Influence of the food protein on the development of hypopharyngeal glands, fat body, quality and lifespan of honeybees

Honey bees collect resin from various plant species and transform it into propolis that is incorporated into the nest. The role of resins in the bee health field is poorly understood. The aim was to evaluate the effects of forced consumption of propolis on the physiological condition and short-term survival of Apis mellifera worker bees. It was tested if the number of circulating hemocytes in hemolymph, the abdominal fat bodies and the hypopharyngeal glands development were affected by the feeding with propolis extracts in laboratory conditions during the warm and the cold seasons. Propolis added to sugar candy was consumed by workers for fourteen days without affecting the bee survival. The number of circulating hemocytes in hemolymph remained constant despite the differential diet during the experiment. However, the development of fat bodies and hypopharyngeal glands was altered by propolis ingestion. The abdominal fat body development in winter bees diminished after fourteen days of propolis consumption, while it increased in summer bees. The hypopharyngeal gland development decreased for the assayed period in workers from both seasons. Our results encourage us to continue exploring this research field and learn how long-term forced ingestion of a plant-derived compound, a non-nutritive substance, can modify physiological bee parameters. A broader understanding of the multiple roles of propolis in the health of the honey bee colonies could be obtained by studying the ways in which it is processed and metabolized and the effect that generates in another physiological responses.

Hypopharyngeal gland (HPG) development in honey bee workers is primarily age-dependent and changes according to the tasks performed in the colony. HPG activity also depends on colony requirements and is flexible in relation to the need for feeding brood. Very little is known about HPG development in the honey bee subspecies found in Southern Africa. We examined HPG development in Apis mellifera scutellata and A. m. capensis, including A. m. scutellata colonies infested with an invasive parasitic clonal lineage of A. m. capensis known to manipulate food provisioning to the parasitic larvae by their A.m. scutellata hosts, under natural in-hive conditions in bees aged 0 to 14days using light microscopy. We found marked differences in acini size (berry-like clusters of secretory cells) and the age at which maximum HPG development occurred between the subspecies and in the presence of the parasite. In A. m. scutellata workers, acini reached maximum size at 6days. The acini of A. m. capensis workers were larger (up to double) than those of A. m. scutellata and reached maximum size at 8days, while the HPG acini in A. m. scutellata workers infested with A. m. capensis clones reached development sizes similar to those of A. m. capensis at day 10 and were 1.5 times larger than those of uninfested A. m. scutellata. This provides foundational insights into a functional response affecting the development of the HPG most likely associated with brood pheromone composition and how this is altered in the presence of a social parasite.

BackgroundThe hypopharyngeal gland of worker bees contributes to the production of the royal jelly fed to queens and larvae. The gland consists of thousands of two-cell units that are composed of a secretory cell and a duct cell and that are arranged in sets of about 12 around a long collecting duct.ResultsBy fluorescent staining, we have examined the morphogenesis of the hypopharyngeal gland during pupal life, from a saccule lined by a pseudostratified epithelium to the elaborate organ of adult worker bees. The hypopharyngeal gland develops as follows. (1) Cell proliferation occurs during the first day of pupal life in the hypopharyngeal gland primordium. (2) Subsequently, the epithelium becomes organized into rosette-like units of three cells. Two of these will become the secretory cell and the duct cell of the adult secretory units; the third cell contributes only temporarily to the development of the secretory units and is eliminated by apoptosis in the second half of pupal life. (3) The three-cell units of flask-shaped cells undergo complex changes in cell morphology. Thus, by mid-pupal stage, the gland is structurally similar to the adult hypopharyngeal gland. (4) Concomitantly, the prospective secretory cell attains its characteristic subcellular organization by the invagination of a small patch of apical membrane domain, its extension to a tube of about 100 μm in length (termed a canaliculus), and the expansion of the tube to a diameter of about 3 μm. (6) Finally, the canaliculus-associated F-actin system becomes reorganized into rings of bundled actin filaments that are positioned at regular distances along the membrane tube.ConclusionsThe morphogenesis of the secretory units in the hypopharyngeal gland of the worker bee seems to be based on a developmental program that is conserved, with slight modification, among insects for the production of dermal glands. Elaboration of the secretory cell as a unicellular seamless epithelial tube occurs by invagination of the apical membrane, its extension likely by targeted exocytosis and its expansion, and finally the reorganisation of the membrane-associated F-actin system. Our work is fundamental for future studies of environmental effects on hypopharyngeal gland morphology and development.

Effect of honeybee race and worker age on development and histological structure of hypopharyngeal glands of honeybee

Consumption rate of some proteinic diets affecting hypopharyngeal glands development in honeybee workers

Simple SummaryThe hypopharyngeal glands (HPGs) are a pair of aciniform glands that are located in the frontal area of the heads of worker bees (Apis mellifera L.) that exhibit age and behavior-dependent development. Little is known about whether/how miRNAs regulate the HPGs development. In this study, small RNA sequencing was employed to analyze the miRNA profiles of HPGs in newly-emerged bees (NEB), nurse bees (NB), and forager bees (FB). We found that there were a total of 31 known miRNAs differentially expressed among the three stages, which might have regulatory roles in the growth and development, protein synthesis, and carbohydrate and energy metabolism in the HPGs. Additionally, the downregulation of ame-miR-184-3p and ame-miR-252a-5p in nurse bees may be involved in royal jelly secretion, while the lower expression of ame-miR-11-3p and ame-miR-281-3p in forager bees are responsible for honey processing.This study aims to investigate the expression differences of miRNAs in the hypopharyngeal glands (HPGs) of honeybees at three developmental stages and to explore their regulation functions in the HPGs development. Small RNA sequencing was employed to analyze the miRNA profiles of HPGs in newly-emerged bees (NEB), nurse bees (NB), and forager bees (FB). Results showed that a total of 153 known miRNAs were found in the three stages, and ame-miR-276-3p, ame-miR-375-3p, ame-miR-14-3p, ame-miR-275-3p, and ame-miR-3477-5p were the top five most abundant ones. Furthermore, the expression of 11 miRNAs, 17 miRNAs, and 18 miRNAs were significantly different in NB vs. FB comparison, NB vs. NEB comparison, and in FB vs. NEB comparison, respectively, of which ame-miR-184-3p and ame-miR-252a-5p were downregulated in NB compared with that in both the FB and NEB, while ame-miR-11-3p, ame-miR-281-3p, and ame-miR-31a-5p had lower expression levels in FB compared with that in both the NB and NEB. Bioinformatic analysis showed that the potential target genes of the differentially expressed miRNAs (DEMs) were mainly enriched in several key signaling pathways, including mTOR signaling pathway, MAPK signaling pathway-fly, FoxO signaling pathway, Hippo signaling pathway-fly. Overall, our study characterized the miRNA profiles in the HPGs of honeybees at three different developmental stages and provided a basis for further study of the roles of miRNAs in HPGs development.

The transgenic Cry1Ac (Bt toxin) + CpTI (Cowpea Trypsin Inhibitor) cotton cultivar CCRI41 is increasingly used in China and potential side effects on the honey bee Apis mellifera L. have been documented recently. Two studies have assessed potential lethal and sublethal effects in young bees fed with CCRI41 cotton pollen but no effect was observed on learning capacities, although lower feeding activity in exposed honey bees was noted (antifeedant effect). The present study aimed at providing further insights into potential side effects of CCRI41 cotton on honey bees. Emerging honey bees were exposed to different pollen diets using no-choice feeding protocols (chronic exposure) in controlled laboratory conditions and we aimed at documenting potential mechanisms underneath the CCRI41 antifeedant effect previously reported. Activity of midgut proteolytic enzyme of young adult honey bees fed on CCRI41 cotton pollen were not significantly affected, i.e. previously observed antifeedant effect was not linked to disturbed activity of the proteolytic enzymes in bees' midgut. Hypopharyngeal gland development was assessed by quantifying total extractable proteins from the glands. Results suggested that CCRI41 cotton pollen carries no risk to hypopharyngeal gland development of young adult honey bees. In the two bioassays, honey bees exposed to 1 % soybean trypsin inhibitor were used as positive controls for both midgut proteolytic enzymes and hypopharyngeal gland proteins quantification, and bees exposed to 48 ppb (part per billion) (i.e. 48 ng g(-1)) imidacloprid were used as controls for exposure to a sublethal concentration of toxic product. The results show that the previously reported antifeedant effect of CCRI41 cotton pollen on honey bees is not linked to effects on their midgut proteolytic enzymes or on the development of their hypopharyngeal glands. The results of the study are discussed in the framework of risk assessment of transgenic crops on honey bees.

Bee bread is the main source of proteins necessary for the development of hypopharyngeal glands in nurse bees. However, the seasonal chemical composition and its effects on honey bee physiology are poorly understood. Thus, this study evaluated how the season and botanical origin of pollen influence the content of crude protein, ash, and total flavonoids, as well as the profile of flavonoids, in Africanized Apis mellifera bee bread. The influence of these factors on the number and area of hypopharyngeal gland acini of nurse bees raised with similar numbers of brood frames over the four seasons of the year were also evaluated. The botanical families found in bee bread and the total flavonoid content and their chemical profile varied markedly with the seasons of the year, with the highest total flavonoid content found in the summer. The percentage of crude protein and ash in bee bread, however, did not change with the seasons. The number and area of acini in the hypopharyngeal gland significantly decreased during autumn and winter. Based on our results, flavonoids rather than crude protein may play a role in hypopharyngeal gland development in honey bees. Further studies to test artificial diets supplemented with flavonoids can improve beekeeping strategies and contribute to colony maintenance in periods of food shortage.

The effect of diet on protein concentration, hypopharyngeal gland development and virus load in worker honey bees ( Apis mellifera L.)

Simple SummaryPollinators adjust their foraging preference based on the pollen cues of foraging plants. Honey bees, for example, prefer to collect one type of pollen from plants that bloom at the same time. In northern China, apricot and pear trees are the two main foraging plants in the early spring. However, honey bees tend to collect pollen from apricot trees. It is interesting to understand what affects the foraging decision of honey bees regarding these two pollen types. In this study, we observed the foraging preference of Apis mellifera workers with respect to apricot and pear pollen under laboratory conditions. The effect of pollen on the development of the hypopharyngeal gland (HG) and ovary was measured. The number of visits made to apricot pollen was significantly higher than that to pear pollen. Furthermore, the response of the HG and ovary to these two pollens was different. The development of the HG was significantly affected by pollen diet treatments. However, there was no significant difference in the ovarian development of caged workers supplied with the two different pollen diets. Overall, honey bees showed a significant preference for apricot pollen over pear pollen. Compared with the ovary, the HG of honey bee workers may be more sensitive to pollen nutrition.With the availability of various plants in bloom simultaneously, honey bees prefer to collect some pollen types over others. To better understand pollen’s role as a reward for workers, we compared the digestibility and nutritional value of two pollen diets, namely, pear (Pyrus bretschneideri Rehd.) and apricot (Armeniaca sibirica L.). We investigated the visits, pollen consumption, and pollen extraction efficiency of caged Apis mellifera workers. Newly emerged workers were reared, and the effects of two pollen diets on their physiological status (the development of hypopharyngeal glands and ovaries) were compared. The choice-test experiments indicated a significant preference of A. mellifera workers for apricot pollen diets over pear pollen diets (number of bees landing, 29.5 ± 8.11 and 9.25 ± 5.10, p < 0.001 and pollen consumption, 0.052 ± 0.026 g/day and 0.033 ± 0.013 g/day, p < 0.05). Both pollen diets had comparable extraction efficiencies (67.63% for pear pollen and 67.73% for apricot pollen). Caged workers fed different pollen diets also exhibited similar ovarian development (p > 0.05). However, workers fed apricot pollen had significantly larger hypopharyngeal glands than those fed pear pollen (p < 0.001). Our results indicated that the benefits conferred to honey bees by different pollen diets may influence their foraging preference.

Insecticides are contributing to global insect declines, thereby creating demand to understand the mechanisms underlying reduced fitness. In the eusocial Hymenoptera, inclusive fitness depends on successful mating of male sexuals (drones) and efficient collaborative brood care by female workers. Therefore, sublethal insecticide effects on sperm and glands used in larval feeding (hypopharyngeal glands (HPG)) would provide key mechanisms for population declines in eusocial insects. However, while negative impacts for bumblebee colony fitness have been documented, the effects of insecticide exposure on individual physiology are less well understood. Here, we show that field-realistic concentrations (4.5–40 ng ml−1) of the neonicotinoid insecticide thiamethoxam significantly impair Bombus terrestris sperm and HPGs, thereby providing plausible mechanisms underlying bumblebee population decline. In the laboratory, drones and workers were exposed to five thiamethoxam concentrations (4.5 to 1000 ng ml−1). Then, survival, food consumption, body mass, HPG development, sperm quantity and viability were assessed. At all concentrations, drones were more exposed than workers due to higher food consumption. Increased body mass was observed in drones starting at 20 ng ml−1 and in workers at 100 ng ml−1. Furthermore, environmentally realistic concentrations (4.5–40 ng ml−1) did not significantly affect survival or consumption for either sex. However, thiamethoxam exposure significantly negatively affected both sperm viability and HPG development at all tested concentrations. Therefore, the results indicate a trade-off between survival and fitness components, possibly due to costly detoxification. Since sperm and HPG are corner stones of colony fitness, the data offer plausible mechanisms for bumblebee population declines. To adequately mitigate ongoing biodiversity declines for the eusocial insects, this study suggests it is essential to evaluate the impact of insecticides on fitness parameters of both sexuals and workers.

Goal. To study the effect of protein feed on the development of the fat body and the life expectancy of bees during the period when an insufficient amount of bee pollen is received in the nest of the bee colony. Methods. Zootechnical — to set up scientific experiments, feed bees, and determine the strength of bee families; micrometric — to study physiological and morphological signs of bee body parts; microscopic — to carry out analysis of fat body; statistical — to process experimental data. Results. It was confirmed that the development of the fat body in bees occurs at a young age when these insects consume the most protein food. The average life expectancy of bees in the case of protein supplementation in laboratory conditions was more than twice the life expectancy under protein starvation. Conclusions. Protein feeding with a mixture of sugar syrup and bee honey in early spring was proven to have a positive effect on the development of fat body and the life expectancy of bees.

Given honey bees’ biological characteristics, none of their individuals can form a new colony independently. In the process of evolution, honey bees have adapted to increase their numbers and spread over space through a complex, multi-stage natural process, such as swarming. Most anti-swarming techniques used in practical beekeeping only partially solve the problem of swarming colonies, not eliminating it. The processes happening in the body of worker bees in preparation for swarming are yet to be studied sufficiently. This work presents data from experimental studies of the mechanism of influence of trophic relationships of bees with honey plants on swarming and productivity of colonies during heavy honey collection. The goal of the work was to study the effect of honey collection from black locusts (Robinia pseudo acacia) on the swarming state and honey and wax productivity of bees. Methods of research. Zootechnical (selection of groups of analogs, accounting for honey and wax), biological (development of fat body, live weight) analytical (analysis of literature data and research results), and statistical (biometric data processing). Biometric data processing was performed using MS Excel software with built-in statistical functions. The average live weight of bees with signs of swarming was 5,6 % higher than the control. The difference in the amount of synthesized wax between the colonies’ groups not stastically significant. The bees in the control group built 11,7 fewer combs than the experimental group of colonies. The honey productivity results of the experimental group colonies were higher - by 12,8 kg, compared to the control group. Providing colonies in swarming conditions with productive honey collection promotes the transition to working conditions. The preparation of a bee colony for swarming, based on the study of the physiological characteristics of bees, can be noticed long before the swarm leaves – 15-20 days prior. In preparation for swarming, the de gree of fat body development among the worker bees and their live weight increase. The increased development of the fat body in bees that will form the basis of the swarm is primarily due to the high energy expenditure involved in creating a new nest and foraging. Monitoring the physiological parameters of worker bees can serve as a bio test of the bee colony’s readiness for swarming long before the swarm emerges. Key words: swarming, Ukrainian steppe breed, fat body, honey collection, honey productivity, trophic relationships.

The hypopharyngeal glands of worker bees located in the head; consist of thousands of two-cell units that are composed of a secretory cell and a duct cell and that are arranged in sets of about 12 around a long collecting duct. The glands contribute to the production of the royal jelly fed to queens and larvae. They are highly sensitive to the quantity and quality of the food as pollen and pollen substitutes that the nurse bee consumes. The role of the worker honey bee Apis mellifera L. changes depending on age after eclosion (age polyethism): young workers (nurse bees) take care of their brood by synthesizing and secreting brood food (royal jelly), while older workers (foragers) forage for nectar and process it into honey. In our experiment, we tested how diets impact hypopharyngeal gland development and their acini size, where our diets compared were (bee bread diet; unfermented diet; fermented diet in a simulation method for nature; and sucrose syrup). Also, we mentioned understanding the role of these glands in hive health. For this study, we have examined the morphogenesis of the hypopharyngeal gland during different ages of workers honeybee Apis mellifera L. that fed on the different diets; we measured the size of glandular acini in a robust measure. These results obtained indicated that the hypopharyngeal gland development has flexibility and can depending on the condition of the colony as the pollen substitute diet we prepared in the periods of food shortage in nature. This described the hypothesis that feeding plays an important role in the development of HG according to diet nutritional values, ensuring the importance of the fermentation process for the better health of honey bees.

Providing bees with benign and sufficient protein feeds is the key to successful beekeeping. Protein feed significantly affects on certain functions of individuals and the vital activity and productivity of the bee colonies. So, as a result of increased consumption of protein feed during the first days of life the supply of proteins in the body significantly increases in young bees, hypopharyngeal glands and other organs become developed, which provides the ability to perform various tasks depending on age and living conditions. Older bees consume protein for tissue renewal with new cells and metabolic processes. Bees raised on low protein feeds become physiologically defective and do not live long. In conditions of protein starvation, brood rearing stops and bees throw larvae out of their cells. The article presents the results of studying the effect of using soy flour and its processed products in bee feeding on the essential amino acids content in their body. It was found that the use of soy peptone in bee feeding contributes to a greater accumulation of essential amino acids in the bees body in different periods of their development, in compared to soy milk, defatted soy flour and roasted soy flour which indicates a higher efficiency of its use as partial substitutes for protein feeds.