Hypopharyngeal Glands Research Articles

Enhancing the Health and Lifespan of Honey Bees (Apis mellifera L.) with an Innovative Soybean-Based Diet Supplemented with Porcine Blood1

Abstract This study investigated the effect of soy-based diets supplemented with porcine blood (PB) on the diet's external morphology, nutritional efficacy, and health impacts on honey bees, Apis mellifera L. (Hymenoptera: Apidae). Scanning electron microscopy revealed that the control diet (maize bee bread) displayed ruptured granules with small particles, whereas the soy-based diets showed varying degrees of protein gelatinization and aggregation depending on the presence of PB. Nutritional analysis indicated that maize bee bread was used as a positive control contained 12.2% protein and provided 312.89 kcal/100 g, whereas the formulated soy-based diets with skim milk or PB showed 13–15% protein, meeting the required needs for optimal honey bee health and development. Survival and longevity assessments showed no significant difference in lifespan between honey bees fed with artificial diets and the control, but all treatment groups outlived the negative control, which fed only on syrup. Moreover, the diameter of the hypopharyngeal gland acini, a key indicator of the nutritional state and health in honey bees, was significantly larger in honey bees fed protein-enriched diets compared with those receiving only syrup. These findings underscore the potential of soy-based artificial protein diets, especially when enhanced with PB, to support honey bee health and longevity, comparable with natural pollen sources.

Gut microbiota influences onset of foraging-related behavior but not physiological hallmarks of division of labor in honeybees.

The honeybee is emerging as a model system for studying gut microbiota-host interactions. Previous studies reported gut microbiota effects on multiple worker bee phenotypes, all of which change during behavioral maturation-the transition from nursing to foraging. We tested whether the documented effects may stem from an effect of the microbiota on behavioral maturation. The gut microbiota only subtly affected maturation: it accelerated the onset of foraging without affecting the overall proportion of foragers or their average output. We also found no effect of the microbiota on host weight, cuticular hydrocarbon (CHC) profile, hypopharyngeal gland size, and the expression of behavioral maturation-related genes. These results are inconsistent with previous studies reporting effects of the gut microbiota on bee weight and CHC profile. Our experiments revealed that co-housed bees tend to converge in behavior and physiology, suggesting that spurious associations may emerge when rearing environments are not replicated sufficiently or accounted for analytically.

Effect of heat stress on Apis mellifera intermissa and Apis mellifera sahariensis in laboratory controlled conditions: a physiological approach

The effects of heat on honey bees have received attention in many subspecies. Nevertheless, some subspecies still remain unexplored. In this study, we investigate for the first time the effect of heat on two subspecies of honey bees endemic to Algeria, Apis mellifera intermissa which is adapted to the Mediterranean climate and Apis mellifera sahariensis, which is adapted to the hot climate of southwest Algeria. We tested the hypothesis that sahariensis is more tolerant to heat stress than intermissa. In two in-vitro tests, we reared workers of both subspecies at 4 temperatures (34 °C, 38 °C, 39 °C, and 40 °C). Then, we determined the hemolymph protein (HP) concentration, hypopharyngeal gland development (HPG), ovary development and survival. We found that the sahariensis had HPG more developed and less affected by heat than intermissa. Conversely, although intermissa had more (HP) at 34 °C, we found a significant increase in (HP) at 38 °C, 39 C° and 40 °C in intermissa than sahariensis. Although ovary development is greater in intermissa than sahariensis at 34 °C and greater at 34 °C than 38 °C, 39 °C and 40 °C, we did not record differences in ovary development between the subspecies at 38 °C, 39 °C, and 40 °C. Finally, in all conditions, sahariensis had a higher survival than intermissa.

Sublethal Effects Induced by a Cyflumetofen Formulation on Honeybee Apis mellifera L. Workers: Assessment of Midgut, Hypopharyngeal Glands, and Fat Body Integrity.

Worldwide, both cultivated and wild plants are pollinated by the honey bee, Apis mellifera. Bee numbers are declining as a result of a variety of factors, including increased pesticide use. Cyflumetofen controls pest mites in some plantations pollinated by bees, which may be contaminated with residual sublethal concentrations of this pesticide, in nectar and pollen. We evaluated the effects of a sublethal concentration of a cyflumetofen formulation on the midgut, hypopharyngeal gland, and fat body of A. mellifera workers orally exposed for 72 h or 10 days. The midgut epithelium of treated bees presented digestive cells with cytoplasm vacuoles and some cell fragmentation, indicating autophagy and cell death. After being exposed to the cyflumetofen formulation for 72 h, the midgut showed a higher injury rate than the control bees, but after 10 days, the organs had recovered. In the hypopharyngeal gland of treated bees, the end apparatus was filled with secretion, suggesting that the acaricide interferes with the secretory regulation of this gland. Histochemical tests revealed differences in the treated bees in both exposure periods in the midgut and hypopharyngeal glands. The acaricide caused cytotoxic effects on the midgut digestive cells, with apical protrusions, plasma membrane rupture, and several vacuoles in the cytoplasm, features of cell degeneration. In the hypopharyngeal glands of the treated bees, the secretory cells presented small electron-dense and large electron-lucent secretory granules. The fat body cells had no changes in comparison with the control bees. In conclusion, the cyflumetofen formulation at sublethal concentrations causes damage to the midgut and the hypopharyngeal glands of honey bee, which may compromise the functions of these organs and colony fitness. Environ Toxicol Chem 2024;43:2455-2465. © 2024 SETAC.

From consumption to excretion: Lithium concentrations in honey bees (Apis mellifera) after lithium chloride application and time-dependent effects on Varroa destructor.

Owing to its systemic mode-of-action and ease of application, lithium chloride (LiCl) is an ideal varroacide for the control of Varroa destructor infestations in honey bee colonies. To better understand how LiCl functions within a colony, we screened different parts of honey bee anatomy for lithium accumulation. We wanted to elucidate the time-dependent effects of LiCl on V. destructor and its metabolism within honey bees when they were fed continuous LiCl treatments, as well as evaluate potential adverse effects such as accumulation in the hypopharyngeal glands of nurse bees, which could negatively impact queens and larvae. Cage experiments reveal rapid acaricidal onset, with >95% mite mortality within 48 h of treatment. Bee hemolymph analysis supports these observations, showing a rapid increase in lithium concentration within 12 h of treatment, followed by stabilization at a constant level. Lithium accumulates in the rectum of caged bees (≤475.5 mg kg-1 after 7 days of feeding 50 mm LiCl), reflecting the bees' metabolic and excretion process. Despite concerns about potential accumulation in hypopharyngeal glands, low lithium levels of only 0.52 mg kg-1 suggest minimal risk to the queen and 1st- and 2nd-instar larvae. Cessation of LiCl treatment results in a rapid decline in mite mortality in the first 5 days, which increases again thereafter, resulting in mite mortality of 77-90% after 10 days. These findings help optimize LiCl application in colonies to achieve high Varroa mortality without unwanted adverse effects and provide important baseline data for future registration. © 2024 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Nutrient Composition and Quality Assessment of Royal Jelly Samples Relative to Feed Supplements.

Royal jelly is a substance secreted by the hypopharyngeal and mandibular glands of nurse honey bees, serving as crucial nutritional source for young larvae, queen honey bees, and also valuable product for humans. In this study, the effect of the feed supplements on the nutritional composition and qualities of royal jelly was investigated. Two types of royal jelly samples were acquired: one from honey bees fed with sugar syrup as a feed supplement and the other from honey bees fed with honey. The production, harvesting, and storage of all royal jelly samples followed standard procedures. Parameters for quality assessment and nutritional value, including stable carbon isotopic ratio, moisture content, 10-hydroxy-2-decenoic acid (10-HDA) level, carbohydrate composition, amino acid composition, and mineral contents, were analyzed. The results revealed that despite variability in moisture content and carbohydrate composition, fructose was lower (2.6 and 4.1 g/100 g as is for sugar-fed and honey-fed royal jelly, respectively) and sucrose was higher (7.5 and 2.7 g/100 g as is for sugar-fed and honey-fed royal jelly, respectively) in the sugar-fed group. The stable isotope ratio (-16.4608‱ for sugar-fed and -21.9304‱ for honey-fed royal jelly) clearly distinguished the two groups. 10-HDA, amino acid composition, and total protein levels were not significantly different. Certain minerals, such as potassium, iron, magnesium, manganese, and phosphorus were higher in the honey-fed group. Hierarchical analysis based on moisture, sugar composition, 10-HDA, and stable carbon isotopes categorized the samples into two distinct groups. This study demonstrated that the feed source could affect the nutritional quality of royal jelly.

IMPACT OF ACARICIDES ON VARROA DESTRUCTOR INFESTATION IN HONEY BEE COLONIES (Apis mellifera L.) AND THEIR HISTOLOGICAL EFFECTS ON HYPOPHARYNGEAL GLANDS

This study aimed to determine the random role of some acaricides, which are the most commonly used in Egypt for controlling Varroa destructor on Hypopharyngeal Glands (HPGs) of honey bees (Apis mellifera L.). The acaricides used in this study were Varroakiller®, Menthocaros®, Amitraz®, Varroby®, and formic acid. Results showed that the total number of fallen mites was 53.5, 47, 28.6, 26, and 24.5 for Varroakiller®, Menthocaros®, Amitraz®, Varroby®, and formic acid, respectively, compared to 16 mites in the control group. For sealed worker brood areas (SWBA), the treatments included Varroakiller® (327.98 cm2), formic acid (228.92 cm2), Varroby® (222.25 cm2), Menthocaros® (129.72 cm2), and Amitraz® (101.73 cm2), while the SWBA for control colonies was 44.83 cm2. Histological studies of the HPGs showed that although Varroakiller® effectively controlled Varroa mites and had no direct impact on worker bees or egg-laying areas, it considerably impacted the HPGs, which could ultimately affect the bee colony. Moreover, formic acid recorded a lower number of fallen Varroa with acceptable outcomes for egg-laying areas and sealed workers. This, coupled with the fact that formic acid was deemed the most significant due to its effectiveness over a short period, resulted in a reduction of 100% after 24 days.

Expression and function of the vitellogenin receptor in the hypopharyngeal glands of the honey bee Apis mellifera (Hymenoptera: Apidae) workers.

The vitellogenin receptor (VgR) is essential for the uptake and transport of the yolk precursor, vitellogenin (Vg). Vg is synthesized in the fat body, released in the hemolymph, and absorbed in the ovaries, via receptor-mediated endocytosis. Besides its important role in the reproductive pathway, Vg occurs in nonreproductive worker honey bee, suggesting its participation in other pathways. The objective was to verify if the VgR occurs in the hypopharyngeal glands of Apis mellifera workers and how Vg is internalized by these cells. VgR occurrence in the hypopharyngeal glands was evaluated by qPCR analyses of VgR and immunohistochemistry in workers with different tasks. The VgR gene is expressed in the hypopharyngeal glands of workers with higher transcript levels in nurse honey bees. VgR is more expressed in 11-day-old workers from queenright colonies, compared to orphan ones. Nurse workers with developed hypopharyngeal glands present higher VgR transcripts than those with poorly developed glands. The immunohistochemistry results showed the co-localization of Vg, VgR and clathrin (protein that plays a major role in the formation of coated vesicles in endocytosis) in the hypopharyngeal glands, suggesting receptor-mediated endocytosis. The results demonstrate that VgR performs the transport of Vg to the hypopharyngeal glands, supporting the Ovary Ground Plan Hypothesis and contributing to the understanding of the role of this gland in the social context of honey bees.

Exploring the effects of the acaricide cyflumetofen on the vital organs of the honey bee Apis mellifera (Hymenoptera: Apidae) workers

Bees are important for maintaining ecosystems, pollinating crops and producing marketable products. In recent years, a decline in bee populations has been reported, with multifactorial causes, including the intensification of pesticide use in agriculture. Among pesticides, cyflumetofen is an insecticide and acaricide used in apple, coffee and citrus crops, whose main pollinator is the honey bee Apis mellifera. Therefore, this bee is a potential target of cyflumetofen during foraging. This study evaluated the histopathological and cytological damage in the midgut, hypopharyngeal glands and fat body of A. mellifera workers exposed to LC50 of cyflumetofen. The midgut epithelium of exposed bees presented cytoplasmic vacuolization, release of vesicles and cell fragments, which indicate autophagy, increased production of digestive enzymes and cell death, respectively. The cytological analysis of the midgut revealed the dilation of the basal labyrinth and the presence of spherocrystals in the digestive cells. The hypopharyngeal glands produced greater amounts of secretion in treated bees, whereas no changes were observed in the fat body. The results indicate that acute exposure to cyflumetofen negatively affect A. mellifera, causing damage to the midgut and changes in the hypopharyngeal glands, which may compromise the survival and foraging of this pollinator.

Production and Quality Characteristics of Royal Jelly in Relation to Available Natural Food Resources

Royal jelly is a secretion produced from the hypopharyngeal glands of worker bees, which requires significant pollen reserves to stimulate gland secretion. The natural sources of food available to the hive during beekeeping season can greatly affect the quantity and quality of produced royal jelly. In this study, samples of royal jelly were collected throughout the beekeeping season, and their physical and chemical characteristics were analyzed to understand how natural variations in bee diet affect royal jelly production. Before each sample collection, the bees’ food reserves were removed from the experimental colonies so that the royal jelly was produced solely from natural sources. The results showed that the production was significantly lower during the summer months compared with spring and autumn. Additionally, the moisture, protein, fructose, and glucose content of fresh royal jelly also showed significant changes in the summer, and all physical and chemical characteristics decreased when the fresh samples were converted into dry matter. It seems that the quality of pollen entering the hives has a direct impact on the physical and chemical properties of the final product, highlighting the crucial role of available resources in stimulating bees to produce royal jelly.

Expression of honey bee (Apis mellifera) sterol homeostasis genes in food jelly producing glands of workers.

Adult workers of Western honey bees (Apis mellifera L.) acquire sterols from their pollen diet. These food sterols are transported by the hemolymph to peripheral tissues such as the mandibular and the hypopharyngeal glands in the worker bees' heads that secrete food jelly which is fed to developing larvae. As sterols are obligatory components of biological membranes and essential precursors for molting hormone synthesis in insects, they are indispensable to normal larval development. Thus, the study of sterol delivery to larvae is important for a full understanding of honey bee larval nutrition and development. Whereas hypopharyngeal glands only require sterols for their membrane integrity, mandibular glands add sterols, primarily 24-methylenecholesterol, to its secretion. For this, sterols must be transported through the glandular epithelial cells. We have analyzed for the first time in A. mellifera the expression of genes which are involved in intracellular movement of sterols. Mandibular and hypopharyngeal glands were dissected from newly emerged bees, 6-day-old nurse bees that feed larvae and 26-day-old forager bees. The expression of seven genes involved in intracellular sterol metabolism was measured with quantitative real-time PCR. Relative transcript abundance of sterol metabolism genes was significantly influenced by the age of workers and specific genes but not by gland type. Newly emerged bees had significantly more transcripts for six out of seven genes than older bees indicating that the bulk of the proteins needed for sterol metabolism are produced directly after emergence.

Changes in Vitellogenin, Abdominal Lipid Content, and Hypopharyngeal Gland Development in Honey Bees Fed Diets with Different Protein Sources.

Honey bees play an important role in the pollination of flowering plants. When honey bee colonies are deficient in pollen, one of their main nutrients, protein supplements are required. In this study, the effects of diets with six different protein sources on the physiological characteristics of worker bees (vitellogenin (Vg), abdominal lipid content (ALC), hypopharyngeal gland (HPG)) and consumption were investigated. The protein sources of the diets (diet I, …, diet VI) included pollen, spirulina dust (Arthrospira platensis Gomont), fresh egg yolk, lyophilized lactose-free skimmed milk powder, active fresh yeast, and ApiProtein. It was identified that consumption by worker bees was highest in the diet group supplemented with spirulina (diet II). Although there was no statistical difference regarding the Vg content in the hemolymph, numerically, the highest content was found in diet group IV (lyophilized lactose-free skimmed milk powder) (4.73 ± 0.03 ng/mL). ALC and HPG were highest in the group fed diet II. These results suggest that offering honey bees diets with certain protein sources can support their physiological traits.

Consumption of Citric Acid by Bees Promotes the Gland Development and Enhances Royal Jelly Quality.

The glands of bees are responsible for generating and secreting various biologically active substances that significantly impact bee physiological health and adaptability. This study aimed to investigate the effects of adding citric acid (CA) to bee feed on gland development and royal jelly quality. By formulating feed with varying proportions of CA, evaluation was undertaken of pollen feeding by honeybees under laboratory conditions, along with the impact of CA on the development of major glands, to determine suitable addition proportions. Further optimization of the CA proportion involved feeding colonies and evaluating royal jelly production and quality. The results indicated that feed containing 0.75% CA significantly extended the lifespan of bees and increased their pollen consumption. Gland development in bees showed a positive correlation with CA addition within the range of 0.25% to 0.75%, especially at 0.50% and 0.75%, which notably accelerated the development of mandibular, hypopharyngeal, and cephalic salivary glands, with active proliferation and differentiation of glandular cells and maintenance of normal gland size and morphology. CA added to feed stimulated vigorous secretion of wax glands in worker bees, resulting in prolific wax construction. Colonies consuming feed containing 0.50% CA produced royal jelly with significantly reduced moisture and total sugar content and increased levels of 10-HDA, total phenolic acids, total proteins, and acidity. These findings demonstrate that CA consumption significantly prolongs bee lifespan, increases consumption, promotes gland development, and enhances royal jelly quality. This research provides theoretical guidance for beekeeping practices and feed development, contributing to the sustainable advancement of apiculture.

Histological Changes Of Bee Worker (Apis mellifera L.) By Bee Venom And Amino Acids Feeding

The effect of feeding honey bee colonies on a sugar solution to which ‎bee ‎venom has been added at a concentration of 5‎‏0‏‎ mg/liter sugar solution ‎and on amino ‎acids extracted from clover 4 cm amino acids/liter sugar ‎solution on the amount of ‎food consumed by honey bee workers as well as ‎the effect on gland tissues ‎Hypopharyngeal and maxillary glands and ‎histological changes that occur in them. ‎Nine honeybee colonies were tested in an apiary of the Bee Research ‎Department, Plant Protection ‎Research Institute, Agricultural Research Center, ‎Ministry of Agriculture, ‎Giza, Egypt, during 2020-2021. Statistically, there are no significant differences in the amount of food consumed by honeybee workers, whether the bee venom solution or the amino acid solution, ‎compared to ‎the control colonies that fed on the sugar solution only. No histological changes were observed in the ‎hypopharyngeal (HPGs) and mandibular‎ ‎glands (MGs) in workers fed sugary solution (control colonies). Feeding ‎with ‎amino acids resulted in histological ‎changes in the HPGs and MGs, ‎ as most of the ‎acini were atrophic and smaller ‎in size than usual, with the loss of vesicles in the ‎cytoplasm. As for the bees ‎that fed on a solution of bee venom, histological changes ‎ in the HPGs and ‎MGs were observed, where severe atrophy and contraction of the ‎acini with ‎an irregular outline were noted. Nuclear thickening of the ‎‎epithelial cell lining was observed. The histological structure of the lower glands ‎showed ‎atrophy in some individual cells, which led to damage to the ‎development of both the ‎HPGs and MGs.‎

Seasonal Changes in Hemolymph Protein Level and Hypopharyngeal Gland Size Depending on Age and In-Nest Location of Honeybee Workers

A honeybee colony, as a super-organism, is regulated through age-polyethism. A honeybee worker’s age is considered by means of a chronological and biological approach. The biological age is estimated with physiologically related biological markers, e.g., total hemolymph protein content (THP) and hypopharyngeal gland size (HGs), which also vary seasonally. Contemporary insights into the age-related spatial workers’ distribution within the hive nest space regarding biological age are insufficiently clarified. This study aimed to monitor changes in selected physiological markers during the entire season in relation to worker age and their spatial position in the hive nest. THP content and HG size analysis was performed in nine colonies for the entire season to compare the physiological markers within and among the groups of the workers whose ages were known and sampled in different hive parts. Seasonal impact on the biomarkers’ development was confirmed in known-age workers. In the case of HGs, this impact was the most apparent in 4- and 5-week-old workers. For THP, the seasonal impact was the most obvious in 2-week-old workers. The highest THP was found in 1- and 2-week-old workers during the entire season. Biologically younger workers of the same age were located predominantly in upper hive parts consistently throughout the year and vice versa. These workers showed significantly higher THP in comparison with those sampled below. Regarding the chronological age, the downwards, spatially shifting mechanism of workers within the hive nest while they aged was characterized. We recommend storage of diluted hemolymph samples up to one month before performing an assay if necessary. The physiological context, relation to division of labor and benefits for beekeeping practices are discussed.

Developmental exposure to hormone-mimicking insect growth disruptors alters expression of endocrine-related genes in worker honey bee (Hymenoptera: Apidae) brains and hypopharyngeal glands.

Division of labor within a honey bee colony creates a codependence between bees performing different tasks. The most obvious example of this is between the reproductive queen and worker bees. Queen bees lay 1,000 or more eggs a day, while young worker bees tend and feed queens. Young workers and queens can be exposed to pesticides when foragers return to the hive with contaminated resources. Previous research has found negative effects of larval exposure to insect-growth disruptors (IGD) methoxyfenozide and pyriproxyfen, on adult responsiveness to artificial queen pheromone. The present work investigates potential physiological and molecular mechanisms underpinning this behavioral change by examining the development of hypopharyngeal glands and ovaries as well as the expression of genes related to reproduction and worker endocrine signaling in the brain and hypopharyngeal gland tissues. Though hypopharyngeal gland and ovary development were not altered by developmental exposure to IGDs, gene expression differed. Specifically, in the brain tissue, ilp1 was downregulated in bees exposed to pyriproxyfen during development, and Kr-h1 was downregulated in both methoxyfenozide- and pyriproxyfen-exposed bees. In the hypopharyngeal glands, Kr-h1, EcR-A, EcR-B, and E75 were upregulated in honey bees exposed to methoxyfenozide compared to those in the pyriproxyfen or control treatments. Here we discuss these results and their potential implications for the health and performance of honey bee colonies.

Sublethal effects of imidacloprid-contaminated honey stores on colony performance, queens, and worker activities in fall and early winter colonies.

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.

Age-dependent hypopharyngeal gland size and protein content of stingless bee workers, Tetragonula pagdeni.

Eusocial insects, such as stingless bees (Meliponini), depend on division of labour, overlapping generations, and collaborative brood care to ensure the functionality and success of their colony. Female workers transition through a range of age-specific tasks during their lifespan (i.e., age-polyethism) and play a central role in the success of a colony. These age-specific tasks (e.g., brood care or foraging) often closely coincide with key physiological changes necessary to ensure optimal performance. However, our understanding of how nutrition, age, and polyethism may affect the development of such physiological traits in stingless bees remains limited. Here we show that pollen consumption and age-polyethism govern hypopharyngeal gland (HPG) acini size and protein content in Tetragonula pagdeni. By conducting a controlled laboratory experiment we monitored the effect of pollen consumption on worker bee survival as well as assessed how a pollen diet and age affected their HPG acini width and protein content. Further, we sampled nurses and foragers from field colonies to measure the effect of age-polyethism on HPG acini width. We found that pollen consumption enhanced survival and led to increased HPG acini width and protein content and that HPG acini were as expected largest in nurse bees. Our findings highlight the beneficial effects of an adequate diet for physiological development and health in stingless bees and reveal that age-polyethism is the key factor governing HPG size in worker bees. As HPGs are imperative for collaborative brood care-an essential component of eusociality-the data provide a foundation for future studies to investigate the impact of potential environmental stressors on a critical physiological trait in stingless bees which may serve as a proxy to understand the effects at the colony level.

Substances in the mandibular glands mediate queen effects on larval development and colony organization in an annual bumble bee.

Social organization is commonly dynamic, with extreme examples in annual social insects, but little is known about the underlying signals and mechanisms. Bumble bee larvae with close contact to a queen do not differentiate into gynes, pupate at an earlier age, and are commonly smaller than siblings that do not contact a queen. We combined detailed observations, proteomics, microRNA transcriptomics, and gland removal surgery to study the regulation of brood development and division of labor in the annual social bumble bee Bombus terrestris. We found that regurgitates fed to larvae by queens and workers differ in their protein and microRNA composition. The proteome of the regurgitate overlaps significantly with that of the mandibular (MG) and hypopharyngeal glands (HPG), suggesting that these exocrine glands are sources of regurgitate proteins. The proteome of the MG and HPG, but not the salivary glands, differs between queens and workers, with caste-specificity preserved for the MG and regurgitate proteomes. Queens subjected to surgical removal of the MG showed normal behavior, brood care, and weight gain, but failed to shorten larval development. These findings suggest that substances in the queen MG are fed to larvae and influence their developmental program. We suggest that when workers emerge and contribute to larval feeding, they dilute the effects of the queen substances, until she can no longer manipulate the development of all larvae. Longer developmental duration may allow female larvae to differentiate into gynes rather than to workers, mediating the colony transition from the ergonomic to the reproductive phase.

Mandibular glands secrete 24-methylenecholesterol into honey bee (Apis mellifera) food jelly

Honey bee (Apis mellifera) workers feed their larvae with food jelly that is secreted by specialized glands in their heads – the hypopharyngeal and the mandibular glands. Food jelly contains all the nutrients the larvae need to develop into adult honey bees, including essential dietary sterols. The main sterol in food jelly, 24-methylenecholesterol (24MC), is pollen-derived and delivered in food jelly to the larvae in a complex with two proteins, major royal jelly protein 1 (MRJP1) and apisim. Whereas the proteins are synthesized in the hypopharyngeal glands, the sterol-secreting gland has not been identified. We here identified the mandibular glands as sterol-secreting gland for food jelly production by direct detection of the four main honey bee sterols (24MC, campesterol, β-sitosterol and isofucosterol). Furthermore, 24MC seems to be specifically enriched in the mandibular glands, thereby ensuring that food jelly contains the amounts of 24MC necessary for complex formation with MRJP1 and apisimin.