Morphology of mandibular and intramandibular glands in workers and virgin queens of Melipona scutellaris Douglas Elias Santos, Edmilson Amaral de Souza, Carlos Ueira Vieira, José Cola Zanuncio, José Eduardo Serrão

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Douglas Elias Santos, Edmilson Amaral de Souza, Carlos Ueira Vieira, José Cola Zanuncio, José Eduardo Serrão. Morphology of mandibular and intramandibular glands in workers and virgin queens of Melipona scutellaris. Apidologie, Springer Verlag, 2015, 46 (1), pp.23-34. ￿10.1007/s13592-014- 0301-6￿. ￿hal-01284420￿

HAL Id: hal-01284420 https://hal.archives-ouvertes.fr/hal-01284420 Submitted on 7 Mar 2016

HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Apidologie (2015) 46:23–34 Original article * INRA, DIB and Springer-Verlag France, 2014 DOI: 10.1007/s13592-014-0301-6

Morphology of mandibular and intramandibular glands in workers and virgin queens of Melipona scutellaris

1 2 3 Douglas Elias SANTOS , Edmilson Amaral de SOUZA , Carlos Ueira VIEIRA , 4 1 José Cola ZANUNCIO , José Eduardo SERRÃO

1Departamento de Biologia Geral, Universidade Federal de Viçosa, Viçosa 36570-900, Brazil 2Departamento de Biologia, Universidade Federal de Viçosa, campus Rio Paranaíba, Rio Paranaíba, MG, Brazil 3Instituto de Genética e Bioquímica, Universidade Federal de Uberlândia, 38400-902, Uberlândia, Brazil 4Departamento de Biologia , Universidade Federal de Viçosa, Viçosa 36570-900, Brazil

Received 16 December 2013 – Revised 20 May 2014 – Accepted 4 June 2014

Abstract – The interactions of societies are associated with chemical signals released by the exocrine glands that control some insect behaviors. Exocrine glands can be classified into different classes: class I release substances that cross the body cuticle and class III glands that have a conducting canal. This study compares the morphology of intramandibular and mandibular glands in virgin queens of different ages, as well as in nurses and forager workers of Melipona scutellaris. We have found the occurrence of two types of glands inside the mandible; glands of class I, corresponding to a well-developed epithelium, secretory only in virgin queens, and glands of class III. On the other hand, mandibular glands have been found to be well-developed in workers and queens and they also have high amounts of mitochondria and smooth endoplasmic reticulum. Together, these data show that the mandibular and intramandibular glands of M. scutellaris virgin queens have morphological features that indicate the synthesis of compounds that are lipid in nature. Furthermore, both glands have a similar developmental degree in newly emerged and 7-day-old ones. head glands / Melipona / communication / behavior / virgin queen

1. INTRODUCTION regard to nestmate recognition, some stingless recognize virgin queens when they are a Maintenance of insect societies is associated few days old, but these virgin queens are with chemical signals released by the exocrine commonly killed by the workers (Kleinert and glands, which are distributed in great numbers Imperatriz-Fonseca 1994; Imperatriz-Fonseca and diversity throughout the insect body and Zucchi 1995; Jarau et al. 2009). (Cruz-Landim 2009). The secretion of exocrine In bees, the exocrine glands are ectodermic in glands can be associated with recognition of origin and can be classified into different classes individuals of the same colony through chem- according to the secretory apparatus. The ical signals, such as those reported in Apis exocrine glands of class I release substances mellifera capensis and Melipona scutellaris that cross the body cuticle via the cuticular pore, (Kerr et al. 2004; Wossler et al. 2006). With which is a result of cuticle deposition after molting. Class III glands have two cell types: secretory cells and cells that form the conducting canal (Noirot and Quennedey Corresponding author: J. Serrão, [email protected] 1991; Abdalla and Cruz-Landim 2002). Among Manuscript editor: James Nieh the exocrine glands of bees, the mandibular, 24 D. Elias-Santos et al. labial, and hypopharyngeal glands form the inferred indirectly on the basis of their cell salivary gland system, which is present in queens, morphology (Abdalla and Cruz-Landim, 2002). males, and workers (Cruz-Landim 1967). In this sense, some glands in the body show The mandibular glands of bees have a bag- variations in size, morphology, and function shaped reservoir and secretory cells of class III according to caste, age, sex, and colony condi- (Nedel 1960). The mandibular glands may act tions (Costa-Leonardo 1981;Cruz-Landim as alarm and defense pheromones (Smith and 1994; Cruz-Landim 2009). The ultrastructure Roubik 1983; van Zweden et al. 2011; Cruz- of the glands in A. mellifera queens, for Lopez et al. 2005, 2007; Schorkopf 2009) and example, has been reported to change according may also participate in a queen’s attractiveness to the pheromone activity, as the queen ages to worker bees in mated A. mellifera queens (Dehazan et al. 1989). Similar age-related (Gary 1961). changes in the cell morphology and secretory The intramandibular glands are epidermal activity of the mandibular glands have also been glands that differentiate during metamorphosis shown to occur in honeybee drones (Lensky et (Martins et al. 2013) and this probably occurs in al. 1985). both sexes of all bee species, with two types of The function of bee’s glands may be inferred glandular cells inside the mandible (Abdalla and from their morphology. Thus, the objective of Cruz-Landim, 2002). The first type is present in this study is to compare the morphology of the region just below the epidermis and consists mandibular and intramandibular glands in virgin of cells of class III (Nedel 1960). The second queens of different ages and in nurse and type is class I formed by a secretory epidermis forager bees of M. scutellaris. We conducted (Cruz-Landim 1967). However, the chemical assays to detect proteins and lipids in these nature and function of the intramandibular gland glands to gain a better understanding of possible compounds are not known (Cruz-Landim et al. gland function. In virgin queens, we hypothe- 2011). size that these glands may be releasing different In the stingless bees Melipona marginata, compounds that allow workers to recognize Melipona quadrifasciata,andMelipona virgin queens. beecheii, the virgin queens, a few days old, are recognized by workers, who may attack them 2. MATERIALS AND METHODS (Kleinert and Imperatriz-Fonseca 1994; Imperatriz-Fonseca and Zucchi 1995; Jarau et 2.1. al. 2009). In this sense, the behavior of the virgin queens may stimulate the workers and the The experiment was conducted at the Apiary of most active queens are persecuted and attacked the Federal University of Viçosa, (20° 45′ S, 42° 52′ by the workers (Jarau et al. 2009). However, W), state of Minas Gerais, Brazil, using four colonies researches involving A. mellifera capensis and of M. scutellaris. Twenty individuals of each caste M. scutellaris suggest that recognition of indi- (nurse and forager workers, newly emerged queens, viduals of the same colony is through chemical and 7-day-old virgin queens) were obtained from signals (Kerr et al. 2004; Wossler et al. 2006). each colony (five from each colony) in the spring and The possible function of the mandibular and summer of 2008, 2009, and 2012. Virgin queens were intramandibular glands in virgin queens of obtained from combs taken from colonies, and were stingless bees, of different ages, may be maintained at 27 °C. We studied 7-day-old virgin obtained by comparing the morphology of these queens, as our preliminary studies showed that this glands in these queens, likely reported for was the age at which these queens were recognized workers in the M. quadrifasciata and killed by the workers (unpublished data). To (Cruz-Landim et al. 2011). obtain 7-day-old virgin queens, the newly emerged The secretory activity and chemical compo- queens were caged in a meshed box together with sition of the secretions from the glands may be some nurse workers and transferred to the colony Head glands in virgin queens of bees 25

with pollen and honey from the storage pots of the cells and nuclei of these gland cells. Measurements same colonies, which was available ad libitum. This were obtained with the aid of the software, Image was a more viable method to collect virgin queens Pro-Plus (Media Cybernetics). with known age, but this might differ from the natural conditions. Forager workers were collected when 2.5. Statistical analysis arriving at the nest entrances with corbiculae loaded with pollen, whereas the nurse workers were collect- The normal distribution of the morphometrical ed inside the nest onto the brood combs. data (total cell and nucleus areas) were tested using the Kolmogorov-Smirnov test, and the data were 2.2. Scanning electron microscopy subjected to one-way analysis of variance (ANOVA) followed by the multiple comparison Tukey’s test at a The mandibles and mandibular glands were 5 % significance level. dissected from two individuals and castes, from each nest, and transferred to Zamboni’s fixative solution 2.6. Transmission electron microscopy (Stefanini et al. 1967), and dehydrated in a graded ethanol series. The samples were then transferred to Three bees of each caste were dissected and the hexamethyldisilazane for 5 min, air dried, and coated mandibles and mandibular glands were removed and with gold (20 nm). The samples were examined using transferred to 2.5 % glutaraldehyde in a sodium a scanning electron microscope (SEM) LEO VP1430. cacodylate buffer of 0.1 M, pH 7.1, for 2 h. Following this, the samples were washed and post- 2.3. Light microscopy fixed in 2 % osmium tetroxide in the same buffer for 2 h and dehydrated in 70 % ethanol. The samples Twelve bees of each caste (3 from each colony), were embedded in LR-White resin and, after poly- were cryoanesthetized for 1 min at −20 °C and merization, were sectioned at 80–90 nm, stained with decapitated shortly thereafter. The mandibles and 1 % aqueous uranyl acetate and lead citrate, and mandibular glands were dissected and transferred to analyzed in a transmission electron microscope Zamboni’s fixative solution for 2 h. The samples (TEM), Zeiss EM 109. The contrast and brightness were then dehydrated in a graded ethanol series and of the micrographs were improved using the soft- embedded in historesin. Slices, 4-μm thin, were ware, Photoshop CS6. stained with hematoxylin and eosin. Finally, ten slices from every bee were submitted for the 3. RESULTS following histochemical tests: periodic acid Schiff (PAS) to detect neutral polysaccharides and 3.1. Intramandibular glands glycoconjugates; Nile blue to detect lipids; mercu- ry–bromophenol blue to detect total protein, accord- Cells typical of class III glands were found ing to Pearse (1985), and tests for acid and alkaline in the intramandibular glands of all the castes phosphatases according to van Noorden and (Figure 1a–d). These glands, which are located Frederiks (1992). under the epidermis, showed different degrees of development between the castes. In the 2.4. Morphometry nurse and forager worker bees, the secretory cells were spherical, with well-developed Longitudinal histological sections of the entire nuclei. The forager worker bees had cells of length of the mandible and mandibular gland were class III glands that were larger (F=7.115, p< randomly selected from ten bees of each caste, and 0.05) than those of the newly emerged and 7- prepared for light microscopy. Ten secretory cells day-old virgin queens (Figure 2a). The class (class III glands) that had an evident nucleus and III cells of the newly emerged virgin queens cytoplasm were selected from each histological slice were spherical, whereas those in the 7-day-old and measurements were taken of the total areas of the virgin queens had an irregular shape and 26 D. Elias-Santos et al.

Figure 1. Light micrographs of intramandibular evident nucleolus (arrows). c Nurse worker showing glands of Melipona scutellaris. a Newly emerged general view of intramandibular gland of class III virgin queen showing the secretory epithelium (g1) (g3) and cubic epithelium (ep). d Nurse worker well developed and the gland of class III (arrow). b showing the well-developed intramandibular gland Seven-day-old virgin queen showing gland of class of class III (g3) and cubic epithelium (ep). cu cuticle, III (g3) with irregular shape and nucleus (n) with fb fat body.

nucleus, but statistically showed a similar size cytoplasmic proteins (Figure 4c). Lipids were (F=0.842, p>0.05; Figure 2a, b). The mandi- also found in these glands (Figure 4d). There bles of the nurse and forager workers had was no significant acid or alkaline phospha- pores on the inner surface, while in the queens tase activity in the cells of the the pores were concentrated at the outer intramandibular glands of bees. surface of the mandible (Figure 3a–c). The ultrastructural analyses of the class III In all the bees analyzed, there was a secretory cells in the intramandibular glands strong positive reaction for neutral polysac- showed a well-developed, smooth endoplas- charides in the excretory canal of the class mic reticulum, as well as mitochondria, in all III gland (Figure 4a, b). Only the nurse and bees. forager workers showed a positive PAS In addition to class III secretory cells, the reaction in the cytoplasm of the secretory virgin queens also had a developed secretory cells. The histochemical test with mercury– epithelium (class I gland), composed of bromophenol blue showed a positive result in columnar cells with spherical nuclei the cytoplasm of secretory cells of all the (Figure 1a), with a predominance of bees analyzed, indicating the presence of decondensed chromatin and a well- Head glands in virgin queens of bees 27

Figure 2. Morphometric data of intramandibular and cell showing difference between foragers and virgin mandibular gland cells of Melipona scutellaris. a queens (asterisk). d Nuclear area of mandibular gland Total cell area of intramandibular class III gland cells showing difference between foragers and queens showing difference between foragers and virgin 0 days old (asterisk). Q0d: newly emerged queen, queens (asterisk). b Nuclear area of intramandibular Q7d: queen 7-days old. Statistical analysis one-way cell of class III gland without significant difference ANOVA followed by multiple comparison Tukey’s. among castes. c Total cell area of mandibular gland

developed nucleolus, with the cytoplasm membrane infoldings associated with mito- strongly PAS positive in the basal region chondria, whereas in the cell apices, the (Figure 4a) and staining positively with plasma membrane had irregular projections mercury–bromophenol blue. Class I glands forming an extensive labyrinth in the were not found along the entire length of the subcuticular space (Figure 5a). The ultra- mandible, being located only at the epitheli- structure of these cells was not analyzed in um lining of the external mandible surface. the workers, as the mandibular epithelium In workers, the mandible epidermis showed was nonglandular in these bees. small cubic cells (Figure 1) with the nuclei containing condensed chromatin. 3.2. Mandibular glands The cells of the class I intramandibular glands in virgin queens had nuclei with Mandibular glands with clustered class III decondensed chromatin in the median cell cells were found in all castes of M. region and a cytoplasm rich in smooth scutellaris. Moreover, there was a large endoplasmic reticulum (Figure 5a, b). The reservoir with a thin wall of flattened cells basal portions of the cells had many plasma and a thin membrane surrounding the secre- 28 D. Elias-Santos et al.

Figure 3. Scanning electronic micrographs of man- showing pores (arrows) in the inner surface of the dible surface of Melipona quadrifasciata. a Schemat- mandible. c Newly emerged queen showing pores ic drawn showing a nomenclature of surface from of (arrows) in the external surface of the mandible. mandible of bee used in this paper. b Nurse worker

tory portion. The secretory cells of the 4. DISCUSSION mandibular glands were smaller in the forag- er workers (F=5.011, p<0.05) than in both 4.1. Intramandibular glands the newly emerged and 7-day-old virgin queens (Figure 2c, d). We found two types of secretory cells in the The histochemical tests showed a strong intramandibular gland, in the mandibles of the positive reaction to neutral polysaccharides in virgin queens of M. scutellaris: class I and class the excretory canals, with the cytoplasm III cells, according to the classification of being weakly reactive in all the bees. Noirot and Quennedey (1991). Both classes of Moreover, there was a strong reaction to glands have been reported to occur in the protein in the cytoplasm of the cells of the mandibles of other bee species as well (Costa- mandibular glands of all the bees analyzed. Leonardo 1978; Nedel 1960; Santos et al. 2009; Tests with Nile blue and acid and alkaline Cruz-Landim et al. 2011). phosphatases were negative in the mandibular Intramandibular secretory cells of class III in glands for all the bees. newly emerged virgin queens differed morpholog- The ultrastructural analyses showed that the ically from those found in 7-day-old virgin queens, cytoplasm of the mandibular glands was rich in which were irregular in shape. This shrink- smooth endoplasmic reticulum and mitochon- ing, which resulted in irregular gland cells, dria (Figure 5c). suggested that these gland cells suffered Head glands in virgin queens of bees 29

Figure 4. Light micrographs of histochemical tests of mic glycogen (arrows) in gland class III (g3); PAS intramandibular glands of Melipona scutellaris. a test. c Forager worker showing protein in glands of Virgin queen 7-days old, showing strong positive class III (g3); mercury-bromophenol test. d Newly reaction for glycogen (arrows) in cells of class III emerged queen showing occurrence of lipid (arrows) (g3) and in the basal portion of secretory epithelium in the gland of class III (g3); Nile blue test. c (g1); PAS test. b Nurse worker evidencing cytoplas- Cytoplasm, cu cuticle, ep epithelium, n nucleus.

morphological age-related changes, but we Santos et al. 2009). This hypothetical function did not find pyknotic nuclei, apoptotic bod- of the intramandibular gland is supported by the ies, or acid phosphatase in the cytoplasm, large size of these cells in forager workers, who which were signs of advanced degenerative collect resins and other resources used in the processes in (Armbruster et al. 1986; nests of stingless bees (Michener 1974). How- Dimitriadis and Kastritsis 1985; Skelton and ever, queens do not participate in any activities, Bowen 1987; Jimenez and Gilliam 1990; Gregorc other than reproduction. We have found lipid and Bowen 1997, 1998; Teixeira et al. 2013). droplets stored in the cells of the Despite the different morphology, the secre- intramandibular glands associated with a tory cells in both ages of the virgin queens show smooth endoplasmic reticulum, suggesting that a similar cellular area. This suggests that similar the class III intramandibular glands play a role amounts of compounds are produced, as the in lipid metabolism. Whether the secretions of area of the glandular cells is associated with the these glands play a role in lubrication of the amount of secretion (Peters et al. 2010). These mouthparts or chemical communication remains cells are thought to be responsible for producing an open question. Chemical signals in social lubricating compounds for the mandibles, and insects, such as cuticular hydrocarbons, are lipid other substances related to the nest building in nature, synthesized from the lipid stores in tasks performed by the workers (Nedel 1960; the glandular cells as well as in the complex 30 D. Elias-Santos et al.

Figure 5. Transmission electron micrographs of Micrograph showing mitochondria (m) and smooth intramandibular and mandibular gland cells of virgin endoplasmic reticulum (ser) in intramandibular gland queens 7 days old of Melipona scutellaris. a Micrograph cell of class I. cu cuticle. c Micrograph of the mandibular showing apical projections of the plasma membrane gland cell showing mitochondria (m), smooth endoplas- forming an extensive labyrinth in the subcuticular space mic reticulum (ser), and intracellular canaliculi (ic). (arrows) in intramandibular gland cell of class I. b

exocrine glands (Peeters et al. 1999; Sledge et secretion (Romani et al. 2003, 2005; Marques- al. 2001; Mant et al. 2005; Vasquez et al. 2008; Silva et al. 2006). Hora et al. 2010). Our findings show that the intramandibular The hypertrophied, epithelial, secretory gland glands of classes I and III have ultrastructural (class I) of the mandible of M. scutellaris virgin features of cells producing lipid compounds and queens is rich in smooth endoplasmic reticulum. the class III cells have a similar size in the The occurrence of this hypertrophied epitheli- virgin queens and nurse workers of M. um, with a well-developed, smooth, endoplas- scutellaris. mic reticulum in virgin queens indicates the production of lipid compounds. However, in the 4.2. Mandibular gland stingless bees Plebeia emerina (Santos et al. 2009), Melipona rufiventris,andM. The mandibular glands of virgin queens of quadrifasciata anthidioides (Costa-Leonardo M. scutellaris, of different ages, showed secre- 1978), only the workers have a hypertrophied tory cells of similar sizes. This suggests that the intramandibular epithelium, suggesting its pos- mandibular glands of virgin queens maintain the sible role in the manipulation of the adhesive same secretory activity at both ages. In the resins and propolis in these insects (Santos et al. virgin queens of Scaptotrigona postica,the 2009). mandibular gland secretion is used as a form In the basal portion of the cells of the of communication with the males during the hypertrophied mandibular epithelium of virgin mating flight (Engels et al. 1997; Gracioli-Vitti queens, the plasma membrane has infoldings et al. 2004a). associated with mitochondria, whereas the cell The ultrastructure of the secretory cells of the apices have projections resulting in a labyrinth mandibular glands of newly emerged and 7- in the subcuticular space. Moreover, histo- day-old M. scutellaris virgin queens is quite chemical analysis shows PAS-positive areas, similar. These cells have spherical nuclei with suggesting storage of glycogen, a molecule decondensed chromatin and prominent nucleoli, that may supply energy. This suggests that the and a cytoplasm with abundant smooth endo- epithelium participates in active transport of plasmic reticulum and mitochondria. These substances between the hemolymph and body features indicate that the secretory cells of the surface. Transport of substances from the mandibular glands of the M. scutellaris virgin hemolymph to the body surface by the queens synthesize compounds that are lipid in hypertrophied epithelium has been suggested nature. In the social insects, like honeybees to occur in ants (Serrão et al. 2009;Horaetal. (Michener 1974), stingless bees (Gracioli-Vitti 2010). et al. 2004b), bumblebees (Cahliková et al. The simultaneous occurrence of 2004), and the ant Polyergus rufescens (Grasso intramandibular glands of classes I and III in et al. 2003), the secretions from the mandibular queens suggests that these two gland classes act glands of queens are pheromones, which are independently in this caste. When different hydrocarbons and esters synthesized from lipids classes of glands are present together in one (Blomquist and Howard, 2003). This contrasts place and do not have a common reservoir, their with the findings for Melipona bicolor, in which secretions may differ in volatility and time of the secretory cells of the mandibular glands of Head glands in virgin queens of bees 31 32 D. Elias-Santos et al. virgin queens show a rough endoplasmic retic- (CNPq) and Minas Gerais State Research Agency ulum and Golgi complexes (Gracioli et al. (FAPEMIG). Authors are grateful to Nucleus of 2004), which are features of cells producing Microscopy and Microanalysis (UFV) for technical proteins other than lipids. assistance. The secretory cells from the mandibular glands of the forager and nurse have a similar Morphologie des glandes mandibulaires et size and the forager worker has smaller man- intramandibulaires chez les ouvrières et les reines dibular gland cells than the virgin queen, vierges de Melipona scutellaris suggesting a decrease in compound production in forager bees. In stingless bees, the mandib- Glandes céphaliques / stingless bee / communica- ular glands of workers are involved in the tion / comportement production of alarm and defense compounds (Smith and Roubik 1983; Hrncir et al. 2004; Cruz-Lopez et al. 2005, 2007; Jarau et al 2006; Morphologie der Mandibeldrüsen und der Schorkopf et al. 2007; 2009; van Zweden et al. Intramandibeldrüsen bei Arbeiterinnen und 2011). jungfräulichen Königinnen von Melipona Similar to those found in intramandibular scutellaris glands, the mandibular ones have the same developmental degree in virgin queens and Kopfdrüsen / Kommunikation / Verhalten / nurse workers, as also a possible role in lipid jungfräuliche Königin metabolism.

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