Histochemical Comparison of the Hypopharyngeal Gland in Apis cerana Fabricius , 1793 Workers and Apismellifera Linnaeus , 1758 Workers

Hypopharyngeal glands of honeybee are age-dependent structures that change with the size of acini and are correlated with various social behaviors. The histochemical structure of Apis cerana and A. mellifera worker hypopharyngeal glands in four different developmental stages wes stained with ninhydrin Schiff ’s and periodic acid Schiff ’s reagents (PAS) for localization of proteins and carbohydrates, respectively, and examined with light microscopy. Nurse bees of both honeybee species had significantly larger glands as compared to guards and forgers, but there were no statistically significant differences between these two species after accounting for caste. Gland protein concentration increased progressively in nurse bees, and this was correlated with the appearance of enriched protein granules in the cytoplasm. In addition, the hypopharyngeal gland protein concentration of A. mellifera was higher than that of A. cerana even though gland size was not significantly different between species. However, gland size was shown to have decreased progressively in foragers and guards.


Introduction
The development of hypopharyngeal glands (HPGs) in dwarf honeybee workers primarily depends on age.These glands begin to differentiate at pupal stage and are largely undeveloped at emergence [1].When workers become nurse bees, they perform brood rearing that is associated with HPGs development.The size of HPGs is correlated with glandular production and generally increases with age from 6 to 18 days in nurse bees [2,3].HPGs synthesize and secrete proteinaceous substances and royal jelly that are fed to the queen and brood [3].The highest rate of protein synthesis occurs during nursing ages from 8 to 16 days [4,5].In bees older than 18 days (guards and foragers), the HPGs decrease considerably in size and secrete enzymes such as α-glucosidases, leucine arylamidase and invertase [6][7][8][9].Forager gland size is reduced and correlated with the gland activity [3,4].We previously demonstrated the location of proteins and carbohydrates within HPGs of dwarf honeybee workers among different ages [1].The glands were composed of several secretory apparati.Each opened into a secretory duct and then passed through the mouthparts.In pupae, the secretory cells were irregular in shape with low concentrations of proteins and carbohydrates while the glands of nurse bees and foragers were fully developed with numerous secretory vesicles [1].For this study, we measured the size of glandular acini, examined protein concentration in hypopharyngeal glands, and identified the location of proteins and carbohydrates in the hypopharyngeal gland workers of Apis cerana and A. mellifera.

Materials and Methods
2.1.Honeybees.Apis cerana and A. mellifera workers of different ages were collected from Samut Songkarm province, Thailand.Pupae (dark brown-eyed stage) were grasped from the cells.Nurse bees were taken from their colony while they were feeding brood.Guards were collected from in front of Psyche

Glandular Size.
Acini of the glands started to develop at the pupal stage and increased in size at the adult stage.The size (width and length) of the acini of both species was the largest in nurse bees and then gradually decreased from nurses to guards (Table 1).However, the acini slightly increased in size when they changed their tasks to become foragers.There was no significant difference in the width of acini between A. cerana and A. mellifera (F 1, 3 = 2.63, P < .1120).However, there were significant width differences among different life stages (F 1, 2 = 31.24,P < .0001,and n = 48).In contrast, the length of the acinus of HPGs showed statistically significant differences between these two species and developmental stages (F 1, 2 = 8.48, 12.92; P < .0048,.0001;n = 48).

Protein Content.
The mean total protein content of the hypopharyngeal glands taken from all three developmental stages of A. mellifera was significantly higher than those of A. cerana (F 1, 2 = 38.88,P < .0001)(Figure 2).The highest protein concentration was found in nurse bees of A. mellifera which were 1389.6 ± 158.9 μg/μL (69.5 ± 8.0 mg/bee).Proteins (revealed with ninhydrin Schiff 's reagent) decreased in concentration in later developmental stages.In contrast, the lowest protein concentrations were found in guards of A. cerana which were 413.8 ± 5.1 μg/μL (20.7 ± 0.3 mg/bee, Figure 2).There was a significant difference in protein concentration among the developmental stages of A. mellifera and A. cerana (F 2.5 = 217.82,P < .001,and n = 49, Figure 1).mellifera was relatively similar among life stages.In both species, several clusters of acini were connected to a long slender secretory duct.Each acinus was composed of 8-10 aggregated secretory cells and the glands consisted of an incomplete irregularly shaped secretory unit with unequal secretory cells.However, there were differences among stages of either A. cerana or A. mellifera.The histochemical staining shows a glandular cell cytoplasm rich in secretory vesicles and presenting glycoprotein secretions.

Pupae.
The hypopharyngeal glands developed and formed an acinus.The glands consisted of an incomplete structure of secretory units which were irregular in shape and composed of 8-10 secretory cells.Each cell had an oval nucleus which stained green.The slightly pink cytoplasm can be seen in this stage, but secretory vesicles do not appear (Figure 2(b)).

Nurse Bees.
Each acinus consisted of numerous vesicles that were not stained with Schiff 's reaction.These white and clear vesicles were found surrounding the nucleus of acinar cells.Strongly positive Schiff 's staining was found in the periphery of secretory cell cytoplasm.Ninhydrin Schiff 's staining clearly distinguished the unstained vesicles from the peripheral area of the cytoplasm that included the nucleus that was strongly positive with pink staining.Nevertheless, areas of some vesicles stained magenta (Figures 2(c) and 2(d)).

Guards.
In the guards, some HPGs demonstrated the beginning of retrogression through the formation of irregular secretory cells.Guard cytoplasm stained weakly with Ninhydrin Schiff 's reagent as compared to the stronger staining of nurse bees and foragers.Even the secretory vesicles of guards were smaller in size than nurse bees or foragers, the vesicles were elongated and cylindrical in shape.
The wider extracellular space between two adjacent secretory cells was clearly seen in this stage (Figure 2(e)).

Foragers.
The glands were composed of several acini smaller than those of nurse bees but larger than those of guards.Each acinus consisted of 8-10 pyramidal secretory cells, with oval nuclei staining positive using Ninhydrin Schiff 's reagent in the area of euchromatin.The cytoplasm of secretory cells, except vesicles, was strongly magenta positive when stained with Ninhydrin Schiff 's reagent (Figure 2(f)).

Histochemical Structure for Localization of Carbohydrate.
Carbohydrate levels in the HPGs of the four stages of A. cerana and A. mellifera workers were similar to each other.
The glands stained strongly positive to PAS, indicating the existence of carbohydrates (Figures 3(a) and 3(b)).However, there were clear differences among developmental stages in both species.

Pupae.
The glands began forming a cluster of paired irregularly shaped secretory units that were composed of 8-10 cell aggregations.The cells gave weak positive staining to PAS with pink in cytoplasm.

Nurse Bees.
The HPGs were fully developed and composed of several secretory units made up of 8-10 aggregated pyramidal acinar cells.Within the acinar cells there were numerous red-pink secretory vesicles indicating a strong positive with PAS reaction and corresponding to glandular sizes (Figure 3(c)) (Table 1).

Guards.
For guards, the glandular structure and histochemistry were slightly different to foragers.For example, each acinus was composed of 8-10 cells with small secretory vesicles and also lower number than those of foragers.However, they showed large extracellular space between adjacent cells, unlike the glands of nurse bees and foragers where there was minimal space (Figures 3(d) and 3(e)).

Foragers.
The glands of foragers showed complete structure consisting of 8-10 aggregated acinar cells.The secretory vesicles of foragers were slightly larger than those of guards.The secretory vesicles of foraging bees gave strong positive staining with PAS which was similar to those of nurse bees (Figure 3(f)).

Discussion
The structure of honeybee hypopharyngeal glands depends on the development and age of individuals, which corresponds with age-specific tasks and is known as age polyethism [3,11].The results of this study showed that the histochemical structure of carbohydrate and protein in A. cerana and A. mellifera was correlated with honeybee Psyche age-specific tasks of the colony.Young worker nurse bees care for and feed their brood with royal jelly that is synthesized and secreted from the hypopharyngeal glands.These hypopharyngeal glands were strongly positive to PAS and Ninhydrin Schiff 's reagent reactions in this study.However older workers, when they became guards, had less positive staining to PAS as compared to nurse bees.This may be related to the development of the hypopharyngeal glands which are fully developed when young workers take care of their brood by synthesizing and secreting royal jelly.Older workers no longer feed the brood, and thus gland atrophy is expected [3,4,8].However, when bees become foragers, the hypopharyngeal gland contains significant amounts of carbohydrate and protein.This finding is consistent with the finding that the hypopharyngeal glands are the site of conversion of nectar to simple sugars by enzymes [6][7][8][9].
The hypopharyngeal gland has been studied in several ways, including its ultrastructure, protein complement, and histochemical structure in Apis andreniformis and A. florea.[1,3,5,8,9,12].In this study, we found similarities in glandular structure between species, and the glands were fully developed in nurses, guards, and foragers.Moreover, the secretory units of the HPGs were filled with numerous vesicles that gave a strong positive staining with PAS and Ninhydrin Schiff 's reagent [1].This indicates that the glands of nurses, guards, and foragers in four species of honeybees (i.e., A. andreniformis, A. florea [1], A. cerana, and A. mellifera,) play an important role not only in secretion of carbohydrate rich substance but also in the secretion of enzymes for converting nectar to honey [1,13,14].However, there were also differences found in structure of the glands between the hive cavity nest honeybees, A. cerana and A. mellifera, and the single open nest honeybees, A. andreniformis and A. florea.The structure of the extracellular space between adjacent cells of A. andreniformis and A. florea was wider than that of A. cerana and A. mellifera.In addition, the secretory units of hypopharyngeal glands of A. mellifera from this study were different from results in the study of Deseyn and Billen [3] who showed that the volume of acini decreased in foragers or displayed degenerative structure while that was not found in this study.
In the present study, the development and histochemical aspects of the hypopharyngeal glands were evaluated in A. cerana and A. mellifera.The findings are in agreement with our previous study [1].Additionally, glandular sizes were the greatest in nurse bees.Hypopharyngeal glandular size is known to be positively correlated with gland activity and is influenced by larval feeding [2,4,15,16].These glands gradually decrease in size when honeybees become guards, cease feeding, and begin defending the colony [3].However, the hypopharyngeal gland size of foragers was significantly larger than that of guards.The results in this study indicate that glandular development corresponded well with total protein synthesis in the hypopharyngeal glands at different adult life stages.A number of reports indicate that the HPGs produce enzymes that are used to hydrolyze nectar into honey, including amylase, α-glucosidases, glucosidase oxidase, galactosidase, esterase, leucine arylamidase, and invertase [4,[6][7][8][9].It can be inferred that the HPGs perform two functions: first producing protein rich royal jelly for the nursing brood (by nurse bees) and then enzyme production (by foragers).However, the function of hypopharyngeal glands has flexibility depending on the colony condition and the need for feeding brood [4,16].The protein concentration of the hypopharyngeal glands peaked in nurse bees and declined in older workers.This corresponds to the degree of protein synthesis and is correlated with the abundance of rough endoplasmic reticulum (RER) found in adult workers.RER of acinar cells of worker glands developed and increased significantly a few days after emergence Most of the cytosol space was filled with RER stacks a few days after emergence, while in foragers the RER decreased [5].However, the total mean protein content of the hypopharyngeal glands of A. mellifera workers was significantly higher than that of A. cerana workers (F 1,2 = 38.88,P < .0001).This is related to glandular sizes.In A. mellifera, the hypopharyngeal glands and average body sizes are larger than those of A. cerana.In summary, the hypopharyngeal glands of A. cerana and A. mellifera workers are fairly similar in terms of the histochemical structure.Each gland begins development as pupae and had a fully forming acinus in nurse bees.Gland regression occurred when the bees developed into foragers and guards.Protein concentration peaked in nurse bees and declined in guards.In the hypopharyngeal glands, the histochemical staining of carbohydrates and protein corresponded well with the glandular size and protein content.

Table 1 :
Hypopharyngeal gland sizes (mean ± S.E.) of A. cerana and A. mellifera workers in different stages of life: nurse, forager, and guard.
Note: Means ± S.E.followed with different letters in the same column denote significant differences (ANOVA-Duncan's multiple range test; F = 24.98,df=5,P< .0001;F=7.32,df= 5, P < .0001).2.2.Glandular Size Measurement.Measurements of glandular size were made from nurse bees, guards, and foragers.Under a stereomicroscope, HPGs of each life stage were removed from the head using modified blades and then transferred into insect saline solution (NaCl 8.766 g., CaCl 2 0.188 g., KCl 0.746 g., MgCl 2 0.407 g., NaHCO 3 0.336 g., sucrose 30.807 g., and trehalose 1.892 g., pH 7.6).Using a micrometer, gland diameters were measured (width and length) under light microscopy.2.3.Preparation of Protein Sample.Ten worker bees were collected to represent each species and anesthetized 2.6.Data Analysis.Statistical differences between hypopharyngeal mean gland size and protein concentration were compared using ANOVA and Duncan's multiple-range test (DMRT).