,Colonies, apiaries, and diet treatments,Colonies used in this study were part of an existing commercial beekeeping operation. The colonies were on pallets containing 4 colonies per pallet. The colonies were not normalized or equalized prior to the start of the experiment. All the colonies had an adult population, open and sealed brood, and no obvious signs of disease at the start of the experiment and were housed in two Langstroth deep boxes with 16-18 frames and 1-2 syrup feeders. Prior to the start of the experiment, the colonies were transported from Fannin County, Texas, USA to North Dakota, USA for honey production on May 20th (Table 1). The colonies were split evenly between two apiaries (“Edmore” and “Loma”; Table 1) and were treated with oxalic acid miticide in early August after the honey was harvested and before the experiment started.,Colonies were fed one of five commercial diets (MegaBee (megabee.com; 60 colonies), BeePro (Mann Lake; 60 colonies), UltraBee (Mann Lake; 56 colonies), AP23 (Dadant; 57 colonies), or Global 4% (base diet + 4% irradiated natural pollen; www.globalpatties.com; 60 colonies)) or a positive control diet (Global base diet + 75% irradiated natural pollen (globalpatties.com; 59 colonies)) in trial 1. The Global 75% diet contained natural pollen that comprised 75% of the dry weight, the maximum amount of dry material that could be used. In trial 2, colonies were fed one of the same five commercial diets, the same positive control diet, or no diet as a negative control (50 colonies per treatment split evenly between the apiaries). All diets came from the manufacturer in patty form. The diet patties weighed 0.454 kg (1lb) except for the MegaBee diet, which came as one solid block of patty that the field crew cut to approximately 0.454 kg patties. The diets were first applied immediately after the August pre-feed colony assessments (Figure 1). Patties were placed under the hive cover on the top bars of the frames weekly for 5 weeks.,In both trials, the colonies were moved in early October from each apiary site to the cold storage facility in Filer, Idaho, USA to overwinter (“post-feed”). In January, the colonies were transported from the cold storage facility to almond orchards in Tulare County, California, USA for pollination (“pre-almonds”). It was logistically unrealistic to assign colonies to specific orchards or spots in the cold storage facility, but the colonies were randomized as much as possible.,Colony assessments,Colonies were assessed at four time points in both trials: a pre-trial assessment before the diet was added (“pre-feed”), after weekly feedings and just prior to cold storage (“post-feed”), after cold storage in the almond orchard before bloom (“pre-almonds”), and during almond bloom (“post-almonds”). Colony assessments took 2 days and were performed using methods described in (DeGrandi-Hoffman et al. 2019). Briefly, the frames of bees (FoBs) in all colonies were measured using a variation of the “California count” that is used to grade colonies for almond pollination (Goodrich and Goodhue 2016). This method enabled the rapid measurement of >300 colonies in 2 days. The top and bottom boxes were separated, tilting the top box against the bottom box. The number of frame tops covered by bees in the bottom box (looking down) and on the frame bottoms in the top box (looking up) were counted. The California count colony size estimates are represented in frames of bees (FoBs).,In trial 1 (2021-2022), at each time point, a more detailed colony assessment was performed in a subset (Nsubset=121) of the experimental colonies located in each apiary (18 BeePro, 19 UltraBee, 17 MegaBee, 15 AP23, 20 Global 4%,16 Global 75%). These data were collected after the California counts were performed. In this more detailed colony assessment, the area of a comb occupied by adult bees was measured as described in Delaplane et al. (2013). Briefly, each frame was divided into five sections per

,The data represent estimates of honey bee colony sizes expressed as combs (frames) of adult bees and combs of brood (eggs, larvae and pupae). Colony sizes were estimated by dividing combs into one-tenth sections and counting the number covered with bees or brood on both sides of a comb. The values from each comb were summed and used as an estimate of combs with bees or brood for each colony. During pre-cold storage measurement in October, colonies were comprised of two deep Langstroth hive boxes. Temperatures were below 10o C so the one-tenth of the comb area method for comb evaluations was not used. Instead, estimates of adult bee populations were made by tilting the top hive body forward on the pallet and frames with at least 75% bee coverage on the top and bottom of the comb were counted as a comb of bees. In the lower hive body, adult populations were estimated from adult bees covering the top of the combs. Combs with bees were totaled for the colony as an estimate of colony size. Estimates of Varroa mites (Varroa destructor Anderson & Trueman) per 100 adult bees were made using approximately 300 worker bees per colony that were brushed from brood comb into jars containing 50 ml of 70% ethanol. Mites were counted by vigorously shaking the sample jars, and pouring the bees and alcohol into a strainer positioned over a pan. The mites that went through the strainer and into the pan were counted. Bees in the strainer also were examined for mites. All the bees in the sample were counted to estimate mites per 100 bees. Nosema (Vairimorpha (Microsporidia: Nosematidae) spore counts per colony were based on samples of 20 bees per colony. Samples were placed in a test tube containing ultra-pure water and homogenized for 5s. Samples equilibrated in 30-60 s, and the supernatant was removed from the center of the sample in the test tube (clear area) and placed into a 1.5ml Eppendorf tube. Spores per colony was estimated by transferring a 15 µl sample of supernatant to a hemocytometer, and examining it using a compound microscope at 400x with phase-contrast lighting. Nosema spores were counted in 16 small squares within five larger squares. The final spore count was calculated by multiplying the hemocytometer counts by 50,000. Fat body metrics (weight and lipid and protein concentrations) are based on pooled samples of 10 bees per colony. Fat bodies were removed by placing an adult worker bee onto a block of dry ice, and removing the abdomen. The entire gut was removed and the remaining abdominal carcass with fat body attached was rinsed to remove remaining gut contents and blotted dry. Fat body weight was estimated after drying the abdominal carcass at 60oC for four days. Fat body protein concentration was estimated with a BCA Protein Assay kit. Samples were analyzed in triplicate and read in a microplate reader at a wave length of 562nm. The absorbance values of blank wells were subtracted from each standard and sample. Protein concentration (µg/µl) was estimated using the absorbance and standard curve. Lipid concentrations were estimated by placing the fat body sample into a tube containing a 2:1 mixture of chloroform:methanol (1 ml) along with 210 µl of 0.25% KCI. The sample was vortexed, and centrifuged at 2,000 rpm for 15 min. The bottom chloroform layer was removed and placed into a 2ml glass screw cap vial. Serial dilutions of corn oil dissolved in chloroform were prepared to construct a standard curve for lipid concentrations. The negative control consisted of 100 µl of chloroform. Samples, standards, and the negative control were dried to completion for approximately 1.5h. Dried samples were reacted with 182 µl of concentrated sulfuric acid at 100oC for 15 min and 1478 µl of vanillin-phosphoric acid for 15 min in the dark at room temperature. Each of the negative controls, standards, and samples (100µl) were plated in triplicate and read using a spectrophotometer at 525nm. The average absorbance value for the negative control

,In this study, we investigated how protein deprivation following adult emergence influences lethal and sublethal effects of boric acid on the pest tephritid melon fly, Zeugodacus cucurbitae. We performed a series of experiments to address the impact of prior diet on mortality, diet consumption, enzymes involved in detoxification and antioxidation, and fly activity. Newly emerged melon fly adults were provided either diet containing 3:1 sucrose:yeast hyrdrolysate or just sucrose for three days prior to bioassays. The raw data from the bioassays and R scripts for the analyses are provided.,

,A native solitary bee to North America, the blue orchard bee (Osmia lignaria Say, Hymenoptera: Megachilidae) is a crucial pollinator for orchard crops such as apples, almonds, and cherries. Osmia lignaria is often managed commercially and sold to complement honey bee pollination services.,We collected data following an accidental drop of developing immature bees inside their cocoons. These bees were part of a larger experiment performed in 2020. On June 17, 2020, bees were dropped approximately one meter onto a linoleum floor at the USDA-ARS-PWA Pollinating Insect Research Unit in Logan, Utah, USA. Developing bees were in gelatin capsules and attached to a sticky board for X-ray imaging. Using a board from the same study that had not fallen, we compared survival, life stages, and bodily injuries to document the effects of dropping immature O. lignaria a short distance.,Our research highlights the risks of handling immature O. lignaria during metamorphosis. Our data provides valuable information for bee managers and researchers about the risks of physical disturbances during critical developmental stages, which could affect bee survival and pollination services in orchards.,Key findings include: (1) Near-complete mortality of developing bees before the adult molt stage, (2) Insights into the vulnerability of O. lignaria during immature developmental stages, even when inside cocoons, and (3) Documentation of how mechanical injury during immature development impacts survival.,The dataset provides counts of bees in different life stages and conditions, including: (1) Life status (alive or dead) at cocoon completion, pupation, and adult molt stages, (2) Sex determination for bees that reached adulthood (male or female), (3) Final life stage reached (prepupa, pupa, or adult), and (4) Body condition after the fall (malformed, melanized, no observable change, or partially melanized).,Additional variables in the dataset include: (1) Sample identifiers, treatment groups, and X-ray board identifiers from the original experiment and (2) Whether the board was dropped or not.,Abbreviations and acronyms in the dataset,,

,Endosymbiont interactions with hosts have important effects on fitness, including the fitness of many pest and beneficial species. Among these interactions, facultative endosymbiotic bacteria can protect aphids from parasitoids. Aphis craccivora and Acyrthosiphon pisum can harbor the symbiotic bacteria Hamiltonella defensa and its bacteriophage APSE. Infection by H. defensa defends these aphids against some but not all parasitoid species in the hymenopteran family Braconidae. Here, we report results on the effect of H. defensa on parasitism of these aphids by species in the other major lineage of aphid parasitoids, Aphelinus species in the family Aphelinidae. Parasitism of aphids infected with H. defensa /APSE by two Aphelinus species did not differ from that of uninfected aphids. While Aphelinus atriplicis showed no difference in fitness components between infected and uninfected aphids, Aphelinus glycinis actually produced more adult progeny and larger female progeny on infected than on uninfected aphids. Aphelinus glycinis may increase host quality for itself by changing the titer of nutritional versus protective bacteria in such a way that aphids infected with H. defensa can be made more suitable for parasitoid development than uninfected aphids. Our results and reasoning suggest that these Aphelinus species may be less prone to harm by H. defensa /APSE that affect eggs because they have anhydropic, heavily chorionated eggs, which may not absorb toxins during embryogenesis.,See the included methods-DefensiveAphidSymbiont.txt file for more information on the methods and design structure of this study.,

,The data set contains two years of data on honey bee colony sizes expressed as frames (combs) of adult bees and brood, Varroa mites counted per 100 bees and Vairimorpha (Nosema) spp. spores in worker bees in each colony. The data set also contains measurement of fat body weights, protein and lipid concentrations made prior to and after colonies were overwintered in cold storage. In the year-2 data set, colony measurements, Nosema and Varroa mite counts and fat body metrics were separated by treatment group. The treatment groups were: colonies fed pollen or protein supplement and treated with Fumagillin for Nosema and colonies fed the same diets but not treated with Fumagillin for Nosema.,

,Experimental Insects,Four to eight-week mixed-sex adults of a field strain from Eastern Kansas (collected in 2022, hereafter FS-22) and pyrethroid-resistant strain collected from Juiz de Fora County in the state of Minas Gerais, southeastern Brazil in 2006 (hereafter, Brazil-resistant) S. zeamais were used in this study. The Brazil-resistant strain exhibits high pyrethroid resistance and low fenitrothion resistance and has been used in prior studies (Guedes et al., 2006a). These strains were reared and maintained on tempered organic maize at 25–27.5 °C, 65% RH, and 16:8 (L:D) h photoperiod.,Synergist-coated glass vials,In this study, we used one of the most effective synergists, piperonyl butoxide (PBO, Tokyo Chemical Industry Co. Ltd., Tokyo, Japan). Briefly, each of 20-ml glass scintillation vials was coated with 0.5 ml of PBO solution in acetone (solvent) at 0.1 mg/ml by using a Roto-Torque Heavy Duty Rotator (Model 7637, Cole-Parmer Instrument Company, Vernon Hills, IL, USA). For the control, vials were treated with 0.5 ml of acetone (solvent) only.,LLIN treatment,We used 0.34% alpha-cypermethrin based LLIN (63.2 mg/m2 active ingredient (a.i.), 40 deniers, 100 holes/cm2; Carifend®, BASF, Ludwigshafen, Germany) and a netting physically identical but without insecticide (Casa Collection, Mesh White, 1721-9668; Jo-Ann's, Hudson, OH, USA) as a control netting in our study.,Effects of synergists on LLIN against S. zeamais,A cohort of 20 mixed-sex S. zeamais adults was first pre-exposed to each scintillation vial coated with PBO or acetone (control) for 60 min (1 h) or 180 min (3 h). The pre-exposed adults were then transferred to each plastic Petri dish (9 × 9 cm square) containing either LLIN or control netting and were exposed for 60 min or 180 min. The inside walls of the dishes were coated with a polytetrafluoroethylene (PTFE) preparation (e.g., fluon, 60 wt% dispersion in water, MilliporeSigma GmbH, Steinheim, Germany) to prevent insects from escaping. After exposure, insects were placed in an environmental chamber under constant conditions (30°C, 65% RH, and 16:8 L:D). A total of n = 5 replicates were performed per treatment combination of strain, exposure time, netting type, and synergist. Immediate mortality was recorded directly after exposure, as well as delayed mortality at 24, 48, 72, and 168 h later. Insect conditions were recorded as alive, affected, or dead as described by Ranabhat et al. (2022). Specifically, insects moving normally were considered alive, whereas they were considered affected if they moved in an uneven pattern and/or exhibited twitching of tarsi or antennae or showed lethargic or drunken movements. On the other hand, insects were considered dead if no visible movement was observed after disturbance with a fine brush.,Lethal exposure assay to determine the susceptibility of S. zeamais,For this assay, a cohort of 20 mixed-sex adults of laboratory (pyrethroid-susceptible, FS-22) or Brazil pyrethroid-resistant strain of S. zeamais was exposed each 20-mL glass scintillation vial coated with a 2 mg/ml deltamethrin solution in acetone (solvent) or acetone only (control) at constant conditions (27.5° ± 0.1 C, 65% RH, 16:8 L:D) in an environmental chamber. Each of the three insect conditions including alive, affected, or dead as described above was recorded at each of 12 time points (i.e., 1, 2, 4, 6, 24, 48, 72, 96, 144, 168, 192, and 216 h) after the exposure. To examine the insect’s conditions, the exposed adults from each vial were transferred to each plastic Petri dish (90 mm in diameter; 59.4 cm2 bottom surface area) with a lining of a filter paper (85 mm D, Grade 1, GE Healthcare, Buckinghamshire, United Kingdom) that was adhered to the bottom using double-sided tape. The inside walls of the dishes were covered with a polytetrafluoroethylene preparation (Fluon, 60 wt% dispersion in water, MilliporeSigma GmbH, Steinheim, Germany) to prevent insects from escaping. The insect conditions were

,Nutritional deprivation is known to contribute to increased honey bee mortality, physiological stress, aberrant behaviors, and disease incidence. To investigate the effect of a realistic nutritional protein deficiency, we simulated a pollen dearth in half of our experimental colonies by robbing incoming foragers of their pollen loads, the primary source of dietary protein, at the colony entrance. We then conducted temperament assays on each colony weekly for pollen deprived and control counterparts. We also identified the plant species bees foraged from and took various physiological measures of honey bee nutritional status including gland size, lipid quantification, and gene expression to further investigate and explain our behavioral results. We found that colonies deprived of pollen reacted by becoming more defensive and that immature bees likely receive cues during rearing which prime their gene expression and behavior as adults, ultimately suggesting that environmental stress caused significant behavioral changes. Temperament is primarily associated with genotype, but there are environmental cues which are less acknowledged and still important. As droughts become increasingly frequent and resource availability therefore changes over time, the impacts on behaviors of agricultural keystone species need additional consideration in order to form scientifically driven best management practices.,

,The data provided is from a study on overwintering honey bee colonies using cold storage. Colonies were summered in two different geographic regions of the USA: South Texas and North Dakota. Colonies summered in North Dakota were placed in cold storage in either October or November of 2019. Placement in cold storage of hives summered in South Texas happened in November of 2019. A second set of colonies overwintered in south Texas. All sets of colonies were evaluated and sampled prior to and after cold storage and again after almond bloom in 2020. Colonies overwintered in South Texas were evaluated during the same periods as those in cold storage. Data are provided for frames of bees and brood pre and post cold storage as well as after almond bloom. The data included in the two files shows: bee frame counts, mite counts and brood counts. Lab data provided shows fat body weight as well as protein and lipid concentrations in worker bees pre and post cold storage /overwintering.,Resources in this dataset:,

,2.1 Source Host and Parasitoid Insects,For all assays, 4–8-week-old R. dominica were reared on wheat, while S. oryzae were reared on wheat tempered to 13% grain moisture. To subculture, a total of 50 individuals were placed on 200 mL of grain in a mason jar (capacity: 473 mL) and given 14 d to mate and lay eggs. At the end of that period, adults were removed by sieving with a #10 sieve (2.00 mm; W.S Tyler Inc., Mentor, Ohio), and colonies were allowed to age for 3-weeks prior to using beetles as hosts for parasitoid rearing. Hosts used for experiments below were 2–3 weeks old. Theocolax elegans were maintained separately on two different hosts, either R. dominica or S. oryzae for at least three full generations. All colonies of hosts and parasitoids were held at 27.5°C, 60% RH, and 14:10 L:D, with parasitoids maintained in a separate environmental chamber than host only colonies to prevent cross-contamination.,2.2 Odor Treatments,Odor treatments included: 13 g of S. oryzae-damaged grain (SO-grain, hereafter) from the non-natal environment, 13 g of R. dominica-damaged grain from the non-natal environment (RD-grain), 13 g of damaged grain + conspecifics from the natal environment (Natal-grain), 10 S. oryzae adults alone (SO), 10 R. dominica adults alone (RD), and a clean (uninfested and undamaged) grain control (Ctrl). Treatments were always freshly sourced from colony material as described above, and adults were sieved out of the insect-damaged grain treatments. Grain was only pulled after colonies were 4-weeks-old. These treatments were used as odor sources for the still-air and four-way olfactometer assay. Odor treatments for the headspace characterization included: clean grain, R. dominica-infested grain, R. dominica-infested grain + T. elegans, S. oryzae-infested grain, S. oryzae-infested grain + T. elegans, psocid-infested grain, and a clean control. We included a psocid-infested grain treatment to rule out the influence of psocids in some of the replicates, which comprised incidental contamination.,2.3 Four Arm Olfactometer,In a four-arm, still-air olfactometer, we evaluated the orientation and taxis of R. dominica- or S. oryzae-reared T. elegans to the odor treatments discussed above (Fig. 2). The custom-built olfactometer consisted of a central, circular (8.26 × 2.54 cm D:H) acrylic release chamber with 12 holes per cm (each of 1.75 mm D), with four abutting rectangular, glass chambers (6.35 × 6.35 × 2.54 cm L:W:H). The bottom of the olfactometer consisted of a single glass sheet (25.4 × 25.4 cm W:L). In each trial, one of the adjacent chambers was randomly selected to contain the odor treatment, while the other three remained empty. A single parasitoid was released in the center of the circular release chamber, and a sheet of glass (25.4 × 25.4 cm W:L) was immediately placed over the top of the olfactometer. The time to response of first decision, and the zone on which adults exited was recorded as either the treatment chamber (stimulus), or empty chamber (non-stimulus; one of the other three edges). Parasitoids were given 3 min to respond to the odors, and non-responders were excluded from analysis. A total of n = 15 replicate wasps were tested per treatment. After each replicate, the olfactometer was wiped down with methanol, then hexane, and allowed to dry. At the end of a day of testing, the whole apparatus was thoroughly washed with soap and water.,,2.4 Headspace Characterization,To characterize the relative difference in volatiles among treatments, a headspace collection system was used (after Van Winkle et al. 2022). Central air was scrubbed using an activated charcoal filter, then pushed through the remaining apparatus. The airflow was restricted to 1 L/min using a flow meter (Volatile Collection Systems, Gainsville, FL, USA) placed directly prior to the sample collection from the headspace chambers (10.2 × 12.7 cm D:H, 500 mL capacity) with an inlet for air and an outlet for a volatile collection trap (VCT).