The venom gland of queens of Apis mellifera (Hymenoptera, Apidae): morphology and secretory cycle

The venom gland of queens of Apis mellifera was examined through light and transmission electron microscopy and subjected to electrophoretic analyses. Virgin queens exhibited prismatic secretory cells containing large amounts of rough endoplasmic reticulum with dilated cisternae, open secretory spaces, numerous vacuoles and granules scattered in the cytoplasm, and spherical nuclei with numerous nucleoli. The secretion produced was non-refringent under polarized light and the electrophoretic analysis of glandular extracts revealed five main protein bands. In mated queens, the venom gland exhibited a high degree of degeneration. Its secretion was refringent under polarized light and one of the main bands was absent in the electrophoretic pattern obtained. The morphological aspects observed are in agreement with the function of this gland in queens, given that virgin queens use venom in battles for the dominance of the colony, a situation that occurs as soon as they emerge, while fertilized queens rarely use venom.     

Temporal and morphological differences in post-embryonic differentiation of the mushroom bodies in the brain of workers, queens, and drones of Apis mellifera (Hymenoptera, Apidae)

The mushroom bodies are structures present in the insect brain described as centers for the neural basis of learning, memory, and other higher functions. Honeybees (Apis mellifera) are insects with a sophisticated system of spatial orientation and possess well-developed learning and memory capabilities, which are associated with neural and brain structures. Thus, the present study aimed to compare the mushroom bodies during post-embryonic development and in newly emerged males, workers, and queens using light and transmission electron microscopy to examine how differential morphological characteristics are established during development. Measurements of structures were also taken in several post-embryonic developmental phases in order to evaluate size differences during the process and in the adult organs. The results show that workers, queens, and males exhibit temporal and size differences during the post-embryonic development of mushroom bodies, probably as adaptations to differences in behavior complexity. The mushroom bodies of workers are precociously formed and are larger than those of queens and drones. Thus, workers have the largest mushroom bodies resulting from differential development during metamorphosis.     

Effect of mating delay on the ovary of Melipona quadrifasciata anthidioides (Hymenoptera: Apidae) queens

The effect of mating delay on the ovary structure of virgin queens of Melipona quadrifasciata anthidioides (Hymenoptera: Apidae) was investigated. Virgin queens 15, 20, 25 and 30 days old were dissected to analyze their degree of ovary degeneration. To verify whether the degeneration would cause permanent sterility, virgin queens of the same ages (15, 20, 25 and 30 days) were mated and accompanied for at least 14 days to verify whether there was physogastry and then dissected. The ovaries were analyzed by histology, histochemical tests and TUNEL to verify programmed cell death. The results showed that mating delay interrupted oogenesis preventing vitellogenesis. Mating delay results in ovary degeneration which increased with queen age. However, even when there was ovariole degeneration, 25-day-old virgin queens after mating presented normal ovariole activation.     

FMRFamide-like midgut endocrine cells during the metamorphosis in Melipona quadrifasciata anthidioides (Hymenoptera, Apidae)

The FMRFamide, gastrin and cholecystokinin (CCK) occurrence in endocrine cells of insects has been described by several authors, although their functions are still not well defined for this group of animals. In the present study, the occurrence of endocrine cells producing FMRFamide, gastrin 1 and CCK-8 in the midgut (ventriculus) of Melipona quadrifasciata anthidioides (Hymenoptera, Apidae), before, during and after the metamorphosis, were investigated by means of pre-embedding immunofluorescence techniques. FMRFamide reactivity was found in the endocrine cells as well as in the nervous fibers and neurons of the intestine of these bees. 'Open' and 'closed' types of FMRFamide-like cells were observed in last instar larvae. In the black eyed pupae the producing cells of FMRFamide seemed to be immature, and, in the workers, where the FMRFamide producing cells were more abundant, the production of this substance seemed to occur only in the open cells. Reactivity of the nervous fibers and neurons were observed, during the prepupae, white eyed pupae, and pink eyed pupae. The same did not occur with the midgut endocrine cells. There were no immunoreactivity observations for gastrin 1 and for CCK-8. The FMRFamide-like cells were present in the midgut of these insects during or close to the period that they were eating, which indicates that the FMRFamide may be involved in the control of the digestive process.     

Alterations induced by the juvenile hormone in glandular cells of the Apis mellifera venom gland: applications on newly emerged workers (Hymenoptera, Apidae)

Histological and histochemical analyses were carried out in order to evaluate the influence of the topical application of a synthetic juvenile hormone on the secretory cycle and degeneration of the venom gland of Apis mellifera. Newly emerged workers received the topical application of synthetic hormone and the results were compared to the normal development of the secretory cycle in virgin and mated queens. The first worker group received the juvenile hormone diluted in hexane (2 microg/microL), the second received only 1 microL of hexane, and the third did not receive any kind of application. After the application the workers were returned to the colony and collected at the ages of 14 and 25 days of adult life. The groups with virgin queens and the other with mated queens, did not receive the treatment. The results show that the individuals treated with juvenile hormone and with pure hexane presented differences in the histological and cytochemical aspects of the secretory cells of the venom gland. The data indicate that both the juvenile hormone and hexane accelerate the activity of the secretory cycle and the degeneration of the venom gland; however, the juvenile hormone proved to be more effective than hexane.     

A comparative study of the proventricular structure in corbiculate apinae (Hymenoptera, Apidae)

The present study compares the proventricular structure, analyzed under scanning electronic microscope (SEM), among tribes of corbiculate Apinae. Fifty-one species of stingless bees (Meliponini), one species of honeybee (Apini), three species of bumblebees (Bombini) and seven species of orchid bees (Euglossini), were analyzed as in-group, and one species of sphecid wasp (Sphecidae) and two species of Halictidae bees, as out-groups. The proventricular bulb presents a basic morphology pattern similar to that of other Hymenoptera such as ants and wasps, being a symplesiomorphy for bees. The shape of proventricular folds constitutes a synapomorphy for Meliponini and an autapomorphy for Apini. The shape of hair-like projections of the cuticle that lines the proventriculus is a synapomorphy for Meliponini and Apini. These proventricular data corroborate the monophyly of the tribe Meliponini and the hypothesis that recognizes only one tribe for stingless bees. In addition, Meliponini+Apini constitutes a monophyletic group and Bombini+Euglossini another monophyletic group. The results confirm that internal morphology is a character that can be used in studies of the phylogeny in insects and the use of SEM as a powerful tool in these analyses.     

Ferritin in iron containing granules from the fat body of the honeybees Apis mellifera and Scaptotrigona postica

It is already known that the behaviour of the honeybee Apis mellifera is influenced by the Earth's magnetic field. Recently it has been proposed that iron-rich granules found inside the fat body cells of this honeybee had small magnetite crystals that were responsible for this behaviour. In the present work, we studied the iron containing granules from queens of two species of honeybees (A. mellifera and Scaptotrigona postica) by electron microscopy methods in order to clarify this point. The granules were found inside rough endoplasmic reticulum cisternae. Energy dispersive X-ray analysis of granules from A. mellifera showed the presence of iron, phosphorus and calcium. The same analysis performed on the granules of S. postica also indicated the presence of these elements along with the additional element magnesium. The granules of A. mellifera were composed of apoferritin-like particles in the periphery while in the core, clusters of organised particles resembling holoferritin were seen. The larger and more mineralised granules of S. postica presented structures resembling ferritin cores in the periphery, and smaller electron dense particles inside the bulk. Electron spectroscopic images of the granules from A. mellifera showed that iron, oxygen and phosphorus were co-localised in the ferritin-like deposits. These results indicate that the iron-rich granules of these honeybees are formed by accumulation of ferritin and its degraded forms together with elements present inside the rough endoplasmic reticulum, such as phosphorus, calcium and magnesium. It is suggested that the high level of phosphate in the milieu would prevent the crystallisation of iron oxides in these structures, making very unlikely their participation in magnetoreception mechanisms. They are most probably involved in iron homeostasis.     

Biochemical and cytochemical studies of the enzymatic activity of the venom glands of workers of honey bee Apis mellifera L. (Hymenoptera,Apidae)

Biochemical studies revealed that the activity of some hydrolytic enzymes from the venom glands of honey bee Apis mellifera was higher in workers of 14 days of age than in those of 40 days. Among these enzymes, the highest activity was recorded for acid phosphatase, which was cytochemically detected throughout the length of the secretory filament and surrounding the canaliculi of the distal region of the reservoir. The acid phosphatase was considered to be a typical secretion product, since it was present in the cytoplasm as well as in the canaliculi of the secretory cells.     

Morphology of the male reproductive system and spermatozoa in Centris Fabricius, 1804 (Hymenoptera: Apidae, Centridini)

The genera Centris and Epicharis constitute the Centridini and are widely distributed in the Neotropical region. Centris is also found in the southern portion of the Neartic region, although both genera are more abundant in the humid tropical regions. To describe the structure of the male reproductive system and spermatozoa, light and transmission electron microscopy were used. The male reproductive system of Centris sp. is formed by a pair of testes, a pair of deferent ducts, a pair of seminal vesicles, a pair of accessory glands and an ejaculatory duct connected to the external genitalia, the aedeagus. In this species, testes and the pre-vesicular deferens ducts as well as the seminal vesicles are encapsulated in a single conjunctive capsule, the scrotal membrane. Each testis consists of four testicular follicles, made up of cysts with up to 64 germinative cells. Histologically, the seminal vesicles are formed by a simple cylindrical epithelium, basal membrane and muscular tunic. The spermatozoa of Centris analis, C. fuscata, C. tarsata and Centris sp. are morphologically similar. They have two easily distinguishable regions: the head and flagellum. The head is formed by the two-layer acrosome, the linear nucleus and the flagellum, the centriole adjunct, the axoneme of pattern 9+9+2 microtubules, two asymmetric mitochondrial derivatives and two accessory bodies. These Centris species share various morphological characteristics of the male reproductive system and spermatozoa with the other bees previously described, indicating that several characteristics are synapomorphic for the family Apidae. Studies on the morphology of the male reproductive system and spermatozoa in Hymenoptera have demonstrated the diversity of the information provided by these reproductive structures, which can be used in taxonomy studies and the phylogeny of this important group of insects.     

Storaged products and presence of acid phosphatase in fat body cells at pre-pupal worker stage of Apis mellifera Linnaeus, 1758 (Hymenoptera, Apidae)

Fat body cells or throphocytes of individuals during beginning of pupation (pre-pupae) of Apis mellifera were collected and studied by routine and cytochemical preparations for transmission electron microscopy (TEM). The results showed that the trophocytes present large reserves of lipids, proteins, and glycogen. Imidazole osmium treatment revealed that lipids are deposited as droplets in the cytoplasm and also within protein granules. Thiery's reaction showed the presence of glycogen inside protein granules. An acid phosphatase reaction was performed to verify the role of this enzyme in the mobilization of stored reserves during metamorphosis. Positive reaction for acid phosphatase was detected at larger protein granules, at the periphery of the large lipid droplets, and free in the cytoplasm. The contents of protein, lipid and glycogen are stored in the trophocytes at larval phase to be used during metamorphosis. The acid phosphatase present in the products stored might be responsible for their metabolization, while acid phosphatase free in cytoplasm might actuates in the trophocytes histolysis that occurs during metamorphosis for energy production.     

Morphology of the male reproductive system and spermiogenesis in Hypanthidium foveolatum (Alfken, 1930) (Hymenoptera: Apidae: Megachilinae)

The morphological aspects of male reproductive tract, spermiogenesis and spermatozoa are typical for each species and reflect its evolution, establishing a unique source of characters, which has been used to help solve phylogenetic problems. In Hypanthidium foveolatum the reproductive tract is composed of the testes comprising 28 testicular tubules, deferent ducts, seminal vesicles, accessory glands and an ejaculatory duct. The differentiation of spermatids occurs within cysts of up to 128 germ line cells each one. During the early spermatid phase, the nucleus resembles that of somatic cells. There follows a gradual chromatin condensation with an increase in nuclear electron density. In the spermatozoon, the nucleus contains heterogeneous chromatin with a loose appearance. The acrosome, shaped with the active participation of the Golgi complex, shows an electron-dense perforatorium involved by four electron-lucent acrosomal vesicle projections. The sperm tail presents an axoneme with a 9 + 9 + 2 microtubule pattern and two mitochondrial derivatives, which appear with different sizes. A dense crystalloid is formed initially in the mitochondrial matrix of the large derivative. The mitochondrial derivatives’ differentiation occurs concomitantly with an axoneme outgrowth. The centriolar adjunct is observed near the axoneme, anterior to the smaller mithocondrial derivative and exhibits an approximately triangular shape in cross-sections. Microtubules were observed around the head region and flagellar components during spermiogenesis.     

Histology and histochemistry of the ventriculus of Dolichoderus (=Monacis) bispinosus (OLIVIER, 1792) (Hymenoptera: Formicidae)

In this study we histologically and histochemically describe the ventriculus of Dolichoderus bispinosus. The epithelium consists of two basic cell types, highly basophilic generative cells, and digestive cells. The latter present several cytoplasmic vesicles, rich in acidic and neutral polysaccharides, and basic proteins. Also, these cells exhibit an apocrine secretion pattern. A mass of fibrous material is observed on the surface of the epithelium. Finally, we discuss the results obtained.     

Circulating hemocytes from larvae of Melipona scutellaris (Hymenoptera,Apidae, Meliponini): Cell types and their role in phagocytosis

Infection in insects stimulates a complex defensive response. Recognition of pathogens may be accomplished by plasma or hemocyte proteins that bind specifically to bacterial or fungal polysaccharides. Several morphologically distinct hemocyte cell types cooperate in the immune response. Hemocytes attach to invading organisms and then isolate them by phagocytosis, by trapping them in hemocyte aggregates called nodules, or by forming an organized multicellular capsule around large parasites. In the current investigation the cellular in the hemolymph third instar larvae of M. scutellaris has been characterized by means of light microscopy analysis and phagocytosis assays were performed in vivo by injection of 0.5 μm fluorescence beads in order to identify the hemocyte types involved in phagocytosis. Four morphotypes of circulating hemocytes were found in 3rd instar larvae: prohemocytes, plasmatocytes, granulocytes and oenocytoids. The results presented plasmatocytes and granulocytes involved in phagocytic response of foreign particles in 3rd instar larvae of M. scutellaris.     

Ultrastructural aspects of the mandibular gland of Melipona bicolor Lepeletier, 1836 (Hymenoptera: Apidae, Meliponi) in the castes

The mandibular gland in Melipona bicolor workers and queens was studied by scanning and transmission electron microscopy. There is no difference in the gland anatomy between the castes, but the transmission electron microscopy showed variation of the cellular ultrastructure according to the secretory phase of the gland in both castes. Smooth endoplasmic reticulum was abundant in the secretory cells of physogastric queens, indicating that these cells produce lipid secretion that is stored in granules with multi-lamellar bodies. Mitochondrial variations during the cell secretory cycle indicates their participation in the lipid synthesis. After secretion, release in the reservoir lumen through the collecting canals, the secretory cells contain many myelinic bodies, indicative of cellular regression.     

Nuclear alterations associated to programmed cell death in larval salivary glands of Apis mellifera (Hymenoptera: Apidae)

The silk glands of bees are a good model for the study of cell death in insects. With the objective to detect the nuclear features during glandular regression stage, larvae at the last instar and pre-pupae were collected and their silk glands were dissected and processed for ultrastructural analysis and histologically for cytochemical and imunocytochemical analysis. The results showed that the cellular nuclei exhibited characteristics of death by atypical apoptosis as well as autophagic cell death. Among the apoptosis characteristic were: nuclear strangulation with bleb formation in some nuclei, DNA fragmentation in most of the nuclei and nucleolar fragmentation. Centripetal chromatin compaction was observed in many nuclei, forming a perichromatin halo differing from typical apoptotic nuclei. With regards to the characteristics of autophagic-programmed cell death, most relevant was the delay in the collapse of many nuclei.     

Morphology and ultrastructure of the venom apparatus in the endoparasitic wasp Pteromalus puparum (Hymenoptera: Pteromalidae)

The venom apparatus of the endoparasitic wasp Pteromalus puparum (Hymenoptera: Pteromalidae) was studied with light and electron microscope and was subjected to the electrophoretic and immunohistochemical analyses. Typically its venom apparatus consists of an unbranched venom gland and a venom reservoir, which is associated with a Dufour gland. The venom gland is lined by a series of secretory units. Each secretory unit comprises a secretory cell and a duct cell. The secretory cell is associated with an end apparatus to collect its secretions into the gland lumen. Secretory cells in the venom gland are characterized by extensive rough endoplasmic reticulum and numerous electron-dense vesicles in the distal and middle parts. They also exhibit several secretory granules and vacuoles. The venom reservoir presents three distinct regions: an external layer, composed by numerous fine muscle fibers; an internal layer, represented by epithelial cell with large nucleus; and an intima portion, represented by thin and uniform organization. The morphological aspect of numerous well-developed organelles responsible for protein generation observed is in agreement with the electrophoretic and immunohistochemical results which reveal that the rich proteinaceous components are present in the venom gland and venom reservoir. The venom proteins are first mainly produced in the secretory unit of venom gland, then drained to the lumen through the end apparatus, and are finally collected and stored in the venom reservoir.     

Antennal morphology and sensilla ultrastructure of the web-spinning sawfly Acantholyda posticalis Matsumura (Hymenoptera: Pamphiliidae)

Acantholyda posticalis (Hymenoptera: Pamphiliidae) is an important pine pest with a world-wide distribution. To clarify the olfactory receptive mechanism of A. posticalis, scanning electron microscopy and transmission electron microscopy were used to examine the morphology, ultrastructure, and distribution of antennal sensilla of adults from two sites in China. The antennae were filiform, and the flagella comprised 32–35 flagellomeres. Six sensillum types were found. Sensilla chaetica were straight setae with sharply pointed tips and without dendrites in the lumen. Sensilla trichodea were characterized by a parallel-grooved wall and one terminal pore and were innervated by four dendrites at the base. Sensilla basiconica I possessed longitudinally grooved surfaces and multiple terminal pores, with five dendrites in the lumen. Sensilla basiconica II not only had a distinct terminal pore but also had numerous tiny wall pores and many dendritic branches within the sensillum lymph. Sensilla coeloconica had deep longitudinal grooves, one terminal pore and six dendrites, while sensilla campaniformia were thick-walled with a terminal opening and sensory nerve bundles in the lumen. Sensilla chaetica and s. trichodea were most abundant and distributed over the entire antennae, while s. basiconica I and II, s. coeloconica, and s. campaniformia were restricted to the ventral flagellar surfaces. Although the shape and structure of antennae were similar in males and females, females had significantly longer antennae than males, and males had significantly more s. basiconica I than females. We compared the morphology and structure of these sensilla to other Hymenoptera and discussed their possible functions.     

Differences in mushroom bodies morphogenesis in workers,queens and drones of Apis mellifera: Neuroblasts proliferation and death

Apis mellifera is an interesting model to neurobiological studies. It has a relatively small brain that commands the complex learning and memory tasks demanded by the social organization. An A. mellifera colony is made up of a queen, thousands of workers and a varying number of drones. The latter are males, whereas the former are the two female castes. These three phenotypes differ in morphology, physiology and behavior, correlated with their respective functions in the society. Such differences include the morphology and architecture of their brains. To understand the processes generating such polymorphic brains we characterized the cell division and cell death dynamics which underlie the morphogenesis of the mushroom bodies, through several methods suitable for evidence the time and place of occurrence. Cell death was detected in mushroom bodies of last larval instar and mainly in black-eyed pupae. Cell division was observed in mushroom bodies, primarily at the start of metamorphosis, exhibiting temporal differences among workers, queens and males.     

Structure and ultrastructure of the spermatozoa of Trypoxylon (Trypargilum) albitarse Fabricius 1804 (Hymenoptera: Apoidea: Crabronidae)

The ultrastructure of the spermatozoa of Trypoxylon (Trypargilum) albitarse is described here for the first time within this genus. Testes and seminal vesicles were dissected and processed for transmission electron microscopy. In the testicular follicles, the spermatids are arranged in a maximum number of 32 for each cyst. The spermatozoa are slender and they measure approximately 150 μm in length. The head is about 17 μm long and is formed by the acrosome and the nucleus. The flagellum consists in an axoneme, two mitochondrial derivatives, two accessory bodies and, at the nucleus–flagellum transition, a symmetric centriolar adjunct. The axoneme presents the typical 9 + 9 + 2 microtubule pattern. In the terminal region, the central microtubules and nine doublets finish first, followed by the accessory microtubules. Both mitochondrial derivatives begin together and are inserted in the base of the centriolar adjunct. Along the middle region, the larger derivative has almost twice the area of the smaller one and includes a discrete paracrystalline region. At the tip, the smaller derivative ends before the larger one and both before the axoneme. The characters derived from the ultrastructure of the spermatozoa of T. albitarse show synapomorphies shared with the Apoidea and present characters that are probably apomorphic for the Crabroninae subfamily.     

Distribution and morphometric studies of flagellar sensilla in Emphorini bees (Hymenoptera, Apoidea)

The tribe Emphorini is a group of pollen-collecting solitary bees with a geographical distribution restricted to the western hemisphere. Most of the Emphorini bees collect Page 10 linepollen from a few specific plant families and display specialized behaviors for constructing their nests. Insect sensilla are the basic structural and functional units of cuticle receptors, serving mainly mechano- and chemo-receptor functions. The external morphology of the antennal sensilla has been well characterized in species of different families of Apoidea, however there is scarce information about this issue in solitary bees of the family Apidae. For a better understanding of the association between the external sensory system and several types of behaviors which emerged along the evolutionary history of bees, it is important to characterize the antennal receptors in several representative species of this tribe. The distribution of the antennal sensilla on the dorsal flagella of 18 taxa was studied in insects of both sexes, using light and scanning electron microscopy. There were six types of sensilla and setae on the antennae, which were identified as sensilla placodea, trichodea, basiconica, coeloconica, coelocapitular and ampullacea. The sensilla trichodea were classified into subtypes, A, B, C-D. Sensilla subtype A were the most abundant sensilla and were distributed over the entire antennae, while sensilla placodea and sensilla trichodea type B, showed a restricted distribution on specific areas of the flagella. We have recognized four patterns of spatial distribution of setae on dorsal flagella. Species having setae on the distal part of the flagellomeres tended to contain a low density of sensilla trichodea type A. Females showed a higher number of sensilla subtypes B and C-D than males; instead sensilla trichodea A were more abundant in males. No significant difference was found in the number of sensilla placodea, ampullacea, coeloconica and coelocapitular. Sensilla basiconica were found only in females. Our results showed that gustative and tactile sensilla were more abundant in female bees, as well as, olfactory receptors predominate in the antennal system of males. The possible coevolution of flagellar sensilla in males and females of solitary bees is discussed in light of previous reports. Patterns of distribution of setae determine the relative abundance of the types of sensilla in the flagellum.