Workers interact directly with their queen by forming a retinue where feeding, grooming, and pheromone communication exchanges occur. Retinue interactions with naturally mated, instrumentally-inseminated (drone sperm and saline) and virgin queens were observed throughout the period of adult queen development from emergence through early oviposition. Retinue observations were recorded in situ on nest frames enclosed by metal observation frames. By the time open mating had ended (9 days after adult emergence), workers formed larger retinues with naturally mated and instrumentally inseminated queens than with virgin queens. At the time when queen oviposition first became widespread (16 days after emergence), more retinue workers were observed attending drone sperm inseminated queens than other treatment queens. Larger retinues may form in support of rapid development after queen mating and insemination. Mated and inseminated queens developed larger, more mature ovaries and greater body masses than virgin queens once oviposition commenced (16 days after emergence). Queen signaling by the major QMP pheromone components differed between virgin queens and mated/inseminated queens. By the time oviposition started (16 days after emergence), virgin queens had less methyl-4-hydroxybenzoate (HOB) than other treatment queens, and less 4-hydroxy-3-methoxyphenylethanol (HVA) than naturally mated or drone sperm inseminated queens. Queens may rely on additional cues present in queens after mating to recruit more retinue workers in support of ovary development and overall fecundity.

Honey bees balance colony populations against available food resources by adjusting brood rearing during nutritionally-stressed periods. Workers limit colony populations primarily through brood cannibalism of eggs and young larvae but often resume brood rearing when conditions improve. However, extended brood cannibalism reduces brood and removes brood signals that mediate brood rearing, such as E-β-ocimene, a volatile pheromone produced by eggs, young larvae, prepupae and ovipositing queens. We examined the effects of pollen supplementation on ocimene signaling in nutritionally-stressed colonies. Pollen-deprived colonies showed declines in ocimene emissions that coincided with sustained brood cannibalism of pheromone-producing brood. In contrast, pollen-supplemented colonies reared more brood and released more ocimene. Twelve day old workers that completed adult development in pollen-deprived colonies had less well developed hypopharyngeal glands and fat bodies than workers that matured in pollen-supplemented colonies. Given that ocimene emissions increased once brood rearing resumed, we considered the possibility that ocimene may help suppress brood cannibalism and support egg retention in nutritionally stressed nuc colonies. Broodless nucleus frames were treated with synthetic ocimene releases equivalent to 3,744 L2-L3 larvae. All ocimene-supplemented nucs retained large numbers of eggs and young larvae four days after initial treatment. By contrast, half of the unsupplemented nucs cannibalized all of their eggs and L1 larvae. Most of the remaining unsupplemented nuc colonies retained fewer eggs and L1 larvae than ocimene supplemented nuc colonies. E-B-ocimene may prime nutritionally stressed workers to increase brood rearing during dearth periods by projecting the presence of healthy eggs and young larvae.

Sunflower production depends on pollination by honey bees, Apis mellifera L., and wild bees. For wild sunflower oligoleges, preference for various sunflower lines seems to reflect the quantity (or accessibility) of floral rewards, with floret size (approximate to accessibility of sunflower nectar) appearing most important. However, it is less clear how honey bees, a key generalist, are affected by sunflower floral traits. The honey bee-sunflower interaction was explored to test if accessibility of nectar (approximate to floret size) and related floral traits explain foraging preferences and to assess potential nutritional variation in sunflower pollen. In the first two years, honey bee foraging preference increased with decreasing floret length similar to previous observations with wild pollinators. In the second year, no similar response was seen, though nectar rewards (by volume or volume x concentration) were remarkably low compared to the previous year. Pollen collected from bagged plants in the second year showed sunflower lines differed in concentrations (mu g/mg +/- SE) of total fatty acids and essential fatty acids. In general, it appears that honey bee responses to floral traits are similar to those of wild bees responsible for pollinating much of the sunflower crop in the central United States and that variation in the nutritional quality of sunflower pollen is greater than previously known. Because of the broad geographic distribution of the sunflower crop, additional research on how the environment influences floral rewards and sunflower-pollinator interactions may be needed.

Neonicotinoid-contaminated sugar stores can have both near term and long term effects on honey bees due to their persistence in honey stores. Effects of imidacloprid food stores contaminants were examined in subtropical colonies that experience reduced brood rearing and foraging during overwintering. Colonies were given treatment sugar syrup containing 0 ppb (control), 20 ppb (field relevant), or 100 ppb (above field relevant) imidacloprid over six weeks to simulate contaminated fall nectar. Colonies were evaluated immediately (post-treatment) and 10 weeks (mid-winter) after treatment to compare proximal and latent effects. Post-treatment 0 ppb and 20 ppb colonies had more workers than 100 ppb colonies while 0 ppb colonies more brood than 20 ppb or 100 ppb colonies. Mid-winter 0 ppb and 20 ppb colonies had more workers than 100 ppb colonies and 0 ppb colonies more brood than 100 ppb colonies. Colonies experienced seasonal declines in stored pollen but no treatment effects. Lower 100 ppb colony performance was associated with reduced effort rather than lifespan. RFID (Radio Frequency Identification) tracking revealed that workers had similar adult lifespans across treatments; however, 100 ppb workers engaged in activities outside the colony for less time than 0 ppb workers. Imidacloprid exposure affected queen but not worker nutritional physiology. Nurses retained well-developed hypopharyngeal glands (as indicated by head protein) across treatments. Mid-winter queens from 0 ppb colonies had marginally higher ovary protein than queens from 100 ppb colonies and more ovary lipids than queens from 20 ppb colonies. However, queen nutrient stores in non-reproductive tissues (fat bodies) did not differ across treatments. Queens from different treatments were attended by comparable numbers of retinue workers and had similar gland contents of four QMP (Queen Mandibular Pheromone) components essential to queen care. High levels of imidacloprid in sugar stores can negatively affect colony performance months after initial storage.

The honey bee parasite, Varroa destructor, is susceptible to removal by dusting agents that physically interfere with its ability to cling to its phoretic hosts. Here we describe modifications to established powdered sugar dusting techniques that modestly increased mite separation from hosts and allowed for greater reductions in whole colony mite infestation rates. These modifications increased body-to-body contact through crowding and mechanical agitation to supplement dusting effects on mite removal. Adult workers were isolated in a screened sugar shake box outside the colony, dusted with powdered sugar, then carefully shaken and bounced in crowded piles for one minute to separate mites from their hosts. Whole colony powdered sugar shake treatments resulted on average in a 92% reduction in post-treatment mite infestation rates over three successive mite treatments. Our modifications provided effective mite control comparable to the miticide Apiguard (thymol) without discernable negative effects on colonies. Four months after treatment, powdered sugar shake treated colonies had relatively similar-sized adult worker and brood populations as Apiguard treated colonies and larger adult populations than controls. Colonies also maintained queens and reared relatively few queen cells across treatments. Further comparisons of method components revealed that mechanical agitation of crowded workers enhanced mite separation from hosts due to powdered sugar dusting. Mechanical agitation alone only modestly increased mite separation compared to controls; however, high levels of mite separation associated with sugar dusting increased with agitation. These modified methods provide a rapid if laborious technique for reducing mite populations without chemical residues from restricted miticides.

This protocol quantifies cysteine residues from small amounts of pollen (10 mg) after phenylisothiocyanate (PITC) derivatization (adapted from Manneberg et al 1995). Quantification of cysteine by other methods is difficult because of its instability during amino acid analysis. Samples of anther pollen, corbicular pollen, or stored pollen from bee colonies are completely digested in strong acid. Cysteine residues are completely oxidized to cysteic acid then derivatized with PITC before HPLC UV VIS analysis. Manneberg, M., Lahm, H., & Fountoulakis, M. (1995). Quantification of cysteine residues following oxidation to cysteic acid in the presence of sodium azide. Analytical Biochemistry, 231, 349-353. https://doi.org/10.1006/abio.1995.9988 National Program Number: NP 305 Project Number: 2022-21000-022-000D, 2022-21000-022-053-I Carl Hayden Bee Research Center, USDA-ARS PWA, Tucson, Arizona, USA Grand Challenge - Assessing the Nutrient Contents of Pollen for Bees

Honey bee colonies maintain viable queens in part through communication with Queen Mandibular Pheromone (QMP), a mixture that signals the queen's presence and reproductive quality to workers. In turn, workers are thought to provide retinue queen care or replace queens partially based on QMP profiles. We examined the effects of seasonal dearth (overwintering in a warm subtropical location) on queen-worker interactions. Retinue worker responses to continuously ovipositing queens were considered in view of QMP signaling and queen reproductive quality. QMP signaling was estimated from QMP residues recovered from nest worker bodies, which is the primary mode of QMP transfer from the queen to the colony at large. QMP residues varied seasonally but not at all with queen reproductive quality (spermatheca sperm storage, ovary protein and lipid contents). 9-HDA and 9-ODA were lower in January than other months. HOB decreased from July to January, while HVA, a component associated with mated queens, increased sharply in January. Despite these seasonal signaling differences, retinue workers attended queens at similar levels through the months. In terms of reproductive quality, queens did not differ over the months in matedness (spermatheca sperm storage) or physiological age (protein carbonyl content), but varied in nutrient allocation to reproductive and non-reproductive tissues. Queen ovaries contained more protein in September than in November, and more lipid in July and September than in November and January. Queen fat bodies had more protein in July than September or November, but less lipid in July and September than November or January. Retinue worker responses did not vary with seasonal QMP changes, but reflected overall continuous brood rearing efforts and queen matedness throughout the year. The absence of seasonal differences in worker responses to QMP should be considered in the broader context of continuous reproductive efforts in warm subtropical colonies.

Nutrition supports social insect colonies by regulating both individual performance and colony growth. In honey bee colonies, task-related behaviors such as nursing and foraging are partially mediated by nutrition. Young workers (nurses) consume almost all of the pollen in the hive, while foragers consume mostly nectar. Pollen provides vital proteins and lipids, consumed by nurse bees for approximately 1 week post-eclosion. The role that lipids play in the physiology and behavior of adult bees is gaining significant attention. Recent research suggests that diets with balanced ratios of fatty acids increase olfactory learning in honey bees. Olfaction is crucial for young worker bees to perform brood care and cell cleaning behaviors, which is important for hive health and disease control. Thus, we targeted the early adult, pollen-feeding stage to examine how fatty acids affect cognition to hive-relevant odors. We fed young workers (days 0-9) diets balanced or unbalanced in their ratio of essential fatty acids (omega-6:3) sourced from pollen or cooking oils. We then measured their ability to learn healthy and damaged brood odors, as well as their ability to discriminate between the two. Workers fed balanced diets could learn and discriminate between brood odors better than workers fed unbalanced diets. Consumption of both diet types decreased with age, but their cognitive effects remained. These results suggest that diet affects young worker cognitive development, which may affect task-related behaviors and colony hygiene.

Honey bees are incidentally exposed to pesticides such as the insect growth regulator methoxyfenozide (MEOF) during crop pollination, exposures that extend into the hive via contaminated stored food. We examined the sublethal effects of MEOF-contaminated pollen and queen cell wax on replacement queen development. MEOF-exposed colonies were largely able to produce replacement queens of similar physiological and reproductive quality as unexposed colonies. Newly established queens did not differ in their body mass, ovariole development, or protein and fatty acid contents in their ovaries and fat bodies. MEOF and control queens had similar glandular contents of queen mandibular pheromone (QMP) and queen retinue pheromone (QRP) compounds. However, MEOF queens stored less sperm in their spermathecae than control queens. Given that queen productivity is ultimately limited by sperm availability, MEOF contamination might shorten the functional lifespan of exposed queens.

Honey bee colony health is a function of the individuals, their interactions, and the environment. A major goal of honey bee research is to understand how colonies respond to stress. Individual-level studies of the bee stress response are tractable, but their results do not always translate to the colony level. Nutritional stress is an important factor in colony declines. Nutrition studies are typically conducted on individual nurse workers (nurses), who are primarily responsible for converting pollen into brood. Nurse physiology is sensitive to both pollen and pheromones, which communicate signals among colony members. Here, we asked whether phero-mones influence nurse nutrient pathways involved in brood care, and whether diet influences colony commu-nication. We exposed caged, nurse-aged workers to different combinations of pheromones and pollen, and measured traits related to brood care. We found that pheromones enhanced pollen-dependent processes such as hypopharyngeal gland growth and mrjp1 expression, and buffered the negative effects of starvation. Pollen also enhanced how nurse phenotypes respond to pheromones. Therefore, diet and pheromones interact to influence nurse nutritional physiology and aspects of brood care. These findings have implications for studying colony function and health in an increasingly stressful climate.

This protocol quantifies total protein contents of small amounts of pollen (10 mg) using the bicinchoninic acid (BCA) assay of the Pierce BCA protein assay kit (Thermo Scientific product number 23225). This protocol adapts the kit protocol for analysis of anther pollen, corbicular pollen, and stored pollen from bee colonies. Pollen samples are subjected to acid hydrolysis to digest refractory proteins and to degrade sugars and other pollen substances that interfere with total protein assays. The acid hydrolysis releases substantially more protein from pollens than occurs with soluble proteins but may result in some protein losses due to the harsh acidic conditions. Similar BCA analysis methods without hydrolysis may be used to quantify soluble proteins (proteins obtained without acid hydrolysis or other digest methods) which may exclude insoluble pollen proteins from full quantification. The total protein contents obtained using BCA analysis of acid hydrosylates represents the maximum protein available to consumers and likely includes some refractory proteins that may not be digested by the consumer. The assay is modified from the original standard protocol to work for pollen analysis: Document Connect (thermofisher.com). Please note that our steps differ from the commercial source protocol. National Program Number: NP 305 Project Number: 2022-21000-022-000D, 2022-21000-022-053-I Carl Hayden Bee Research Center, USDA-ARS PWA, Tucson, Arizona, USA Grand Challenge - Assessing the Nutrient Contents of Pollen for Bees

This protocol uses FAME (Fatty Acid Methyl Esterification) techniques adapted from Seppanen-Laasko et al., 2002 to quantify fatty acid contents in small samples (10 mg) of anther pollen, corbicular pollen, or stored pollen. Pollen is initially fractured by cell disruption and fatty acids are extracted by Folch extraction/partition. Fatty acids are converted to their fatty acid methyl ester (FAME) equivalents by acid methylation then separated and analyzed by GCMS in SIM mode. Seppänen-Laasko, T., Laakso, I., & Hiltunen, R. (2002). Analysis of fatty acids by gas chromatography and its relevance to research on health and nutrition. Analytica Chimica Acta, 465, 39-62. https://doi.org/10.1016/S0003-2670(02)00397-5 National Program Number: NP 305 Project Number: 2022-21000-022-000D, 2022-21000-022-053-I Carl Hayden Bee Research Center, USDA-ARS PWA, Tucson, Arizona, USA Grand Challenge - Assessing the Nutrient Contents of Pollen for Bees

Honey bees obtain lipids from pollen or commercial supplements. These supplements do not fully support colony health. We tested the hypothesis that supplements are deficient because they lack essential fatty acids (EFAs). The five supplements we tested had low linolenic (⍵3) acid and were unbalanced (⍵6:⍵3 > 6) compared to natural pollen. We selected two of these supplements for further study because they had different levels of individual EFAs and different ⍵6:⍵3 ratios. Bees from hives fed these different supplements had equivalent tissue EFA levels. In choice assays, hives fed these different supplements were presented with flours with various absolute and relative levels of EFAs. We saw no difference in foraging preference. Rather, all hives preferred flours with small grain size and high protein to lipid ratios. We conclude that bees balance their internal EFAs and that differential colony nutrition does not affect foraging preference. The data also argue for more linolenic (⍵3) acid in commercial supplements.

This protocol quantifies total lipid contents of pollen through colorimetric oxidation of lipids by chromic acid (adapted from Amenta, 1970). Small pollen samples (10 mg) are fractured by cell homogenization and lipids are extracted by Folch extraction. Lipid amounts are measured by colorimetric changes when hexavalent (+6 oxidation state) chromium ion is reduced to a trivalent (+3 oxidation state) state during lipid oxidation. The accuracy of the assay depends on how thoroughly the initial extraction excludes non-lipid compounds such as sugars and proteins. A considerable drawback of this method is the toxicity, carcinogenicity, and environmental toxicity of the hexavalent chromium reagents used in this assay. Amenta, J.S. (1970). A rapid extraction and quantification of total lipids and lipid fractions in blood and feces. Clinical Chemistry, 16, 339-346. National Program Number: NP 305 Project Number: 2022-21000-022-000D, 2022-21000-022-053-I Carl Hayden Bee Research Center, USDA-ARS PWA, Tucson, Arizona, USA Grand Challenge - Assessing the Nutrient Contents of Pollen for Bees

Honey bee colonies have a yearly cycle that is supported nutritionally by the seasonal progression of flowering plants. In the spring, colonies grow by rearing brood, but in the fall, brood rearing declines in preparation for overwintering. Depending on where colonies are located, the yearly cycle can differ especially in overwintering activities. In temperate climates of Europe and North America, colonies reduce or end brood rearing in the fall while in warmer climates bees can rear brood and forage throughout the year. To test the hypothesis that nutrients available in seasonal pollens and honey bee responses to them can differ we analyzed pollen in the spring and fall collected by colonies in environments where brood rearing either stops in the fall (Iowa) or continues through the winter (Arizona). We fed both types of pollen to worker offspring of queens that emerged and open mated in each type of environment. We measured physiological responses to test if they differed depending on the location and season when the pollen was collected and the queen line of the workers that consumed it. Specifically, we measured pollen and protein consumption, gene expression levels (hex 70, hex 110, and vg) and hypopharyngeal gland (HPG) development. We found differences in macronutrient content and amino and fatty acids between spring and fall pollens from the same location and differences in nutrient content between locations during the same season. We also detected queen type and seasonal effects in HPG size and differences in gene expression between bees consuming spring vs. fall pollen with larger HPG and higher gene expression levels in those consuming spring pollen. The effects might have emerged from the seasonal differences in nutritional content of the pollens and genetic factors associated with the queen lines we used.

Honey bees (Apis mellifera) collect and store both honey and pollen in preserved forms. Pollen storage involves the addition of honey or nectar and oral secretions to pollen granules. It is controversial whether the duration of pollen storage alters the palatability or nutritive value of the pollen storage medium. We examined how bees utilize different-aged stored pollen during an extended pollen flow. The deposition of pollen into wax cells and subsequent consumption were monitored daily on 18 brood frames from 6 colonies over an 8d observation period. Despite a greater abundance of older stored pollen cells on brood frames, bees showed a marked preference for the consumption of freshly-stored pollen. Two to four day-old pollen cell contents were significantly more likely to be consumed, while pollen cell contents more than seven days old were eaten at much lower rates. Similar experiments that controlled for cell abundance and spatial effects using cage assays yielded the same result. One day-old stored pollen was consumed approximately three times more often than 10d-old stored pollen, and two times more often than 5d-old stored pollen. These consumption preferences for freshly-stored pollen occurred despite a lack of clear developmental advantages. Young adult workers reared for 7 days on 1d-, 5d-, or 10d-old stored pollen showed no difference in body mass, stored pollen consumption, hindgut fecal material accumulation, or hypopharyngeal gland (HPG) protein titers, suggesting that different-aged pollen stores did not vary in their nutritional value to adult bees. These findings are inconsistent with the hypothesis promoting a period of microbially-mediated, "beebread maturation" that results in greater palatability or nutritive value for aged pollen stores. Rather, stored pollen that is not eaten in the first few days accumulates as excess stores preserved in a less preferred, but nutritionally-similar state.

Pollen nutrition is necessary for proper growth and development of adult honey bees. Yet, it is unclear how pollen affects the honey bee brain and behavior. We investigated whether pollen affects amino acids in the brains of caged, nurse-aged bees, and what the behavioral consequences might be. We also tested whether parasitic stress altered this relationship by analyzing bees infected with prevalent stressor, Nosema ceranae. Levels of 18 amino acids in individual honey bee brains were measured using Gas Chromatography - Mass Spectrometry at two different ages (Day 7 and Day 11). We then employed the proboscis extension reflex to test odor learning and memory. We found that the honey bee brain was highly responsive to pollen. Many amino acids in the brain were elevated and were present at higher concentration with age. The majority of these amino acids were non-essential. Without pollen, levels of amino acids remained consistent, or declined. Nosema-infected bees showed a different profile. Infection altered amino acid levels in a pollen-dependent manner. The majority of amino acids were lower when pollen was given, but higher when pollen was deprived. Odor learning and memory was not affected by feeding pollen to uninfected bees; but pollen did improve performance in Nosema-infected bees. These results suggest that pollen in early adulthood continues to shape amino acid levels in the brain with age, which may affect neural circuitry and behavior over time. Parasitic stress by N. ceranae modifies this relationship revealing an interaction between infection, pollen nutrition, and behavior.

Methoxyfenozide is an insect growth regulator (IGR) commonly used in agriculture to simultaneously control pests and preserve beneficial insect populations; however, its impact on honey bees in not fully understood. We conducted field and laboratory experiments to investigate bee health in response to field-relevant concentrations of this pesticide. Significant effects were observed in honey bee colony flight activity and thermoregulation after being exposed over 9 weeks to supplemental protein patty containing methoxyfenozide. Compared to bee colonies in the control group, colonies fed pollen patty with 200 ppb methoxyfenozide (as measured by residue analysis) had: 1) a significantly reduced rate of weight loss due to forager departure in the morning; and 2) higher temperature variability during the winter. Colonies in the 100 ppb (as measured by residue analysis) treatment group had values between the 200 ppb group and control for both response variables. The dusk break point, which is the time associated with the end of forager return, differed among all treatment groups but may have been confounded with direction the hives were facing. Bee colony metrics of adult bee mass and brood surface area, and measurements of bee head weight, newly-emerged bee weight, and hypopharyngeal gland size were not significantly affected by methoxyfenozide exposure, suggesting that there may be significant effects on honey bee colony behavior and health in the field that are difficult to detect using standard methods for assessing bee colonies and individuals. The second experiment was continued into the following spring, using the same treatment groups as in the fall. Fewer differences were observed among groups in the spring than the fall, possibly because of abundant spring forage and consequent reduced treatment patty consumption. Residue analyses showed that: 1) observed methoxyfenozide concentrations in treatment patty were about 18-60% lower than the calculated concentrations; 2) no residues were observed in wax in any treatment; and 3) methoxyfenozide was detected in bee bread only in the 200 ppb treatment group, at about 1-2.5% of the observed patty concentration.

Pollinator populations are declining (Biesmeijer et al., 2006; Brodschneider et al., 2018; Cameron et al., 2011; Goulson, Lye, & Darvill, 2008; Kulhanek et al., 2017; National Research Council, 200...

Nosema sp. is an internal parasite of the honey bee, Apis mellifera, and one of the leading contributors to colony losses worldwide. This parasite is found in the honey bee midgut and has profound consequences for the host's physiology. Nosema sp. impairs foraging performance in honey bees, yet, it is unclear whether this parasite affects the bee's neurobiology. In this study, we examined whether Nosema sp. affects odor learning and memory and whether the brains of parasitized bees show differences in amino acids and biogenic amines. We took newly emerged bees and fed them with Nosema ceranae At approximate nurse and forager ages, we employed an odor-associative conditioning assay using the proboscis extension reflex and two bioanalytical techniques to measure changes in brain chemistry. We found that nurse-aged bees infected with N. ceranae significantly outperformed controls in odor learning and memory, suggestive of precocious foraging, but by forager age, infected bees showed deficits in learning and memory. We also detected significant differences in amino acid concentrations, some of which were age specific, as well as altered serotonin, octopamine, dopamine and l-dopa concentrations in the brains of parasitized bees. These findings suggest that N. ceranae infection affects honey bee neurobiology and may compromise behavioral tasks. These results yield new insight into the host-parasite dynamic of honey bees and N. ceranae, as well as the neurochemistry of odor learning and memory under normal and parasitic conditions.