Honey Bee (Apis mellifera) Exposure to Pesticide Residues in Nectar and Pollen in Urban and Suburban Environments from Four Regions of the United States.

The risk of honey bee (Apis mellifera L.) exposure to pesticide residues while foraging for nectar and pollen is commonly explored in the context of agroecosystems. However, pesticides are also used in urban and suburban areas for vegetation management, vector control, and the management of ornamental plants in public and private landscapes. The extent to which pesticides pose a health risk to honey bees in these settings remains unclear. We addressed this at a landscape scale by conducting pesticide residue screening analyses on 768 nectar and 862 pollen samples collected monthly over 2 years from honey bee colonies located in urban and suburban areas in eight medium to large cities in California, Florida, Michigan, and Texas (USA). A risk assessment was performed using the US Environmental Protection Agency's BeeREX model whenever an oral toxicity value was available for a compound. Chemical analyses detected 17 pesticides in nectar and 60 in pollen samples during the survey. Approximately 73% of all samples contained no detectable pesticide residues. Although the number of detections varied among the sampled regions, fewer pesticides were detected in nectar than in pollen. Per BeeREX, four insecticides showed a potential acute risk to honey bees: imidacloprid, chlorpyrifos, and esfenvalerate in nectar, and deltamethrin in nectar and pollen. In general, exposure of honey bees to pesticides via nectar and pollen collection was low in urban and suburban areas across the United States, and no seasonal or spatial trends were evident. Our data suggest that honey bees are exposed to fewer pesticides in developed areas than in agricultural ones. Environ Toxicol Chem 2022;41:991-1003. © 2022 SETAC.

The Utility of a Bumble Bee (Bombus spp. [Hymenoptera: Apidae]) Brood Test for Evaluating the Effects of Pesticides.

Risk assessment for chemicals in the United States relies upon the honey bee (Apis meliffera L. [Hymenoptera: Apidae]) as a surrogate for other bee species. There is uncertainty in extrapolating honey bee toxicity data to bumble bees due to differences in life history strategies, food consumption, and nest structure. Here we evaluated the design of a queenless bumble bee microcolony test that could be considered for generating larval toxicity data. Three microcolony studies were conducted with Bombus impatiens to evaluate the effects of exposure to 1) diflubenzuron in pollen, 2) dimethoate in pollen, and 3) dimethoate in sucrose. Immature drone bee emergence, worker survival, pollen, and sucrose utilization were measured throughout the study duration. For dimethoate, a 10-d chronic adult bumble bee study was also conducted to compare microcolony endpoints to toxicity endpoints on individual adults. Microcolonies exposed to 10 mg diflubenzuron/kg pollen produced fewer adult drones despite no effects on worker survival. Microcolonies treated with dimethoate at ≥3 mg a.i./kg pollen and ≥0.1 mg a.i./kg sucrose solution produced fewer drones. Exposure to dimethoate in the 10-d chronic adult study resulted in direct mortality to the adult workers at ≥0.1 mg a.i./kg diet. Results from the 10-d study suggest direct effects of dimethoate on workers in the microcolony will alter provisioning of diet to the brood, resulting in lower drone production in the microcolony. Our data suggest that the microcolony study is only appropriate to assess brood effects to bumble bees for substances with low toxicity to adults, as demonstrated with diflubenzuron.

A Comparison of Acute Toxicity Endpoints for Adult Honey Bees with Technical Grade Active Ingredients and Typical End-use Products as Test Substance.

The honey bee, Apis mellifera L. (Hymenoptera: Apidae), is a model organism for pollinators in risk assessment frameworks globally. The acute toxicity tests with adult honey bees for contact and oral exposure are part of the requirements for pesticide registration and are typically conducted with the active ingredient. A question often asked is if the typical end-use product (TEP) is more toxic than the technical grade active ingredient (TGAI) to honey bees. We explored this question by mining publicly available databases from regulatory agencies worldwide, where testing with the TEP is required. The objective of this study was to determine whether TEPs are comparable in toxicity to the TGAI. The dataset was analyzed via a 3 × 3 contingency table with toxicity categories, as the data cannot be computed for regression analysis. Of the 151 active ingredients with reported endpoints for contact exposure, 28 were classified as either moderately or highly toxic, 123 were classified as practically nontoxic, and 3 were inconclusive. Only two (1.3%) were reclassified from nontoxic to moderately toxic as the TEP. Of the 141 active ingredients with reported endpoints for oral exposure, 23 were classified as moderately or highly toxic, 113 were classified as practically nontoxic, and 5 were inconclusive. Only five (3.6%) were reclassified from nontoxic to moderately toxic as the TEP. Fewer than 5% of the total TEPs evaluated (contact and oral) were shown to be more toxic than the TGAI, suggesting that the risk assessments of TGAIs would be sufficiently protective to pollinators at the screening laboratory level.

Seasonal variation of pollen collected by honey bees (Apis mellifera) in developed areas across four regions in the United States.

For honey bees (Apis mellifera), colony maintenance and growth are highly dependent on worker foragers obtaining sufficient resources from flowering plants year round. Despite the importance of floral diversity for proper bee nutrition, urban development has drastically altered resource availability and diversity for these important pollinators. Therefore, understanding the floral resources foraged by bees in urbanized areas is key to identifying and promoting plants that enhance colony health in those environments. In this study, we identified the pollen foraged by bees in four developed areas of the U.S., and explored whether there were spatial or temporal differences in the types of floral sources of pollen used by honey bees in these landscapes. To do this, pollen was collected every month for up to one year from colonies located in developed (urban and suburban) sites in California, Texas, Florida, and Michigan, except during months of pollen dearth or winter. Homogenized pollen samples were acetolyzed and identified microscopically to the lowest taxonomic level possible. Once identified, each pollen type was classified into a frequency category based on its overall relative abundance. Species richness and diversity indices were also calculated and compared across states and seasons. We identified up to 64 pollen types belonging to 39 plant families in one season (California). Species richness was highest in CA and lowest in TX, and was highest during spring in every state. In particular, "predominant" and "secondary" pollen types belonged to the families Arecaceae, Sapindaceae, Anacardiaceae, Apiaceae, Asteraceae, Brassicaceae, Fabaceae, Fagaceae, Lythraceae, Myrtaceae, Rhamnaceae, Rosaceae, Rutaceae, Saliaceae, and Ulmaceae. This study will help broaden our understanding of honey bee foraging ecology and nutrition in urban environments, and will help promote the use of plants that serve the dual purpose of providing aesthetic value and nutritious forage for honey bee colonies placed in developed landscapes.

Comparison of Pesticide Exposure in Honey Bees (Hymenoptera: Apidae) and Bumble Bees (Hymenoptera: Apidae): Implications for Risk Assessments.

To date, regulatory pesticide risk assessments have relied on the honey bee (Apis mellifera L.) (Hymenoptera: Apidae) as a surrogate test species for estimating the risk of pesticide exposure to all bee species. However, honey bees and non-Apis bees may differ in their susceptibility and exposure to pesticides. In 2017, a workshop ('Pesticide Exposure Assessment Paradigm for Non-Apis Bees') was held to assess if honey bee risk assessment frameworks are reflective of non-Apis bee pesticide exposure. In this article, we summarize the workshop discussions on bumble bees (Bombus spp.). We review the life history and foraging behavior of bumble bees and honey bees and discuss how these traits may influence routes and levels of exposure for both taxa. Overall, the major pesticide exposure routes for bumble bees and honey bees are similar; however, bumble bees face additional exposure routes (direct exposure of foraging queens and exposure of larvae and adults to soil residues). Furthermore, bumble bees may receive comparatively higher pesticide doses via contact or oral exposure. We conclude that honey bee pesticide risk assessments may not always be protective of bumble bees, especially queens, in terms of exposure. Data needed to reliably quantify pesticide exposure for bumble bees (e.g., food consumption rates, soil residue levels) are lacking. Addressing these knowledge gaps will be crucial before bumble bee exposure can be incorporated into the pesticide risk assessment process. Because bumble bees exhibit appreciable interspecific variation in colony and behavioral characteristics, data relevant to pesticide exposure should be generated for multiple species.

A feeding protocol for delivery of agents to assess development in Varroa mites.

A novel feeding protocol for delivery of bio-active agents to Varroa mites was developed by providing mites with honey bee larva hemolymph supplemented with cultured insect cells and selected materials delivered on a fibrous cotton substrate. Mites were starved, fed on treated hemolymph to deliver selected agents and then returned to bee larvae. Transcript levels of two reference genes, actin and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), as well as for nine selected genes involved in reproductive processes showed that the starvation and feeding protocol periods did not pose a high level of stress to the mites as transcript levels remained comparable between phoretic mites and those completing the protocol. The feeding protocol was used to deliver molecules such as hormone analogs or plasmids. Mites fed with Tebufenozide, an ecdysone analog, had higher transcript levels of shade than untreated or solvent treated mites. In order to extend this feeding protocol, cultured insect cells were incorporated to a final ratio of 1 part cells and 2 parts hemolymph. Although supplementation with Bombyx mori Bm5 cells increased the amount of hemolymph consumed per mite, there was a significant decrease in the percentage of mites that fed and survived. On the other hand, Drosophila melanogaster S2 cells reduced significantly the percentage of mites that fed and survived as well as the amount of hemolymph consumed. The feeding protocol provides a dynamic platform with which to challenge the Varroa mite to establish efficacy of control agents for this devastating honey bee pest.

An Evaluation of the Honey Bee (Hymenoptera: Apidae) Safety Profile of a New Systemic Insecticide, Flupyradifurone, Under Field Conditions in Florida.

Flupyradifurone (Sivanto) is a novel systemic insecticide from the butenolide class developed by Bayer. Based on available data (USEPA 2014), this insecticide appears to have a favorable safety profile for honey bee colonies. As a result, the label permits the product to be applied during prebloom and bloom in various crops, including citrus, except when mixed with azole fungicides during the blooming period. We placed 24 honey bee (Apis mellifera L.) colonies adjacent to eight flowering buckwheat (Fagopyrum esculentum Moench) fields that either had been sprayed with the maximum label rate of flupyradifurone or with water only (control fields), with three colonies placed adjacent to each field. We conducted colony strength assessments during which the number of adult bees, eggs, uncapped brood cells, capped brood cells, food storage cells, and weights of honey supers and brood chambers were determined prior to, during, and after the flowering period. We also analyzed bee-collected pollen and nectar for flupyradifurone residues. Overall, there were no differences in any colony strength parameter for colonies placed at control and flupyradifurone-treated buckwheat fields. Residue analyses showed that pollen (x = 565.8 ppb) and nectar (x = 259.4 ppb) gathered by bees on fields treated with flupyradifurone contained significantly higher flupyradifurone residues than did bee bread and unprocessed nectar collected by bees from control fields (75% of samples

Initial recommendations for higher-tier risk assessment protocols for bumble bees, Bombus spp. (Hymenoptera: Apidae).

Global declines of bumble bees and other pollinator populations are of concern because of their critical role for crop production and maintenance of wild plant biodiversity. Although the consensus among scientists is that the interaction of many factors, including habitat loss, forage scarcity, diseases, parasites, and pesticides, potentially plays a role in causing these declines, pesticides have received considerable attention and scrutiny. In response, regulatory agencies have introduced more stringent pollinator testing requirements for registration and reregistration of pesticides, to ensure that the risks to pollinators are minimized. In this context, guidelines for testing bumble bees (Bombus spp.) in regulatory studies are not yet available, and a pressing need exists to develop suitable protocols for routine higher-tier studies with these non-Apis sp., social bees. To meet this need, Bayer CropScience LP, Syngenta Crop Protection LLC US, and Valent USA. Corporation organized a workshop bringing together a group of global experts on bumble bee behavior, ecology, and ecotoxicology to discuss and develop draft protocols for both semi-field (Tier II) and field (Tier III) studies. The workshop was held May 8-9, 2014, at the Bayer Bee Care Center, North Carolina, USA. The participants represented academic, consulting, and industry scientists from Europe, Canada, the United States, and Brazil. The workshop identified a clear protection goal and generated proposals for basic experimental designs, relevant measurements, and endpoints for both semifield (tunnel) and field tests. These initial recommendations are intended to form the basis of discussions to help advance the development of appropriate protocol guidelines.

Mi-1-Mediated Nematode Resistance in Tomatoes is Broken by Short-Term Heat Stress but Recovers Over Time.

Tomato (Solanum lycopersicum L.) is among the most valuable agricultural products, but Meloidogyne spp. (root-knot nematode) infestations result in serious crop losses. In tomato, resistance to root-knot nematodes is controlled by the gene Mi-1, but heat stress interferes with Mi-1-associated resistance. Inconsistent results in published field and greenhouse experiments led us to test the effect of short-term midday heat stress on tomato susceptibility to Meloidogyne incognita race 1. Under controlled day/night temperatures of 25°C/21°C, 'Amelia', which was verified as possessing the Mi-1 gene, was deemed resistant (4.1 ± 0.4 galls/plant) and Rutgers, which does not possess the Mi-1 gene, was susceptible (132 ± 9.9 galls/plant) to M. incognita infection. Exposure to a single 3 hr heat spike of 35°C was sufficient to increase the susceptibility of 'Amelia' but did not affect Rutgers. Despite this change in resistance, Mi-1 gene expression was not affected by heat treatment, or nematode infection. The heat-induced breakdown of Mi-1 resistance in 'Amelia' did recover with time regardless of additional heat exposures and M. incognita infection. These findings would aid in the development of management strategies to protect the tomato crop at times of heightened M. incognita susceptibility.

Examining the role of foraging and malvolio in host-finding behavior in the honey bee parasite, Varroa destructor (Anderson & Trueman).

When a female varroa mite, Varroa destructor (Anderson & Trueman), invades a honey bee brood cell, the physiology rapidly changes from feeding phoretic to reproductive. Changes in foraging and malvolio transcript levels in the brain have been associated with modulated intra-specific food searching behaviors in insects and other invertebrates. Transcription profiles for both genes were examined during and immediately following brood cell invasion to assess their role as potential control elements. Vdfor and Vdmvl transcripts were found in all organs of varroa mites with the highest Vdfor transcript levels in ovary-lyrate organs and the highest Vdmvl in Malpighian tubules. Changes in transcript levels of Vdfor and Vdmvl in synganglia were not associated with the cell invasion process, remaining comparable between early reproductive mites (collected from the pre-capping brood cells) and phoretic mites. However, Vdfor and Vdmvl transcript levels were lowered by 37 and 53%, respectively, in synganglia from reproductive mites compared to early reproductive mites, but not significantly different to levels in synganglia from phoretic mites. On the other hand, in whole body preparations the Vdfor and Vdmvl had significantly higher levels of transcript in reproductive mites compared to phoretic and early reproductive, mainly due to the presence of both transcripts accumulating in the eggs carried by the ovipositing mite. Varroa mites are a critical component for honey bee population decline and finding varroa mite genes associated with brood cell invasion, reproduction, ion balance and other physiological processes will facilitate development of novel control avenues for this honey bee parasite.

Novel adult feeding disruption test (FDT) to detect insecticide resistance of lepidopteran pests in cotton.

BACKGROUND: Resistance monitoring is an important aspect of insect resistance management and the preservation of insecticide efficacy. The adult vial test (AVT) is most often used for resistance monitoring for a variety of insects. A potential alternative method is feeding disruption where resistant insects are distinguished from susceptible insects on the basis of their ability to feed on insecticide in nectar containing a colorimetric marker to measure feeding. The advantages of a feeding disruption test (FDT) for lepidopteran adults might include a more rapid assay than AVT, an assay format easier to prepare, a bioassay applicable to both oral and contact insecticides and the provision of food and water during the course of the test. The objective of the present work was to determine the feasibility of an adult FDT. RESULTS: Heliothis virescens moths fed permethrin and spinosad in dyed nectar yielded dose-dependent ingestion, fecal production and mortality data. A permethrin diagnostic dose distinguished pyrethroid-resistant from pyrethroid-susceptible moths, based on fecal production. CONCLUSION: Proof of concept was demonstrated for an adult FDT in which resistant moths were distinguished from susceptible moths on the basis of the ability of the insect to feed on insecticide in dyed nectar and produce dyed feces.

New approach for the study of mite reproduction: The first transcriptome analysis of a mite, Phytoseiulus persimilis (Acari: Phytoseiidae).

Many species of mites and ticks are of agricultural and medical importance. Much can be learned from the study of transcriptomes of acarines which can generate DNA-sequence information of potential target genes for the control of acarine pests. High throughput transcriptome sequencing can also yield sequences of genes critical during physiological processes poorly understood in acarines, i.e., the regulation of female reproduction in mites. The predatory mite, Phytoseiulus persimilis, was selected to conduct a transcriptome analysis using 454 pyrosequencing. The objective of this project was to obtain DNA-sequence information of expressed genes from P. persimilis with special interest in sequences corresponding to vitellogenin (Vg) and the vitellogenin receptor (VgR). These genes are critical to the understanding of vitellogenesis, and they will facilitate the study of the regulation of mite female reproduction. A total of 12,556 contiguous sequences (contigs) were assembled with an average size of 935bp. From these sequences, the putative translated peptides of 11 contigs were similar in amino acid sequences to other arthropod Vgs, while 6 were similar to VgRs. We selected some of these sequences to conduct stage-specific expression studies to further determine their function.

Regulation of female reproduction in mites: a unifying model for the Acari.

It is well established in the literature that circulating high levels of juvenile hormone (JH) are responsible for the initiation of vitellogenesis and female reproduction in most insects studied so far. Exceptions include some Diptera, Lepidoptera and Hymenoptera. The current view is that JH also regulates yolk protein (vitellogenin, Vg) synthesis and female reproduction in mites. However, there is no published evidence that mites have the common insect JHs at any stage of their development. Also, research on the effects of exogenous applications of JH and JH analogs on the reproduction of mites is contradictory. Significant information is available on the life history of mite reproduction, and new information has become available on mite storage proteins including Vg. Although initial studies suggested that ticks may respond to exogenously applied juvenile hormone or anti-JHs, current research shows that ticks cannot synthesize the common insect JHs and have no detectable levels of these hormones in their hemolymph during female reproduction. In ticks, it appears that ecdysteroids, and not JH, regulate expression of the Vg gene and the synthesis and release of Vg protein into the hemolymph. In fact within the Arthropoda, JH has been found only in insects. Methyl farnesoate and not JH regulates Vg synthesis in the Crustacea, the sister group to the insects. Based on this evidence, a new working hypothesis is proposed, i.e., that ecdysteroids and not the JHs regulate vitellogenesis in the Acari including both ticks and mites. To the present, the role of neuropeptides in the regulation of female reproduction in mites is not known.

Characterization of vitellin protein in the twospotted spider mite, Tetranychus urticae (Acari: Tetranychidae).

In mites, vitellogenin synthesis, regulation and uptake by the oocytes as vitellin remain practically unknown. Although a partial sequence of the gene is now available, no previous studies have been conducted that describe the native vitellin protein in mites. The objective of this study was to characterize vitellin in the twospotted spider mite, Tetranychus urticae. The native twospotted spider mite vitellin migrated as a single major band with a molecular weight of 476+/-14.5 kDa as compared to 590+/-25.5 kDa for vitellin from the American dog tick, Dermacentor variabilis. However, isoelectric focusing analysis of native spider mite vitellin showed five bands with pI values slightly acidic to neutral (pH 5.8, 6.2, 6.7, 7.0 and 7.2), as is the case for insect and tick vitellins. Reducing conditions (SDS-PAGE) also revealed multiple subunits ranging from 290.9 to 3.6 kDa and was similar to that found in D. variabilis. Spider mite vitellin weakly bound lipids and carbohydrates compared to the tick. Unlike D. variabilis, the spider mite egg yolk protein does not bind heme. The significance of non-heme binding in mites is discussed.

Development and reproduction of Stratiolaelaps scimitus (Acari: Laelapidae) with fungus gnat larvae (Diptera: Sciaridae), potworms (Oligochaeta: Enchytraeidae) or Sancassania aff. sphaerogaster (Acari: Acaridae) as the sole food source.

Stratiolaelaps (=Hypoaspis) miles (Berlese) (Acari: Mesostigmata: Laelapidae) is a polyphagous soil-dwelling predatory mite that is widely marketed for use in greenhouse production systems to manage populations of dark-winged fungus gnats, Bradysia spp. (Diptera: Sciaridae) and for supplemental control of thrips. The suggestion by Walter and Campbell (2003, Biol. Control 26: 253-269) that North American commercial cultures of S. miles may actually be S. scimitus was confirmed. The development and reproduction at 21-23 degrees C of S. scimitus provided ad libidum with one of three different prey--the fungus gnat Bradysia aff. coprophila (Lintner), potworms (Enchytraeidae), or Sancassania aff. sphaerogaster (Zachvatkin, 1937) (Acari: Astigmata: Acaridae)--were compared. Developmental duration of the egg and non-feeding larval stages were 2.47 and 1.11 days, respectively; mortalities were 8.3 and 5.5%. Stratiolaelaps scimitus failed to develop beyond the protonymphal stage when provided with S. aff. sphaerogaster alone, although some feeding was observed. Development and reproduction of S. scimitus was successful on both fungus gnat larvae and enchytraeids, with no influence of prey on protonymphal duration (4.70 days) and mortality (8.3%), or on deutonymphal duration (4.61 days) and mortality (6.1%). Adult female S. scimitus feeding on potworms, compared to those feeding on fungus gnat larvae, had a significantly shorter pre-oviposition period (2.69 vs. 4.59 days). However, diet did not influence other adult female developmental or reproductive characteristics (oviposition period, 18.6 days; post-oviposition period, 6.2 days; total adult longevity, 27.3 days; total number of eggs, 26.5). S. scimitus reared on potworms tended (p = 0.06) to have a higher intrinsic rate of increase, a higher finite rate of increase and a shorter doubling time (rm = 0.142 day(-1), lambda = 1.153, Dt = 4.85 days) than those reared on fungus gnat larvae (rm = 0.105 day(-1), lambda = 1.110, Dt = 6.58 days), but differences in net reproductive rate (R0) and generation time (G) were not significant.

Effects of greenhouse pesticides on the soil-dwelling predatory mite Stratiolaelaps scimitus (Acari: Mesostigmata: Laelapidae) under laboratory conditions.

Knowledge of the effects of pesticides on biological control agents is required for the successful implementation of integrated pest management (IPM) programs in greenhouse production systems. Laboratory assays were conducted to assess the effects of an acaricide (dicofol), two insecticides (chlorpyrifos and pyriproxyfen), and two fungicides (fosetyl-Al and mefenoxam) on Stratiolaelaps scimitus (Womersley), a soil-dwelling predatory mite widely marketed in North America under the name Hypoaspis miles (Berlese) as a biological control agent of dark-winged fungus gnats (Bradysia spp.). Eggs, larvae, protonymphs, deutonymphs, and adult male and female mites were first assayed using dicofol, an acaricide used in the experiments as a positive control, applied to filter paper in an enclosed arena. Protonymphs were assayed for lethal and sublethal effects against the remaining pesticides at maximum label-recommended rates applied to filter paper, by using dicofol as a positive control and water as a negative control. The larva and protonymph were the life stages most susceptible to dicofol, with estimated 24-h LC50 values of 9 and 26 mg m(-2), respectively. Chlorpyrifos was highly toxic to the protonymphs of S. scimitus, causing >95% mortality after 24-h exposure and 100% mortality after 48 h. In contrast, the insect growth regulator (IGR) pyriproxyfen was much less toxic to protonymphs of S. scimitus; pyriproxyfen caused no significant mortality, compared with <5% mortality in the water control. Mortality caused by the fungicides was also relatively low; 72-h exposure to fosetyl-Al and mefenoxam resulted in 17.4 and 27.5% mortality, respectively. The IGR and fungicides increased the duration of the protonymphal stage by 1.2-1.8-fold, but they had no effect on the duration of subsequent life stages, nor on the duration of preoviposition, oviposition, and postoviposition periods of adult females. Total numbers and viability of eggs laid by mites exposed to the IGR and fungicides did not differ from the negative control, although the average rate of egg production during the oviposition of mites exposed to fosetyl-Al was increased. Pyriproxyfen, fosetyl-Al, and mefenoxam are likely to be compatible with S. scimitus under field conditions, because these pesticides caused little mortality of protonymphs, and they did not negatively affect the development and reproduction of S. scimitus under extreme laboratory conditions. In contrast, the use of chlorpyrifos in conjunction with S. scimitus is not recommended unless more comprehensive testing under semifield or field conditions demonstrates compatibility.