the bee nests. We found that nest microbial communities were significantly different between treatments, indicating that bee nests become inoculated with environmentally derived microbes. We did not find evidence of interactions between the fungi and bacteria within our samples. Furthermore, both the bacterial and fungal communities were quite diverse and contained numerous exact sequence variants (ESVs) of known plant and bee pathogens that differed based on treatment. Our research indicates that bees deposit plant-associated microbes into their nests, including multiple plant pathogens such as smut fungi and bacteria that cause blight and wilt. The presence of plant pathogens in larval pollen provisions highlights the potential for bee nests to act as disease reservoirs across seasons. We therefore suggest that future research should investigate the ability of bees to transmit pathogens from nest to host plant.

Workshop on Pesticide Exposure Assessment Paradigm for Non-Apis Bees: Foundation and Summaries.

Current pesticide risk assessment practices use the honey bee, Apis mellifera L., as a surrogate to characterize the likelihood of chemical exposure of a candidate pesticide for all bee species. Bees make up a diverse insect group that provides critical pollination services to both managed and wild ecosystems. Accordingly, they display a diversity of behaviors and vary greatly in their lifestyles and phenologies, such as their timing of emergence, degree of sociality, and foraging and nesting behaviors. Some of these factors may lead to disparate or variable routes of exposure when compared to honey bees. For those that possess life histories that are distinct from A. mellifera, further risk assessments may be warranted. In January 2017, 40 bee researchers, representative of regulatory agencies, academia, and agrochemical industries, gathered to discuss the current state of science on pesticide exposure to non-Apis bees and to determine how well honey bee exposure estimates, implemented by different regulatory agencies, may be protective for non-Apis bees. Workshop participants determined that although current risk assessment procedures for honey bees are largely conservative, several routes of exposure are unique to non-Apis bees and warranted further investigation. In this forum article, we discuss these key routes of exposure relevant to non-Apis bees and identify important research gaps that can help inform future bee risk assessment decisions.

Genomes of the Hymenoptera.

Hymenoptera is the second-most sequenced arthropod order, with 52 publically archived genomes (71 with ants, reviewed elsewhere), however these genomes do not capture the breadth of this very diverse order (Figure 1, Table 1). These sequenced genomes represent only 15 of the 97 extant families. Although at least 55 other genomes are in progress in an additional 11 families (see Table 2), stinging wasps represent 35 (67%) of the available and 42 (76%) of the in progress genomes. A more comprehensive catalog of hymenopteran genomes is needed for research into the evolutionary processes underlying the expansive diversity in terms of ecology, behavior, and physiological traits within this group. Additional sequencing is needed to generate an assembly for even 0.05% of the estimated 1 million hymenopteran species, and we recommend premier level assemblies for at least 0.1% of the >150,000 named species dispersed across the order. Given the haplodiploid sex determination in Hymenoptera, haploid male sequencing will help minimize genome assembly issues to enable higher quality genome assemblies.

Colony Collapse Disorder (CCD) and bee age impact honey bee pathophysiology.

Honey bee (Apis mellifera) colonies continue to experience high annual losses that remain poorly explained. Numerous interacting factors have been linked to colony declines. Understanding the pathways linking pathophysiology with symptoms is an important step in understanding the mechanisms of disease. In this study we examined the specific pathologies associated with honey bees collected from colonies suffering from Colony Collapse Disorder (CCD) and compared these with bees collected from apparently healthy colonies. We identified a set of pathological physical characteristics that occurred at different rates in CCD diagnosed colonies prior to their collapse: rectum distension, Malpighian tubule iridescence, fecal matter consistency, rectal enteroliths (hard concretions), and venom sac color. The multiple differences in rectum symptomology in bees from CCD apiaries and colonies suggest effected bees had trouble regulating water. To ensure that pathologies we found associated with CCD were indeed pathologies and not due to normal changes in physical appearances that occur as an adult bee ages (CCD colonies are assumed to be composed mostly of young bees), we documented the changes in bees of different ages taken from healthy colonies. We found that young bees had much greater incidences of white nodules than older cohorts. Prevalent in newly-emerged bees, these white nodules or cellular encapsulations indicate an active immune response. Comparing the two sets of characteristics, we determined a subset of pathologies that reliably predict CCD status rather than bee age (fecal matter consistency, rectal distension size, rectal enteroliths and Malpighian tubule iridescence) and that may serve as biomarkers for colony health. In addition, these pathologies suggest that CCD bees are experiencing disrupted excretory physiology. Our identification of these symptoms is an important first step in understanding the physiological pathways that underlie CCD and factors impacting bee health.

An Inert Pesticide Adjuvant Synergizes Viral Pathogenicity and Mortality in Honey Bee Larvae.

Honey bees are highly valued for their pollination services in agricultural settings, and recent declines in managed populations have caused concern. Colony losses following a major pollination event in the United States, almond pollination, have been characterized by brood mortality with specific symptoms, followed by eventual colony loss weeks later. In this study, we demonstrate that these symptoms can be produced by chronically exposing brood to both an organosilicone surfactant adjuvant (OSS) commonly used on many agricultural crops including wine grapes, tree nuts and tree fruits and exogenous viral pathogens by simulating a horizontal transmission event. Observed synergistic mortality occurred during the larval-pupal molt. Using q-PCR techniques to measure gene expression and viral levels in larvae taken prior to observed mortality at metamorphosis, we found that exposure to OSS and exogenous virus resulted in significantly heightened Black Queen Cell Virus (BQCV) titers and lower expression of a Toll 7-like-receptor associated with autophagic viral defense (Am18w). These results demonstrate that organosilicone spray adjuvants that are considered biologically inert potentiate viral pathogenicity in honey bee larvae, and guidelines for OSS use may be warranted.

Characterization of viral siRNA populations in honey bee colony collapse disorder.

Colony Collapse Disorder (CCD), a special case of collapse of honey bee colonies, has resulted in significant losses for beekeepers. CCD-colonies show abundance of pathogens which suggests that they have a weakened immune system. Since honey bee viruses are major players in colony collapse and given the important role of viral RNA interference (RNAi) in combating viral infections we investigated if CCD-colonies elicit an RNAi response. Deep-sequencing analysis of samples from CCD-colonies from US and Israel revealed abundant small interfering RNAs (siRNA) of 21-22 nucleotides perfectly matching the Israeli acute paralysis virus (IAPV), Kashmir virus and Deformed wing virus genomes. Israeli colonies showed high titers of IAPV and a conserved RNAi-pattern of matching the viral genome. That was also observed in sample analysis from colonies experimentally infected with IAPV. Our results suggest that CCD-colonies set out a siRNA response that is specific against predominant viruses associated with colony losses.

Cross-species transmission of honey bee viruses in associated arthropods.

There are a number of RNA virus pathogens that represent a serious threat to the health of managed honey bees (Apis mellifera). That some of these viruses are also found in the broader pollinator community suggests the wider environmental spread of these viruses, with the potential for a broader impact on ecosystems. Studies on the ecology and evolution of these viruses in the arthropod community as a whole may therefore provide important insights into these potential impacts. We examined managed A. mellifera colonies, nearby non-Apis hymenopteran pollinators, and other associated arthropods for the presence of five commonly occurring picorna-like RNA viruses of honey bees - black queen cell virus, deformed wing virus, Israeli acute paralysis virus, Kashmir bee virus and sacbrood virus. Notably, we observed their presence in several arthropod species. Additionally, detection of negative-strand RNA using strand-specific RT-PCR assays for deformed wing virus and Israeli acute paralysis virus suggests active replication of deformed wing virus in at least six non-Apis species and active replication of Israeli acute paralysis virus in one non-Apis species. Phylogenetic analysis of deformed wing virus also revealed that this virus is freely disseminating across the species sampled in this study. In sum, our study indicates that these viruses are not specific to the pollinator community and that other arthropod species have the potential to be involved in disease transmission in pollinator populations.

Rethinking vector immunology: the role of environmental temperature in shaping resistance.

Recent ecological research has revealed that environmental factors can strongly affect insect immunity and influence the outcome of host-parasite interactions. To date, however, most studies examining immune function in mosquitoes have ignored environmental variability. We argue that one such environmental variable, temperature, influences both vector immunity and the parasite itself. As temperatures in the field can vary greatly from the ambient temperature in the laboratory, it will be essential to take temperature into account when studying vector immunology.

Lack of evidence for an association between Iridovirus and colony collapse disorder.

Colony collapse disorder (CCD) is characterized by the unexplained losses of large numbers of adult worker bees (Apis mellifera) from apparently healthy colonies. Although infections, toxins, and other stressors have been associated with the onset of CCD, the pathogenesis of this disorder remains obscure. Recently, a proteomics study implicated a double-stranded DNA virus, invertebrate iridescent virus (Family Iridoviridae) along with a microsporidium (Nosema sp.) as the cause of CCD. We tested the validity of this relationship using two independent methods: (i) we surveyed healthy and CCD colonies from the United States and Israel for the presence of members of the Iridovirus genus and (ii) we reanalyzed metagenomics data previously generated from RNA pools of CCD colonies for the presence of Iridovirus-like sequences. Neither analysis revealed any evidence to suggest the presence of an Iridovirus in healthy or CCD colonies.

Weighing risk factors associated with bee colony collapse disorder by classification and regression tree analysis.

Colony collapse disorder (CCD), a syndrome whose defining trait is the rapid loss of adult worker honey bees, Apis mellifera L., is thought to be responsible for a minority of the large overwintering losses experienced by U.S. beekeepers since the winter 2006-2007. Using the same data set developed to perform a monofactorial analysis (PloS ONE 4: e6481, 2009), we conducted a classification and regression tree (CART) analysis in an attempt to better understand the relative importance and interrelations among different risk variables in explaining CCD. Fifty-five exploratory variables were used to construct two CART models: one model with and one model without a cost of misclassifying a CCD-diagnosed colony as a non-CCD colony. The resulting model tree that permitted for misclassification had a sensitivity and specificity of 85 and 74%, respectively. Although factors measuring colony stress (e.g., adult bee physiological measures, such as fluctuating asymmetry or mass of head) were important discriminating values, six of the 19 variables having the greatest discriminatory value were pesticide levels in different hive matrices. Notably, coumaphos levels in brood (a miticide commonly used by beekeepers) had the highest discriminatory value and were highest in control (healthy) colonies. Our CART analysis provides evidence that CCD is probably the result of several factors acting in concert, making afflicted colonies more susceptible to disease. This analysis highlights several areas that warrant further attention, including the effect of sublethal pesticide exposure on pathogen prevalence and the role of variability in bee tolerance to pesticides on colony survivorship.

Contributions of immune responses to developmental resistance in Lymantria dispar challenged with baculovirus.

How the innate immune system functions to defend insects from viruses is an emerging field of study. We examined the impact of melanized encapsulation, a component of innate immunity that integrates both cellular and humoral immune responses, on the success of the baculovirus Lymantria dispar multiple nucleocapsid nucleopolyhedrovirus (LdMNPV) in its host L. dispar. L. dispar exhibits midgut-based and systemic, age-dependent resistance to LdMNPV within the fourth instar; the LD(50) in newly molted larvae is approximately 18-fold lower than in mid-instar larvae (48-72h post-molt). We examined the role of the immune system in systemic resistance by measuring differences in hemocyte immunoresponsiveness to foreign targets, hemolymph phenoloxidase (PO) and FAD-glucose dehydrogenase (GLD) activities, and melanization of infected tissue culture cells. Mid-instar larvae showed a higher degree of hemocyte immunoresponsiveness, greater potential PO activity (pro-PO) at the time the virus is escaping the midgut to enter the hemocoel (72h post-inoculation), greater GLD activity, and more targeted melanization of infected tissue, which correlate with reduced viral success in the host. These findings support the hypothesis that innate immune responses can play an important role in anti-viral defenses against baculoviruses and that the success of these defenses can be age-dependent.

Pathogenesis of Lymantria dispar multiple nucleopolyhedrovirus in L. dispar and mechanisms of developmental resistance.

Lymantria dispar has a long historical association with the baculovirus Lymantria dispar multiple nucleopolyhedrovirus (LdMNPV), which is one of the primary population regulators of L. dispar in the field. However, host larvae exhibit strong developmental resistance to fatal infection by LdMNPV; the LD50 in newly moulted fourth instars is 18-fold lower than in the middle of the instar (48-72 h post-moult). Using a recombinant of LdMNPV expressing lacZ, we examined the key steps of pathogenesis in the host to explore mechanisms of developmental resistance. At the midgut level, we observed reduced primary midgut infections in mid-fourth instars, indicating increased sloughing of infected cells. Additional barriers were observed as the virus escaped the midgut. Mid-fourth instars had higher numbers of melanized foci of infection associated with the midgut, apoptotic tracheal epidermal cells and haemocytes, and reduced numbers of infected haemocytes later in infection. Our results show that the co-evolutionary relationship between L. dispar and LdMNPV has resulted in both midgut-based and systemic antiviral defences and that these defences are age-dependent within the instar. This age-related susceptibility may contribute to how the virus is maintained in nature and could influence management of L. dispar by using the virus.