Eusocial insect declines: Insecticide impairs sperm and feeding glands in bumblebees

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

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.

One hundred and fifty newly emerged worker bees were transferred into Pain-type cages. The experiment included five replicates per batch: five cages for the control, five for bees treated with 10 mM lithium chloride (LiCl), and five for bees treated with 25 mM LiCl. Pollen paste and syrup consumption were measured at 7 and 14 days. Worker bees were sampled at 0, 7, and 14 days for hemolymph extraction (to measure protein, lipid, and sugar content) and for dissection of hypopharyngeal glands (HPGs) and ovaries. Oxidative stress marker levels were also quantified. The results revealed that LiCl treatment did not significantly affect food consumption (pollen paste and syrup). Protein, sugar, and lipid levels in the hemolymph increased with age (p < 0.001) without any effect from LiCl. HPGs development was significantly reduced by 25 mM LiCl at 7 days (p = 0.003) and by both concentrations at 14 days (p < 0.001). Ovarian development was significantly inhibited by 25 mM LiCl at 7 days (p < 0.001) and 14 days (p = 0.016), whereas 10 mM had no significant effect. Malondialdehyde (MDA) levels and antioxidant capacity were not affected by LiCl but varied with age. These results suggest that a high dose of LiCl (25 mM) disrupts HPG and ovarian development in honey bees without inducing measurable oxidative stress.

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

Research on the anatomical and physiological characteristics of bees (the state of fat body, hypopharyngeal glands) in connection with changes in natural and climatic conditions (soil composition, prolonged droughts, prolonged rains, cold weather, environmental disasters) impoverishment of fodder base for bees due to the decrease of sown areas of honey crops leading to the use of bees’ feeding, is relevant. The research aims to study the influence of food protein on the development of hypopharyngeal glands, fat body and life expectancy of honeybees. The zootechnical (the setting of the scientific research, the feeding of the bee families, the power of the bee families, productivity), ethological (orientation in the bee-entrance area), micrometrical (physiological and morphological characteristics of the parts of the bee’s body), microscopic (analysis of hypopharyngeal glands and fat body of bees) and statistical (biometric data processing) methods were used in the study. The significant difference in their degree of development depending on the carbohydrate or protein feeding was identified. After feeding sugar syrup to bees, signs of protein malnutrition and the state of development of hypopharyngeal glands corresponding to the І-ІІ degrees were noted. Alveoli of hypopharyngeal glands were reduced, and underdeveloped, with marked spaces, whereas after feeding protein food in the form of sugar syrup with bee pollen the bees had well-developed hypopharyngeal glands. The alveoli of the hypopharyngeal glands of the researched bees filled out, grew milky, with no space between them, and were able to produce larval food. Such a state of the glands’ development corresponds to the ІV degree of the development of hypopharyngeal glands. The research results indicate that the development of the fat body of bees happens at a young age when bees consume protein food the most. It has been proven that protein feeding of the bee colonies of experimental groups contributed to the better development of hypopharyngeal glands in both old and young bees, which resulted in better provision of food for larvae and more active development of the bee colonies. Protein feeding of bees in early spring under conditions of limited supply of protein food contributes to the production of larger larvae, which in turn contributes to the production of more complete bees

Buffered fitness components: Antagonism between malnutrition and an insecticide in bumble 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.

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.

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

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.

Hypopharyngeal glands (HGs) are the most important organ of hymenopterans which play critical roles for the insect physiology. In honey bees, HGs are paired structures located bilaterally in the head, in front of the brain between compound eyes. Each gland is composed of thousands of secretory units connecting to secretory duct in worker bees. To better understand the recent progress made in understanding the structure and function of these glands, we here review the ontogeny of HGs, and the factors affecting the morphology, physiology, and molecular basis of the functionality of the glands. We also review the morphogenesis of HGs in the pupal and adult stages, and the secretory role of the glands across the ages for the first time. Furthermore, recent transcriptome, proteome, and phosphoproteome analyses have elucidated the potential mechanisms driving the HGs development and functionality. This adds a comprehensive novel knowledge of the development and physiology of HGs in honey bees over time, which may be helpful for future research investigations.

Regulation of hypopharyngeal gland activity and oogenesis in honey bee ( Apis mellifera) workers

Emerging infectious diseases can have a major impact on fitness of novel hosts, thereby contributing to ongoing species declines. In social insects, collaborative brood care by workers and successful mating of male sexuals are key to colony fitness. The microsporidian endoparasite Nosema ceranae has spread almost globally, shifting across honeybee species and now to bumblebees. However, despite N. ceranae being linked to recent population declines, its possible impact on bumblebee colony fitness remains poorly understood. Here, we show that N. ceranae infections can significantly impact Bombus terrestris worker feeding glands, as well as longevity, sperm quality and mating abilities of drones. In the laboratory, workers and drones were either exposed to the parasite or not. Then, parasite infection rates and loads, as well as lethal and sublethal parameters, were assessed. Infected drones revealed higher parasite infection rates and spore titres, as well as mortality compared with female workers, suggesting sex-specific susceptibility. Furthermore, infections impaired feeding glands, affected sperm traits and altered mating behaviour, all of which are key to colony fitness. Our findings provide a mechanistic explanation on how N. ceranae contributes to the ongoing decline of wild bumblebee populations, calling for respective mitigation measures.