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The pharyngeal plate is a morphological complex with extensive anatomical variation among bees and, therefore, potential as a source of phylogenetic information. The pharyngeal plate of bees is divided into four morphologically distinct regions: sitophore, hypopharyngeal lobe, pharyngeal rods, and median oral plate. In this work we illustrate and document in detail for the first time the pharyngeal plate of 43 bee species, providing descriptions of the morphological variation and contrasting these findings with representatives of apoid wasps (Crabronidae and Sphecidae). We evaluate and discuss the potential of this structure as a rich source of morphological information in the context of bee phylogeny and any research potentially impacted by comparative morphological data. The shape of the hypopharyngeal lobe is highly variable among suprageneric taxa of bees and can be readily employed to characterise taxa at various levels. We argue that the global patterns in the variation of the pharyngeal plate can provide information for phylogenetic inference within bees and constructed and coded 10 characters that encompass the most noticeable morphological differences discussed herein.

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Many insects have been associated with actinobacteria in protective symbiosis where antimicrobial metabolites inhibit host pathogens. However, the microbiota of neotropical insects such as the stingless-bee Tetragonisca angustula is poorly explored. T. angustula is a meliponid bee widely distributed in Latin America, its honey is traditionally exploited because of its ethno-pharmacological properties and its antimicrobial activity has been demonstrated. Also, the well-structured nest of this species allows exploration of the microbiota of its different components. Even though Streptomyces spp. have been cultured from stingless-bees, little is known about their role in this insect-microbe relationship. In this study, we examined the association between culturable actinobacteria and T. angustula, and evaluated the isolates' potential as antimicrobial producers. We isolated 51 actinobacteria from adult bees and different substrates of the hive of T. angustula (pollen and honey storage, garbage pellets and cerumen). We then performed a 16S rRNA phylogenetic analysis that clusters the bacteria to previously described lineages of host-associated Streptomyces. In addition, all the isolates were classified according to their antibacterial activity against human pathogens, measured by a growth inhibition test based on diffusion in agar. More than 50 % of our isolates exhibit antimicrobial activity, mainly to Gram-positive bacteria and fungi and only two against Gram-negative bacteria. Additionally, we obtained electron micrographs of adult bees with what appears to be patches of hyphae with Streptomyces-like cell morphology on their body surface. Our results suggest that T. angustula possibly uptakes and transfers actinobacteria from the environment, acting as vectors for these potentially beneficial organisms. This research provides new insights regarding the microbiota associated with T. angustula and justify future studies exploring the full diversity of the microbial community associated with the hive and the possible exchange of microbes with the crops they pollinate.

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The honeybee Apis mellifera has ecological and economic importance; however, it experiences a population decline, perhaps due to exposure to toxic compounds, which are excreted by Malpighian tubules. During metamorphosis of A. mellifera, the Malpighian tubules degenerate and are formed de novo. The objective of this work was to verify the cellular events of the Malpighian tubule renewal in the metamorphosis, which are the gradual steps of cell remodeling, determining different cell types and their roles in the excretory activity in A. mellifera. Immunofluorescence and ultrastructural analyses showed that the cells of the larval Malpighian tubules degenerate by apoptosis and autophagy, and the new Malpighian tubules are formed by cell proliferation. The ultrastructure of the cells in the Malpighian tubules suggest that cellular remodeling only occurs from dark-brown-eyed pupae, indicating the onset of excretion activity in pupal Malpighian tubules. In adult forager workers, two cell types occur in the Malpighian tubules, one with ultrastructural features (abundance of mitochondria, vacuoles, microvilli, and narrow basal labyrinth) for primary urine production and another cell type with dilated basal labyrinth, long microvilli, and absence of spherocrystals, which suggest a role in primary urine re-absorpotion. This study suggests that during the metamorphosis, Malpighian tubules are non-functional until the light-brown-eyed pupae, indicating that A. mellifera may be more vulnerable to toxic compounds at early pupal stages. In addition, cell ultrastructure suggests that the Malpighian tubules may be functional from dark-brown-eyed pupae and acquire greater complexity in the forager worker bee.

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Abstract The antennal sensilla are sensory organs formed by a group of neurons and accessory cells, which allow perception of environmental cues, which play a role as mechanoreceptors and chemoreceptors. This study describes the post-embryonic development of the antennal sensilla of the stingless Tetragonisca angustula (Hymenoptera: Meliponini) workers. The development of the antennal sensilla begins in the transition stage of the pre-pupae to white-eyed pupae. The sensilla are completely developed at the black-eyed pupae stage, but they are covered by the old cuticle. The sensilla are exposed to the environment only in newly emerged workers of T. angustula, but it is possible that environmental stimuli can be recognized due to the pores in the old cuticle.

Resumo As sensilas antenais são órgãos sensoriais formados por um conjunto de neurônios que captam estímulos ambientais e células acessórias, desempenhando as funções de mecanorreceptores e quimiorrecepterores. Este trabalho descreve o desenvolvimento pós-embrionário das sensilas antenais de operárias das abelhas sem ferrão Tetragonisca angustula (Hymenoptera: Meliponini). O desenvolvimento das sensilas tem início na transição de pré-pupa para pupa de olho branco e estão completamente desenvolvidas no estágio de pupa de olho preto, mas ainda estão cobertas pela cutícula velha. As sensilas estão completamente expostas em operárias recém-emergidas de T. angustula, mas é possível que estímulos ambientais sejam percebidos em estágios anteriores devido aos poros presentes na cutícula velha.

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The antennal sensilla are sensory organs formed by a group of neurons and accessory cells, which allow perception of environmental cues, which play a role as mechanoreceptors and chemoreceptors. This study describes the post-embryonic development of the antennal sensilla of the stingless Tetragonisca angustula (Hymenoptera: Meliponini) workers. The development of the antennal sensilla begins in the transition stage of the pre-pupae to white-eyed pupae. The sensilla are completely developed at the black-eyed pupae stage, but they are covered by the old cuticle. The sensilla are exposed to the environment only in newly emerged workers of T. angustula, but it is possible that environmental stimuli can be recognized due to the pores in the old cuticle.

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A new stingless bee genus, Plectoplebeia gen. nov., is proposed to accommodate a peculiar Neotropical species currently placed in Plebeia Schwarz, 1938. The brief, but convoluted taxonomic history of its type species, Trigona nigrifacies Friese, 1900, is documented, with Trigona (Plebeia ) intermedia Wille, 1960 being placed as its junior synonym. Plectoplebeia gen. nov. resembles an enlarged species of Plebeia , differing by its larger body size, wider distance between the clypeus and the eye, presence of a shallow depression on the frons above the antennal sockets, coarser punctures on the head and mesosoma, a shorter, convex scutellum, an elongate propodeum and metapostnotum, long wings, with a higher number of hamuli, and by an elongate metasoma, with an unusually long first tergum. The single included species, Plectoplebeia nigrifacies comb. nov., is known only from the Bolivian Yungas, an ecoregion extending from west-central Bolivia to southeastern Peru.(AU)

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A new stingless bee genus, Plectoplebeia gen. nov., is proposed to accommodate a peculiar Neotropical species currently placed in Plebeia Schwarz, 1938. The brief, but convoluted taxonomic history of its type species, Trigona nigrifacies Friese, 1900, is documented, with Trigona (Plebeia ) intermedia Wille, 1960 being placed as its junior synonym. Plectoplebeia gen. nov. resembles an enlarged species of Plebeia , differing by its larger body size, wider distance between the clypeus and the eye, presence of a shallow depression on the frons above the antennal sockets, coarser punctures on the head and mesosoma, a shorter, convex scutellum, an elongate propodeum and metapostnotum, long wings, with a higher number of hamuli, and by an elongate metasoma, with an unusually long first tergum. The single included species, Plectoplebeia nigrifacies comb. nov., is known only from the Bolivian Yungas, an ecoregion extending from west-central Bolivia to southeastern Peru.

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An undescribed microsporidium was detected and isolated from the South American bumble bee Bombus atratus collected in the Pampas region of Argentina. Infection intensity in workers averaged 8.2 × 10(7)spores/bee. The main site of infection was adipose tissue where hypertrophy of adipocytes resulted in cyst-like body formation. Mature spores were ovoid and monomorphic. They measured 4.00 µm × 2.37 µm (fresh) or 3.98 µm × 1.88 µm (fixed). All stages were diplokariotic and developed in direct contact with host cytoplasm. Isofilar polar filament was arranged in 16 coils in one or, posteriorly, two layers. Coiling angle was variable, between perpendicular and almost parallel to major spore axis. Late meronts and sporogonial stages were surrounded by vesicles of approximately 60 nm in diameter. Based on both new and already designed primers, a 1827 bp (SSUrRNA, ITS, LSUrRNA) sequence was obtained. Data analyses suggest that this microsporidium is a new species of the genus Tubulinosema. The name Tubulinosema pampeana sp. n. is proposed.

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BACKGROUND: Studies on stingless bees are scarce, and little is known about these insects, especially regarding the effects of contamination by neurotoxic insecticides, which can cause damage to important structures of the insect brain. This study evaluated the morphological changes in the intrinsic neurons of the protocerebral mushroom bodies (Kenyon cells) of the stingless bee Scaptotrigona postica after exposure to different doses of fipronil, using light microscopy and transmission electron microscopy. This region of the brain was selected for analysis because of its importance as a sensory integration centre. RESULTS: In both oral and topical treatments, Kenyon cells presented pyknotic profiles, suggesting cell death. Statistical analysis showed significant differences among doses and exposure times. Transmission electron microscopy revealed changes in the nucleus and cellular organelles. Depending on the dose, the characteristics observed suggested apoptotosis or necrosis. CONCLUSION: This study demonstrates the toxic effects of fipronil. An increase in the number of pyknotic profiles of Kenyon cells of mushroom bodies was observed even at the sublethal doses of 0.27 ng AI bee(-1) and 0.24 ng AI µL(-1) in the topical and oral treatments respectively. Also, differences in the number of pyknotic profiles were dose and time dependent.

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Bumblebees are widely distributed across the world and have great economic and ecological importance as pollinators in the forest as well as in agriculture. The insect midgut consists of three cell types, which play various important roles in digestion, absorption, and hormone production. The present study characterized the anterior and posterior midgut regions of the bumblebee, Bombus morio. The digestive, regenerative and endocrine cells in the midgut showed regional differences in their number, nuclear size, as well as the size of the striated border. Ultrastructurally, the digestive cells contained many mitochondria and long microvilli; however, in the anterior midgut region, these cells showed dilated basal labyrinths with a few openings for the hemocoel, whereas the labyrinths of the basal posterior region remained inverse characteristics. Thus, the characterization of the midgut of B. morio supported an ecto-endoperitrophic circulation, contributing to a better understanding of the digestive process in this bee.

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Bumblebees need to keep bodily homeostasis and for that have an efficient system of excretion formed by the Malpighian tubules, ileum, and rectum. We analyzed the excretory organs of Bombus morio, a bee without rectal pads. In addition, we analyzed the rectal epithelium of Melipona quadrifasciata anthidioides which has rectal pads. The Malpighian tubules exhibited two cell types and the ileum four types. However, comparative analysis of the rectum showed that only cells of the anterior region of the rectal epithelium of B. morio are structurally distinct. We suggest that cells of the Malpighian tubules of B. morio have an excretory feature and that cells of ileum have different functions, such as ion absorption and water, organic compound, and protein secretion. In addition, only the anterior region of the rectum of B. morio showed characteristic absorption. We suggest that Malpighian tubules participate in the excretion of solutes and that the ileum and rectal epithelium are responsible for homeostasis of water and solutes, compensating for the absence of rectal papillae. These results contribute to our understanding of the morphophysiology of the excretory organs of bees without rectal pads.

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Studies of sub-lethal effects of pesticide residues on stingless bees are scarce and morphological analysis of organs would add information to toxicological analysis in order to clarify the continuous exposure of Scaptotrigona postica to insecticides. The aim of this study was to evaluate the morphology and histochemistry of the Malpighian tubules (excretory organ) of S. postica exposed to fipronil or boric acid to detect cellular responses that indicate toxicity or adaptative mechanisms to stress induced by exposure of worker bees to low doses of these chemical compounds. Newly emerged bees were submitted to toxicological bioassays and morphological analyses by optical microscopy and Transmission Electron Microscopy, as well as histochemical methods, were performed to detect proteins and glycoconjugates. Additionally, immunohistochemical detection of DNA fragmentation and HSP70 (70-kDa Heat shock protein) were performed to detect cell death and stress response, respectively. Statistical analysis, for the bioassays conducted with ingestion of contaminated diet with boric acid at 0.75% (w/w) or with fipronil at 0.1µg/kg of food, showed that the survival of bees that ingested the contaminated diets were significantly different to the survival rate presented by the control group (P<0.0001). Although some characteristics indicative of initiation of cell death were observed, the cells remained metabolically active in the processes of excretion and inactivation of chemical compounds. The data from this study reinforce the importance of research on sublethal effects of low doses of pesticides on bees in an attempt to assess a possible realistic dose and evaluate the risk assessment of stingless bee S. postica foraging in the vicinity of cultivated fields and/or in green urban areas.

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The fat body (FB) consists of two types of cells: throphocytes and oenocytes. Throphocytes are related to intermediary metabolism storing lipids, carbohydrates, and proteins while oenocytes play role in the lipids and lipoproteins production. The vitellogenin is the precursor of egg yolk (vitelline) and is synthesized on FB. The aim of this work was to analyze the effects of hormones acting in bee reproduction, as juvenile hormone (JH) and ecdisteroids (20 HE) on FB cells, where vitellogenin is synthesized. For the study were chose nurse workers that in Melipona quadrifasciata anthidioides present activated ovaries and produce eggs, and virgin queens whose ovaries are not yet activated, presenting only previtellogenic follicles. FB trophocytes from these classes of bees were cultivated in media containing different amounts of JH and 20-HE. The effects on trophocytes cytoplasm reserves of lipids, proteins, and activity of acid phosphatase were compared by observing preparations from cultured FB, treated and control, by transmission electron microscopy (TEM). The results showed that the hormones effects are related to the bee's caste and functional ovary stage. The role of acid phosphatase on mobilization of the trophocyte reserves was also determined.

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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.

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Ovaries of newly emerged virgin queens, aged 1, 3, 5, and 10 days, were submitted to an atmosphere of CO2 for 1 min, and studied by transmission electron microscopy. Newly emerged control and experimental queens were kept caged during 15 days with six nurse workers (changed every 2 days) and fed with sugar candy and water ad libitum. For the ultrastructural studies, the ovaries were collected when the queens were 3, 5, 10, and 15 days old. The effect of the treatment on the ovary was mainly evaluated by cell death incidence and acid phosphatase detection. Neither the confinement nor CO2 treatment interfered with the pattern of queen ovarian development for up to 5 days; that is, no differences were observed in ovarian ultrastructure or acid phosphatase activity between control and treated queens. Beyond the age of 10 days, treated queens showed a lower rate of cell death than controls, but the positivity to acid phosphatase reaction remained similar in both. The protective effect of CO2 on cells, however, did not persist, and 15-day-old treated queens showed no differences compared to the controls in the rates of cell death or enzyme reactivity. In conclusion, treatment with CO2 did not significantly change the ultrastructure of ovarian cells, or acid phosphatase activity in them, during the caging time of the queens. From an applied perspective, the short-term imprisonment of newly emerged queens outside of the colony, as is done by beekeepers, does not seem to affect their performance in terms of future fertility, although the narcosis, as applied in this study, may negatively affect their long-term performance.

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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.

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Morphology of spermatozoa in bees has provided promising results for phylogenetic analyses. In this work, the structure and ultrastructure of spermatozoa from Thygater (Thygater) analis and Melitoma segmentaria were characterized and the synapomorphies shared in the family Apidae are discussed. In these species, spermatozoa bundles which are undone in the seminal vesicle possess, on average, 50 cells. Spermatozoa consist of a head and a flagellar region. The head includes an acrosome containing the perforatorium, covered by the acrosomal vesicle and a nucleus. The flagellum is formed by two mitochondrial derivatives, which are asymmetric in diameter and length, with one centriolar adjunct, one axoneme (9 + 9 + 2), and two accessory bodies. In cross section the centriolar adjunct is asymmetric and the accessory bodies are triangular in shape. In the distal region of the flagellum, the derivative terminates before the axoneme and the small derivative terminates first. The axoneme is gradually disorganized and the accessories microtubules are the last to terminate. In these two species, spermatozoa share diverse synapomorphies with those of other bee species previously described in the literature, which allows for the establishment of a morphological pattern for spermatozoa of the family Apidae.

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The intramandibular glands of workers and queens of Melipona quadrifasciata Lepeletier (Hymenoptera: Apidae), at different ages and from different functional groups, were studied using light and transmission electron microscopy. The results demonstrated that these glands are composed of two types of secretory structures: 1.A hypertrophied epidermis on the dorsal side of the mandible that is an epithelial gland. 2. Free secretory cells filling the inner spaces of the appendices that constitute a unicellular gland. The epithelial gland is larger in the young (1-2-day-old workers), and the gland becomes involuted during the nurse worker stage. The unicellular glands of the workers posses some secretion during all of the studied phases, but secretory activity is more intensive in the foraging workers. Vesicles of secretion are absent in the unicellular glands of queens. These results demonstrate that these glands show functional adaptations in different castes corresponding to the functions of each caste.

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The present paper shows through conventional light microscopy, silver nitrate impregnation and Transmission Electron Microscopy, the changes in nuclear activity that occurs in the ovary cells during vitellogenesis of Apis mellifera (Linnaeus, 1758). The material impregnated by silver nitrate was detected in nurse cells, oocyte germinal vesicle and follicular cells, whose amounts are greater in phases corresponding to ovarian cells more intense activity. The silver impregnation permitted to demonstrate that the oocyte germinal vesicle synthezises ribonucleoproteins (RNP) during the beginning of the vitellogenesis. The ultrastructure show nucleolar-like material corresponding to nuclear sites impregnated by silver.

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The present paper shows through conventional light microscopy, silver nitrate impregnation and Transmission Electron Microscopy, the changes in nuclear activity that occurs in the ovary cells during vitellogenesis of Apis mellifera (Linnaeus, 1758). The material impregnated by silver nitrate was detected in nurse cells, oocyte germinal vesicle and follicular cells, whose amounts are greater in phases corresponding to ovarian cells more intense activity. The silver impregnation permitted to demonstrate that the oocyte germinal vesicle synthezises ribonucleoproteins (RNP) during the beginning of the vitellogenesis. The ultrastructure show nucleolar-like material corresponding to nuclear sites impregnated by silver.

Este estudo mostra, por meio de microscopia convencional, impregnação por nitrato de prata e microscopia eletrônica de transmissão, as mudanças que ocorrem na atividade nuclear de células do ovário durante a vitelogênese de Apis mellifera (Linnaeus, 1758). Material impregnado por nitrato de prata foi detectado no núcleo de células nutridoras, na vesícula germinal do ovócito e no núcleo das células foliculares. As maiores quantidades de material impregnado pela prata foram observadas durante as fases de maior atividade dessas estruturas. A impregnação por prata permitiu demonstrar que a vesícula germinal sintetiza ribonucleoproteínas (RNP) durante o início da vitelogênese. A ultraestrutura mostrou que o material impregnado apresenta características morfológicas de nucléolos.

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