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Conselho Nacional de Desenvolvimento Científico e Tecnológico The eusocial stingless bee Melipona quadrifasciata anthidioides belongs to the Meliponini, a tribe that comprises important pollinators of tropical and subtropical plants. Within a typical eusocial colony there are drones and females that include different casts, i.e. workers and queen(s). In this species, the caste differentiation can be influenced by the Juvenile Hormone (JH). When JH is topically applied in last instar male larvae, they originate individuals with queen s phenotype in which the male-like reproductive system persists. Take into account that different individuals play different roles in the colony, it is reasonable to assume that they can be distinguished by their sensorial organs. Sensilla are basic integumental unit that are important for the insect thermo, mechanical, higro, CO2 and chemical perceptions. In addition, these sensory structures may be distinguished by their morphologies and functions. The objective of this study was to compare the antennal sensilla of individuals of different sexes and castes, including queen-like males of M. quadrifasciata anthidioides, showing for the first time details of their antennal topographies. Images of antenna dorsal surfaces were taken with the scanning electron microscope. Different types of antennal sensilla can be classified as follows: trichoid (ST, subtypes I to VII), placoid, basiconic, coeloconic and ampullacea. The the ST I and placoid sensilla were the most abundant sensillum types found in all individuals, even more in males. On the other hand, basiconic sensilla were only found in females, i.e. in workers and queens. The ST II is the third most abundant type of sensilla found in M. quadrifasciata anthidioides and the ST III and IV occurred only in the first flagellomere, being the first one bigger in females in comparison to males and the second one capable of separating the castes according to their types and length, though their numbers are not different. Moreover, the ST V and VI and the ampullacea types occurred only at the antenna s tip, with ST V being lower in number and smaller. ST VI is also larger in queens, separating them from other individuals, and greater in females. On the other hand, the other sensillae were seen along all antennas surface. The chaetic sensillum had apical pore and this pore was only found in females. Despite queen-like males had queen s phenotype, according to the Morisita similarity index, they resembled more the males, especially 2n ones, regarding the antenna phenotype and the composition of their sensilla. Generally speaking, irrespective of gender and cast, individuals of the stingless bee M. quadrifasciata anthidioides have similar sensillum types. However, they differed significantly in respect to the sensilla number and length and these differences were discussed here according to the gender and cast of this species. A abelha sem ferrão Melipona quadrifasciata anthidioides é conhecida popularmente como mandaçaia e pertence à tribo Meliponini que inclui importantes insetos eusociais polinizadores de espécies de plantas tropicais e subtropicais. Em um ninho típico de abelhas sociais há indivíduos de diferentes sexos e castas. Esses são compostos pelos zangões e pelas castas femininas que incluem as operárias e a(s) rainha(s). Entretanto, quando o hormônio juvenil (HJ) é aplicado topicamente em larvas de machos, eles desenvolvem fenótipo da rainha. Entretanto, esses rainhóides ainda retêm o sistema reprodutivo masculino. Considerando que os indivíduos de diferentes castas e sexos desempenham papéis diferentes no ninho, podemos afirmar que os seus órgãos sensoriais como, por exemplo, as sensilas antenais também o sejam. Essas estruturas sensoriais podem ser diferenciadas tanto pela sua morfologia quanto pela função que desempenham como, por exemplo, a quimio- e mecanorrecepção. O objetivo deste trabalho foi estudar comparativamente as sensilas antenais de indivíduos de diferentes sexos e castas, incluindo os machos 2n e rainhóides, de M. quadrifasciata anthidioides, através de imagens da superfície dorsal das antenas, que foram feitas ao microscópio eletrônico de varredura. As diferentes sensilas antenais foram identificadas como sendo: sensilas tricóides (ST, dos subtipos I ao VI), placóides, basicônicas, céticas, celocônicas e ampuláceas. As sensilas basicônicas possuem um poro apical e são exclusivas das fêmeas (rainhas e operárias). As ST I e as placóides são as mais abundantes em todos os indivíduos, sendo encontradas em maior quantidade nos machos. Já as sensilas basicônicas possuem um poro apical e são exclusivas das fêmeas (rainhas e operárias). As ST II são o terceiro tipo de sensilas mais abundante em M. quadrifasciata anthidioides e as ST III e IV ocorrem apenas no primeiro flagelômero, sendo o primeiro tipo de sensila maior nas fêmeas que nos machos e o segundo tipo capaz de separar as castas femininas quanto ao seu comprimento, mas não diferem à quantidade. Por outro lado, as ST V e VI e ampuláceas ocorrem apenas na extremidade do último flagelômero antenal, com ST V sendo menor nos machos de M. quadrifasciata anthidioides tanto em quantidade quanto em comprimento, mas sendo úteis ainda para a distinção das castas femininas. As ST VI são mais compridas nas rainhas, separando-as dos demais indivíduos, e maiores nas fêmeas. Apesar dos rainhóides se assemelharem às rainhas quanto à morfologia externa, uma análise utilizando o índice de similaridade de Morisita mostrou que eles se assemelham mais aos machos, principalmente os 2n, quanto às suas antenas e a composição das sensilas, colocando-os em um grupo separado das fêmeas. Embora de maneira geral os mesmos tipos de sensilas tenham sido encontrados nas diferentes classes de indivíduos de M. quadrifasciata anthidioides, diferenças significativas foram observadas no número e no comprimento dos morfotipos das mesmas. Essas diferenças foram discutidas de acordo com a função do sexo e das castas dessa espécie.

Declines in pollinator colonies represent a worldwide concern. The widespread use of agricultural pesticides is recognized as a potential cause of these declines. Previous studies have examined the effects of neonicotinoid insecticides such as imidacloprid on pollinator colonies, but these investigations have mainly focused on adult honey bees. Native stingless bees (Hymenoptera: Apidae: Meliponinae) are key pollinators in neotropical areas and are threatened with extinction due to deforestation and pesticide use. Few studies have directly investigated the effects of pesticides on these pollinators. Furthermore, the existing impact studies did not address the issue of larval ingestion of contaminated pollen and nectar, which could potentially have dire consequences for the colony. Here, we assessed the effects of imidacloprid ingestion by stingless bee larvae on their survival, development, neuromorphology and adult walking behavior. Increasing doses of imidacloprid were added to the diet provided to individual worker larvae of the stingless bee Melipona quadrifasciata anthidioides throughout their development. Survival rates above 50% were only observed at insecticide doses lower than 0.0056 µg active ingredient (a.i.)/bee. No sublethal effect on body mass or developmental time was observed in the surviving insects, but the pesticide treatment negatively affected the development of mushroom bodies in the brain and impaired the walking behavior of newly emerged adult workers. Therefore, stingless bee larvae are particularly susceptible to imidacloprid, as it caused both high mortality and sublethal effects that impaired brain development and compromised mobility at the young adult stage. These findings demonstrate the lethal effects of imidacloprid on native stingless bees and provide evidence of novel serious sublethal effects that may compromise colony survival. The ecological and economic importance of neotropical stingless bees as pollinators, their susceptibility to insecticides and the vulnerability of their larvae to insecticide exposure emphasize the importance of studying these species.

The fat body (FB) of insects is where yolk proteins are synthesized. Therefore, relationships between the FB and oogenesis were studied in nurse workers, virgins, and physogastric queens of Melipona quadrifasciata anthidioides, a stingless bee in which the workers produce and lay eggs while provisioning brood cells. The relationships between FB and oogenesis, as well as the routes of materials from hemolymph to the oocytes, were studied through the cytochemical detection of lipids by osmium imidazole (OI), carbohydrates by ruthenium red (RR) and basic proteins by ammoniacal silver (AS). The results show differences in the presence of the studied materials in FB trophocytes and ovary of the classes of females studied and oogenesis phases. Material that tested positive for the treatments was detected among the classes of individuals studied in both, trophocytes and oocytes, and in the route of those materials from hemolymph to the oocytes. The differences found among the individual classes indicate relationships with the nutrition and adaptation to the parsimonious use of nutrients in the metabolism of reproduction.

Universidade Federal de Minas Gerais Research on stimulus equivalence constitutes an experimental approach to assess variables underlying the development of symbolic behavior, which depends on the establishment of conditional relations between stimuli. Matching-to-sample procedure is typically used for teaching conditional discriminations, and it has been also used in early studies with honeybees. Bees are capable of learning conditional relations between stimuli, such as specific identity relations, arbitrary relations between visual stimuli, arbitrary relations between colors and odors and generalized identity matching. Therefore, basic processes involved in symbolic behavior could be studied using honeybees as an experimental model. The main objective of the present study was to establish conditional relations in honeybees. Another goal was to test for select and reject-control relations in simple discrimination baselines, since these controlling relations are involved in responding by exclusion. In Experiment 1, eleven bees Melipona quadrifasciata and Melipona rufiventris were individually trained to perform an identity matching-to-sample task. Identity relations were demonstrated by most of the bees. In Experiment 2, four M. rufiventris were individually trained on arbitrary relations. One of these bees demonstrated an increasing trend in accuracy in an arbitrary MTS task over the last sessions. In Experiment 3, twenty Apis mellifera and four M. rufiventris were trained to perform arbitrary relations. Additionally, Apis mellifera bees were presented with a symmetry test. Apis mellifera bees had acquired arbitrary MTS, but Melipona rufiventris performed at chance level. In the symmetry test, Apis mellifera performed at chance level. In Experiment 4, twenty M. rufiventris were individually trained to perform a simple discrimination task. Subsequently, each honeybee was presented to a select and reject control test. Select as much reject control had developed in simple discrimination training with M. rufiventris. On one hand, these data indicate that in most cases the arbitrary matching performance was not accurate. Moreover, symmetry relations did not emerge from training. On the other hand, most subjects responded highly accurately in simple discrimination and identity matching tasks. Most subjects had also demonstrated select and reject-control relations. These advancements in research on basic learning processes in bees may be a promising starting point for investigating the prerequisite repertoires involved in symbolic behavior. Pesquisas sobre formação de classes de equivalência constituem uma abordagem experimental sobre o desenvolvimento do comportamento simbólico, o qual depende essencialmente do desenvolvimento de relações condicionais entre estímulos. O procedimento de emparelhamento com o modelo (MTS) é tipicamente usado para ensinar relações condicionais e tem sido usado também em pesquisas com abelhas. Abelhas são capazes de aprender discriminações condicionais, como relações de identidade, relações arbitrárias entre estímulos visuais, relações arbitrárias entre cores e odores e responder generalizado por identidade. Abelhas poderiam, então, ser usadas como um modelo experimental para o estudo de processos básicos envolvidos no desenvolvimento de precursores do comportamento simbólico. O principal objetivo da presente pesquisa foi estabelecer discriminações condicionais em abelhas. Outro objetivo foi testar o controle por seleção e controle por rejeição em linhas de base de discriminação simples, tendo em conta que essas relações de controle estão envolvidas no responder por exclusão. No Experimento 1, onze abelhas M. quadrifasciata e M. rufiventris foram treinadas em uma tarefa de emparelhamento por identidade. A tarefa foi aprendida pela maioria das abelhas. No Experimento 2, quatro M. rufiventris foram individualmente treinadas em uma tarefa de MTS arbitrário. Uma dessas abelhas apresentou indícios de aprendizagem da tarefa. No Experimento 3, vinte Apis mellifera e quatro M. rufiventris foram treinadas em uma tarefa de MTS arbitrário. Além disso, foi conduzido um teste de simetria com as Apis mellifera. A tarefa de MTS arbitrário foi aprendida pelas A. mellifera, mas não pelas M. rufiventris. No teste de simetria, as A. mellifera apresentaram desempenho com nível de precisão ao acaso, isto é, o comportamento não ficou sob controle da nova função de modelo. No Experimento 4, vinte M. rufiventris foram treinadas em uma tarefa de discriminação simples. Em seguida, foi conduzido um teste de controle por seleção e rejeição. Foi demonstrado tanto o controle por seleção quanto por rejeição. Por um lado, esses dados mostram que não foram produzidas discriminações condicionais estáveis. Além disso, não ocorreu emergência de simetria. Por outro lado, foram sistemática e fidedignamente produzidas discriminações simples, MTS por identidade e relações de controle por seleção e por rejeição em abelhas. Esses avanços no estudo de processos básicos de aprendizagem em abelhas podem ser um ponto de partida seguro para o estudo de repertórios relacionais complexos.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior In the genus Melipona, a bee Melipona quadrifasciata is one of the best known species and is divided into two subspecies M. q. quadrifasciata and M. q. anthidioides. This study aimed to evaluate the identity of bees mandaçaia the northern coast of Sergipe through morphometric and molecular techniques, and verify the possible geographic barrier represented by the Rio São Francisco. The survey was conducted in a total area of 30ha subdivided into three fragments in the municipalities of Piaçabuçu - AL, Brejo Grande SE, Ilha da Criminosa - SE. Were recorded in 14 colonies mandaçaia Brejo Grande and collected 10 specimens of each colony. No nest was located on the Ilha da Criminosa and Piaçabuçu, which reinforces the hypothesis that the Rio São Francisco could're representing a geographical barrier to the spread of the colonies. The spatial distribution of nests on a scale of 10ha, can be considered as random. Morphometry were used in the wings of the specimens collected and identified three other specimens of M. q. quadrifasciata, M. q. anthidioides and M. mandacaia. The data were subjected to cluster analyzes, principal component analysis, canonical variate analysis, Procrustes analysis of variance test and correlation matrices. Analyses of morphometric data are congruent and identified the M. q. quadrifascita as the identity of the species collected. The analysis of cross-validation and correlation matrices showed no significance, showing great similarity intrapopulation. Total DNA from each sample was subjected to analysis of PCR / RFLP (enzymes: Taq I, Vsp I and Mbo II), identifying a unique pattern of haplotypes for the bee population of the mouth, showing the high genetic similarity. All nests were located in palm trees that would be condemned by the low production due to damage caused by pests, besides the very low supply of other sites for nesting, which sets very high risk of population extinction. No gênero Melipona, a abelha Melipona quadrifasciata é uma das espécies mais conhecida e está dividida em duas subespécies M. q. quadrifasciata e M. q. anthidioides. O presente trabalho teve como objetivo avaliar a identidade das abelhas mandaçaia do litoral norte do Estado de Sergipe por meio de técnicas moleculares e morfométrica, e verificar a possível barreira geográfica representada pelo Rio São Francisco. A pesquisa foi realizada em uma área total 30ha subdividida em três fragmentos nos municípios de Piaçabuçu AL, Brejo Grande SE e Ilha da Criminosa SE. Foram registradas 14 colônias de mandaçaia em Brejo Grande e coletados 10 espécimes de cada colônia. Nenhum ninho foi localizado na Ilha da Criminosa e em Piaçabuçu, o que reforça a hipótese de que o Rio São Francisco poderia está representando uma barreira geográfica para a dispersão das colônias. A distribuição espacial de ninhos em uma escala de 10ha, pode ser considerada como aleatória. Na morfometria foram utilizadas as asas dos espécimes coletados e de 3 outras amostras identificadas de M. q. quadrifasciata, M. q. anthidioides e M. mandacaia. Os dados foram submetidos a Análises de Agrupamento, Análise de Componentes Principais, Análise de Variáveis Canônicas, Análise de Variância de Procrustes e teste de correlação de matrizes. As análises dos dados morfométricos são congruentes e apontaram a M. q. quadrifascita como a identidade da espécie coletada. A análise de validação cruzada e a correlação de matrizes não apresentou significância, denotando grande similaridade intrapopulacional. O DNA total de cada amostra foi submetido a análise de PCR/RFLP (enzimas: Taq I, Vsp I e Mbo II), identificando um único padrão de haplótipos para a população de abelhas da foz, evidenciando a grande similaridade genética. É importante ressaltar que todos os ninhos estavam localizados em coqueiros que seriam condenados pela baixa produção ocasionada pelos danos decorrente de pragas, além da baixíssima oferta de outros locais para nidificação, o que configura altíssimo risco de extinção populacional.

Males, queens and workers of stingless bees show differences in external morphology, behaviour and roles within a colony. In addition, each individual has a cuticular chemical signature responsible for mutual communication that is essential for maintaining the integrity of the colony. In this paper we characterize the cuticular hydrocarbon composition of newly emerged diploid and haploid males, workers and virgin queens of Melipona quadrifasciata by gas chromatography-mass spectrometry (GC/MS) analysis. This is the first time that the cuticular profile of diploid males in a species of stingless bee has been characterized. We found differences in the cuticular hydrocarbon composition among males, workers and virgin queens, recording both qualitative and quantitative differences among individuals of different phenotypes. However, no compound was found exclusively in diploid males. The cuticular chemical profiles of haploid and diploid males were very similar to those of workers. Moreover, the cuticular lipids of males and workers were significantly different from those of queens. Tricosane, pentacosene-2 and 7-methyl-heptacosane were the compounds responsible for this significant separation. This result correlates with the behavioural and morphological differences among these phenotypes.

International audience; The first studies on the genome size of stingless bee species showed a range from 0.27 pg (Melipona subnitida and Melipona quadrifasciata) to 1.38 pg (Melipona capixaba). Considering this variation, we quantified the DNA content of 26 species of Meliponini, in order to provide input for future comparative studies in this tribe. Haploid genome size (1C) estimates, using flow cytometry analyses (FCM), ranged from 0.26 ± 0.003 pg (Paratrigona subnuda) to 0.98 ± 0.023 pg (Melipona flavolineata), with an average of 0.54 ± 0.17 pg. FCM analyses also demonstrated a small difference in the haploid genome size between males and females of the same species, with the males generally having a smaller genome than females. Our data also evidenciated that variations in the genome size of stingless bees do not correlate with changes in chromosome number and that in some genera the DNA content is more variable than in others.

Study of the epithelial morphology of a stingless bee ileum from the pyloric valve to the last portion of high absorptive cells shows that although the bee ileum is an anatomically undifferentiated tube, four types of epithelial cells along the tube (in addition to the valve cells) indicate physiological differentiation. The anterior end seems to be less active in reabsorption, while the posterior region contains cells with typical morphology of an ion pump and permits conclusions about the mechanisms of absorption in the posterior end of the intestine. © 1994 Wiley-Liss, Inc.

In Hymenoptera, homozygosity at the sex locus results in the production of diploid males. In social species, these pose a double burden by having low fitness and drawing resources normally spent for increasing the work force of a colony. Yet, diploid males are of academic interest as they can elucidate effects of ploidy (normal males are haploid, whereas the female castes, the queens and workers, are diploid) on morphology and life history. Herein we investigated expression levels of ten caste-related genes in the stingless bee Melipona quadrifasciata, comparing newly emerged and 5-day-old diploid males with haploid males, queens and workers. In diploid males, transcript levels for dunce and paramyosin were increased during the first five days of adult life, while those for diacylglycerol kinase and the transcriptional co-repressor groucho diminished. Two general trends were apparent, (i) gene expression patterns in diploid males were overall more similar to haploid ones and workers than to queens, and (ii) in queens and workers, more genes were up-regulated after emergence until day five, whereas in diploid and especially so in haploid males more genes were down-regulated. This difference between the sexes may be related to longevity, which is much longer in females than in males.

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Conselho Nacional de Desenvolvimento Científico e Tecnológico The present study had focused to address different aspects of gene expression and cuticular hydrocarbons composition of queens, workers, diploid and haploid males of Melipona quadrifasciata, as well as trying to understand the molecular mechanisms involved in the process of differentiation of male. The profile of gene expression of ten caste-specific genes was evaluated comparatively among newly emerged and 5 day-old workers, queens, diploid and haploid males. In general, the abundance of transcripts of diploid males was similar to that of haploid males and workers than to queens for the two age groups analyzed. After five days of emergence there were significant increases in gene expression levels of queens and workers, while opposite results were observed in male. These differences may be related to differential longevity between males and females. Despite haploid and diploid males are not easily distinguished visually, differentially expressed genes between these two phenotypes were identified by the technique of RDA (Representational Difference Analysis). Overall, the ESTs sequenced from the haploid male RDA library showed similarity with sequences of Drosophila (32.5%), Apis (25.6%) and stingless bees (7%). In the diploid male library, the percentage of ESTs with similarity to Drosophila and Apis sequences were slightly higher, being 41.8% and 30.2%, respectively. Both subtracted libraries also presented considerable proportions of ESTs without significant match to sequences in the nr database (34.9% for the haploid and 28% for the diploid male libraries). Differential gene expression was confirmed by real time PCR for ten of seventeen selected genes. Comparative gene expression analysis on samples of males with five days of age showed changes in abundance of transcripts in the phase of sexual maturation and early migration of sperm from the testicles to the seminal vesicle. Males and females of M. quadrifasciata had different chemical cuticular signatures, and the chemical standard of haploid and diploid males are more similar to workers than those of queens, corroborating the morphological and behavioral similarities between males and workers. No compound was found exclusively in the cuticle of newly emerged diploid males, indicating the absence of a substance that activates the agressive behavior of workers when these males are detected in inbred colonies. No presente trabalho procurou-se abordar diferentes aspectos relacionados a expressão gênica e composição dos hidrocarbonetos cuticulares de rainhas, operárias, machos haplóides e machos diplóides de Melipona quadrifasciata, assim como se procurou compreender os mecanismos moleculares envolvidos nos processos de diferenciação dos machos desta abelha. O perfil de expressão gênica de dez genes casta específicos foi avaliado de forma comparativa entre operárias, rainhas, machos haplóides e diplóides recém-emergidos e com cinco dias de idade adulta. De modo geral, a abundância de transcritos de machos diplóides foi mais similar à de machos haplóides e operárias do que à de rainhas, para as duas classes etárias analisadas. Após cinco dias de emergência verificaram-se aumentos significativos nos níveis de expressão gênica de rainhas e operárias, enquanto que resultados opostos foram observados nos machos. Estas diferenças podem estar relacionadas à longevidade diferencial de machos e fêmeas. Apesar dos machos haplóides e diplóides não serem facilmente distinguidos visualmente, genes diferencialmente expressos entre estes dois fenótipos foram identificados por meio da técnica de RDA (Representational Difference Analysis). Na biblioteca obtida de machos haplóides, observou-se que 32.5% das ESTs apresentaram similaridade às sequências de Drosophila, 25.6% às sequências de Apis e 7% são similares à meliponíneos. Na biblioteca de machos diplóides, as porcentagens de similaridade às sequências de Drosophila e Apis foram levemente maiores, sendo 41.8% e 30.2%, respectivamente. Em ambas as bibliotecas subtrativas, algumas ESTs não apresentaram similaridade significativa com nenhuma sequência depositada em bancos de dados públicos sendo consideradas como produtos preditos (34.9% e 28% para as bibliotecas de machos haplóides e diplóides, respectivamente). A expressão gênica diferencial foi confirmada por PCR em tempo real para dez de dezessete genes selecionados. Análises de expressão gênica comparativa com amostras de machos com cinco dias de idade revelaram mudanças na abundância dos transcritos neste período em que ocorre a maturação sexual e o início da migração dos espermatozóides dos testículos para a vesícula seminal. Machos e fêmeas de M. quadrifasciata apresentaram diferentes assinaturas química cuticulares, sendo o padrão químico de machos haplóides e diplóides mais similares aos de operárias do que aos de rainhas, corroborando as semelhanças morfológicas e comportamentais entre machos e operárias. Nenhum composto foi encontrado exclusivamente na cutícula dos machos diplóides recém-emergidos, indicando a inexistência de uma substância que ative o comportamento agressivo das operárias quando estes machos são detectados em colônias endogâmicas.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior The bees of Melipona genus have trofogenetic mechanism to determination of caste. The level of juvenile hormone (JH) in several stages of larval development determines the characteristics of females. Diploid males and females of Melipona quadrifasciata that, in last instar larvae, received topical JH develop external morphological characteristics of queen. This work was based on morphometric data to investigate the effect of JH III in the development of haploid and diploid males of Melipona quadrifasciata and compare morphological characteristics of them with morphological characteristics already known in females of this species. Ten individuals were used for each treatment; workers, natural queens, queens produced from the topical application of JH, natural haploid males, haploid males received application of JH but did not differ, haploid males that received topical application of JH and developed features of queens (QLM - Queen-Like Male), natural diploid males, diploid males that received topical application of JH but did not differ and diploid QLM. The anatomical averages analyzed were: width of compound eyes, width of tibia, length of mouthparts, length of mandible, intertegule distance and interorbit distance. All averages allowed the separation of individuals into groups. However, the lengths of the mouthpart averages were differences most significantly between treatments. Natural queens and queens produced from the effect of JH had similar averages. Diploid and haploid QLM showed average variables with similar size in the natural queens and queens produced from topical application of JH. Workers were more similar to males than the queens in the most averages variables and, in general, the workers presented average size more than queens. Natural diploid and haploid males had close averages, which does not allow visual differentiation of these individuals. The dates confirm that the JH is primarily responsible for the differentiation of specific characteristics of each caste, and, in the high levels of it causes the appearance of the characteristics of queen, independent on genotype. Abelhas do gênero Melipona possuem mecanismo de determinação das castas trofogenético. O nível de hormônio juvenil (HJ) em certas fases do desenvolvimento larval determina as características das fêmeas. Fêmeas e machos diplóides de Melipona quadrifasciata que, na fase pré defecante, recebem aplicação tópica de HJ desenvolvem características morfológicas externas de rainha. Este trabalho baseou-se em dados morfométricos para investigar o efeito do HJ III no desenvolvimento de machos haplóides e diplóides de Melipona quadrifasciata e comparar estes efeitos com os efeitos já conhecidos em fêmeas desta espécie. Foram utilizados dez indivíduos de cada tratamento, sendo operárias, rainhas naturais, rainhas produzidas a partir da aplicação tópica de HJ, machos haplóides naturais, machos haplóides que receberam aplicação de HJ mas não se diferenciaram, machos haplóides que receberam aplicação tópica de HJ e desenvolveram características de rainha (QLM – Queen-Like Male), machos diplóides naturais, machos diplóides que receberam aplicação tópica de HJ mas não se diferenciaram e QLM diplóides. As medidas anatômicas analizadas foram largura do olho composto, largura da tíbia, comprimento do aparelho bucal, comprimento da mandíbula, distância intertégula e distância interorbital. Todas as medidas possibilitaram a separação dos indivíduos em grupos, no entanto o comprimento do aparelho bucal foi a medida com diferenças mais significativas entre os tratamentos. Rainhas naturais e rainhas produzidas a partir do efeito do HJ possuem medidas semelhantes. QLM haplóides e diplóides apresentaram as variáveis medidas com tamanhos semelhantes as das rainhas naturais e produzidas a partir da aplicação tópica de HJ. Operárias se mostraram mais semelhantes aos machos do que as rainhas quanto a maior parte das maioria das variáveis medidas e, em geral, as operárias apresentam médias de tamanho maiores e as rainhas menores. Macho diplóides e haplóides naturais possuem medidas próximas, o que não permite a diferenciação visual desses indivíduos. Os dados corroboram que o HJ é o principal responsável pela diferenciação das características específicas de cada castas, assim, em altos níveis ele acarreta o aparecimento das características de rainha, independente do genótipo.

Submitted by Ricardo Cedraz Duque Moliterno (ricardo.moliterno@uefs.br) on 2020-05-07T23:27:26Z No. of bitstreams: 1 DISSERTA?AO VERS?O FINAL - MONIQUE.pdf: 898669 bytes, checksum: bef0b2306c85e9175dd549714b82b4fc (MD5) Made available in DSpace on 2020-05-07T23:27:26Z (GMT). No. of bitstreams: 1 DISSERTA?AO VERS?O FINAL - MONIQUE.pdf: 898669 bytes, checksum: bef0b2306c85e9175dd549714b82b4fc (MD5) Previous issue date: 2011-02-17 Conselho Nacional de Pesquisa e Desenvolvimento Cient?fico e Tecnol?gico - CNPq The stingless bees (Meliponina) are eusocial bees, living mainly in tropical and subtropical regions and often building their nests in pre-existing cavities. The worker classes of these bees normally have internal and external activities to the nest. The external activities are related to the collection of pollen and nectar, the collection of nest-building material (resin or clay) and the exit with garbage from the colony. This study aimed the investigatation and comparison of the daily outside activities of thre classes of stingless bees, namely the Melipona scutellaris, Melipona quadrifasciata and Frieseomelitta doederleini and to evaluate the presence of the daily rhythmic patterns in different months of the year, as well as the influence of environmental and meteorological factors in these activities. The largest number of activities of the species M. scutellaris and F. doederleini was observed from January to March, both considered respectively as strong and medium sized colonies. M. quadrifasciata showed a greater activity in July and August and was considered a weak sized colony. The initial activities of Melipona spp. a larger bee, generally occurred at sunrise and earlier than F. doederleini, a smaller bee. Melipona spp. showed preferential times (acrophase) of entrances, exits, entrances with pollen and resin during the mornings. F. doederlein differently, prefered usually the late mornings or early afternoons, during the different months that occurred at higher temperature values than those for Melipona spp. During the month of March, the wettest month, almost all acrophase activities for Melipona spp occurred later. The daily repetition of the first and last activities in the colony and the presence of significant acrophase for the activities of the bees in different months of the year, suggest that the presence of a daily rhythm of activity is probably influenced by the light / dark cycle, by rain and temperature. The final results of the role of environmental and meteorological factors in the external activities rhythms of the species of Meliponina studied, can only be obtained in a controlled laboratory situation. Os melipon?neos (Meliponina) s?o abelhas eussociais, principalmente tropicais e subtropicais e geralmente constroem seus ninhos em cavidades pr?-existentes. As oper?rias destas abelhas apresentam atividades internas e externas ao ninho. As atividades externas de v?o est?o relacionadas ? coleta de p?len e n?ctar, de material de constru??o do ninho (resina ou barro) e sa?da com lixo da col?nia. O presente estudo teve como objetivo investigar e comparar as atividades di?rias externas de Melipona scutellaris, Melipona quadrifasciata e Frieseomelitta doederleini, avaliando a presen?a de padr?es r?tmicos di?rios em diferentes meses do ano e a influ?ncia dos fatores ambientais e meteorol?gicos nestas atividades. O maior n?mero de atividades foi observado nos meses de janeiro e mar?o para M. scutellaris e F. doederleini, consideradas col?nias forte e m?dia e M. quadrifasciata apresentou a maior atividade nos meses de julho e agosto e foi considerada uma col?nia fraca. As atividades iniciais de Melipona spp., abelha de maior tamanho, ocorreram geralmente no nascer do sol e mais cedo do que F. doederleini, abelha de menor tamanho. Melipona spp., apresentaram hor?rios preferenciais de atividade (acrofases) de entradas, sa?das, entradas com p?len e entrada com resina durante a manh? e F. doederleini, geralmente no final da manh?, ou in?cio da tarde, nos diferentes meses, que ocorriam a valores de temperatura mais elevados do que para Melipona spp. Durante o m?s de mar?o, o mais chuvoso, quase todas as acrofases das atividades para Melipona spp. ocorreram mais tarde. As repeti??es di?rias das primeiras e ?ltimas atividades na col?nia e a presen?a de acrofases significativas para as atividades das abelhas, em diferentes meses do ano, sugerem a presen?a de ritmo di?rio de atividade influenciado provavelmente pelos ciclo claro/escuro, pela chuva e pela temperatura. Os resultados definitivos do papel destes fatores ambientais e meteorol?gicos nos ritmos das atividades externas das tr?s esp?cies de Meliponina estudadas s? poder?o ser obtidos em situa??o controlada de laborat?rio.

The workers of the stingless bee, Melipona quadrifasciata, assume different tasks during their adult life. Newly emerged individuals remain inside the nest, without contact with the external environment. Maturing workers go to more peripheral regions and only the oldest, the foragers, leave the nest. As this diversity of activities implies different metabolic patterns, oxygen consumption has been measured in workers of three different ages: 24–48 h (nurses), 10–15 days (builders), and older than 25 days (foragers). Oxygen consumption of individually isolated workers was determined by intermittent respirometry, under constant darkness and temperature of 25 ± 1°C. Sets of 24-h measurements were obtained from individuals belonging to each of the three worker groups. Rhythmicity has been assessed in the daily (24 h) and ultradian (5–14 h) domains. This experimental design allowed detection of endogenous rhythms without the influence of the social group and without inflicting stress on the individuals, as would be caused by their longer isolation from the colony. Significant 24-h rhythms in oxygen consumption were present in nurses, builders and foragers; therefore, workers are rhythmic from the age of 24–48 h. However, the amplitude of the circadian rhythm changed according to age: nurses showed the lowest values, while foragers consistently presented the largest ones, about ten times larger than the amplitude of nurses’ respiratory rhythm. Ultradian frequencies were detected for all worker groups, the power and frequencies of which varied little with age. This means that the ultradian strength was relatively larger in nurses and apparently maintains some relationship with the queen’s oviposition episodes.

Competition for floral resources is a key force shaping pollinator communities, particularly among social bees. The ability of social bees to recruit nestmates for group foraging is hypothesized to be a major factor in their ability to dominate rich resources such as mass-flowering trees. We tested the role of group foraging in attaining dominance by stingless bees, eusocial tropical pollinators that exhibit high diversity in foraging strategies. We provide the first experimental evidence that meliponine group foraging strategies, large colony sizes and aggressive behavior form a suite of traits that enable colonies to improve dominance of rich resources. Using a diverse assemblage of Brazilian stingless bee species and an array of artificial "flowers" that provided a sucrose reward, we compared species' dominance and visitation under unrestricted foraging conditions and with experimental removal of group-foraging species. Dominance does not vary with individual body size, but rather with foraging group size. Species that recruit larger numbers of nestmates (Scaptotrigona aff. depilis, Trigona hyalinata, Trigona spinipes) dominated both numerically (high local abundance) and behaviorally (controlling feeders). Removal of group-foraging species increased feeding opportunities for solitary foragers (Frieseomelitta varia, Melipona quadrifasciata and Nannotrigona testaceicornis). Trigona hyalinata always dominated under unrestricted conditions. When this species was removed, T. spinipes or S. aff. depilis controlled feeders and limited visitation by solitary-foraging species. Because bee foraging patterns determine plant pollination success, understanding the forces that shape these patterns is crucial to ensuring pollination of both crops and natural areas in the face of current pollinator declines. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00040-009-0055-8) contains supplementary material, which is available to authorized users.

International audience; The stingless bees are among the most abundant and ecologically important social invertebrates in tropical communities. The Neotropical stingless bee Melipona quadrifasciata has two subspecies: M. quadrifasciata quadrifasciata and M. quadrifasciata anthidioides. The main difference between subspecies are the yellow metassomal stripes, which are continuous in M. q. quadrifasciata and discontinuous in M. q. anthidioides. Recently, two populations were described with continuous stripes and inhabiting clearly disjunct areas in relation to M. q. quadrifasciata. We sequenced 852 bp of the mtDNA COI gene from 145 colonies from 56 localities, and for the first time performed a detailed phylogeographic study of a neotropical stingless bee. Phylogenetic analyses revealed the existence of two clades exhibiting a south to north distribution: southern populations comprise the subspecies M. q. quadrifasciata, and northern populations are composed of M. q. anthidioides and two disjunct populations with continuous stripes. The divergence time of these two phylogroups was estimated between 0.233 and 0.840 million years ago in the Pleistocene, a period of climatic changes and geomorphological alterations in the Neotropical region. No evidence of genetic structure in relation to the tergal stripes was found, indicating that the morphological trait regarding the pattern of stripes on tergites is not an accurate diagnostic for the subspecies of M. quadrifasciata.

The stingless bee species Melipona quadrifasciata includes two subspecies, Melipona quadrifasciata anthidioids and Melipona quadrifasciata quadrifasciata. The morphological difference between the two subspecies is the presence of three to five continuous yellow stripes on the terga on the 3rd to 6th segments in workers and males of M. q. quadrifasciata, and two to five interrupted bands in M. q. anthidioides. We identified a DNA marker which is present in M. q. quadrifasciata and absent in M. q. anthidioides. Only one among the M. q. quadrifasciata colonies did not present the marker. It was also absent in bees collected in northern Minas Gerais State (Brazil), despite their morphological resemblance to M. q. quadrifasciata. The marker can be used for studying the genetic structure of the hybridization zone formed by the intercrossing of the two subspecies.A espécie de abelha sem ferrão Melipona quadrifasciata apresenta duas subespécies, Melipona quadrifasciata quadrifasciata Lep. e Melipona quadrifasciata anthidioides Lep. A diferença morfológica entre as duas subespécies é a presença de três a cinco bandas tergais amarelas do 3º ao 6º segmentos em operárias e machos de M. q. quadrifasciata e duas a cinco bandas interrompidas em M. q. anthidioides. Nós identificamos um marcador de DNA que está presente em M. q. quadrifasciata e ausente em M. q. anthidioides. Este marcador está ausente em abelhas coletadas no norte do Estado de Minas Gerais (Brasil), embora esses indivíduos apresentem morfologia similar à de M. q. quadrifasciata. Este marcador poderá ser utilizado em estudos da zona de hibridação entre as subespécies.

O forideo P. kerteszi é uma praga que ataca principalmente as crias de abelhas sociais. As conseqüências do ataque vão desde o enfraquecimento até o extermínio total da colônia de abelhas. O objetivo deste trabalho foi a criação de uma metodologia que permita a erradicação deste parasita quando estiver atacando colônias de abelhas melipona. Três colônias de M. quadrifasciata parasitadas com o forídeo foram tratadas com solução de mel de meliponídeos ou vinagre associada ao veneno de mosca oktrine. Um frasco contendo essa solução foi colocado entre o ninho e a lixeira em colmeias do modelo Uberlândia. Uma tela de malha que não permitisse a passagem das abelhas foi colocada entre o ninho e a lixeira. Por um período de três a sete dias foram encontrados forídeos mortos dentro ou próximo à solução.The forid fly P. kerteszi is a pest that mainly attacks the brood of social bees. The consequences of this attack range from weakening to total extermination of bee colonies. The objective of the present study was to develop a methodology that would eradicate this pest when P. kerteszi is parasitizing colonies of Melipona bees. Three M. quadrifasciata colonies parasitized with the forid fly were treated with a solution of meliponid honey or vinegar in combination with the oktrine fly poison. A flask containing this solution was placed between the nest and a garbage tray separated by a mesh net that would permit the passage of forid flies but not of bees. Dead forid flies inside or close to the solution were found daily for a period of three to seven days.

The pollination effectiveness of the stingless bee Melipona quadrifasciata and the honey bee Apis mellifera was tested in tomato plots. The experiment was conducted in four greenhouses as well as in an external open plot in Ribeirao Preto, SP, Brazil. The tomato plants were exposed to visits by M. quadrifasciata in one greenhouse and to A. mellifera in another; two greenhouses were maintained without bees (controls) and an open field plot was exposed to pollinators in an area where both honey bee and stingless bee colonies are abundant. We counted the number of tomatoes produced in each plot. Two hundred tomatoes from each plot were weighed, their vertical and transversal circumferences were measured, and the seeds were counted. We collected 253 Chrysomelidae, 17 Halictidae, one Paratrigona sp, and one honey bee from the flowers of the tomato plants in the open area. The largest number of fruits (1414 tomatoes), the heaviest and largest tomatoes, and the ones with the most seed were collected from the greenhouse with stingless bees. Fruits cultivated in the greenhouse with honey bees had the same weight and size as those produced in one of the control greenhouses. The stingless bee, M. quadrifasciata, was significantly more efficient than honey bees in pollinating greenhouse tomatoes.

Melipona quadrifasciata Lepeletier, 1836, regionally known as "mandaçaia", has been traditionally divided in two distinct subspecies: M. quadrifasciata anthidioides and M. quadrifasciata quadrifasciata. The main difference between the subspecies refers to the yellow metasomal stripes which are continuous in M. q. quadrifasciata and discontinuous in M. q. anthidioides. This study investigated the geographic differentiation in the metasomal stripes and characterized the restriction sites in the mtDNA of both chromatic types. Specimens from 198 localities were examined, and the variation observed in the pattern of stripes was grouped into distinct classes. The distribution pattern found in the present work agrees with the previously reported pattern: M. q. quadrifasciata inhabits the southern portion of the distribution, from Misiones, Argentina, southeastern Paraguay and Rio Grande do Sul to southern São Paulo, and M. q. anthidioides ranges from northeastern São Paulo to the northern Diamantina Plateau, Bahia, and westwards to the central portion of the Goiás state. It is documented for the first time the occurrence of two populations with continuous stripes inhabiting disjunct areas in relation to M. q. quadrifasciata - one in northern Minas Gerais and another in northeastern Bahia and Sergipe. The data of RFLP showed two restriction patterns, one present in M. q. quadrifasciata, and another in M. q. anthidioides and in populations with continuous metasomal stripes from northern Minas Gerais and northeastern Bahia and Sergipe. The observed patterns of geographic differentiation of M. quadrifasciata suggests the occurrence of repeated events of geographical isolation, followed by range expansion, that occurred probably during the cycles of climatic changes in the Pleistocene.

The Melipona quadrifasciata (Lepeletier, 1836) is considered a potential pollinator of tomatoes in greenhouse. However, this pollinator may be directly affected by toxic compounds sprayed on the crop to control pests and diseases. The objective was to evaluate the lethal effects (LD50) of the insecticides abamectin, deltamethrin, spinosad and methamidophos to the stingless bee M. quadrifasciata (Lepeletier, 1836) and the Africanized honeybee Apis mellifera (Linnaeus, 1758) (Hymenoptera: Apidae), using methods adapted from tests with A. mellifera by different routes of exposure. In tests of oral exposure, the insecticide formulations of abamectin, deltamethrin, spinosad and methamidophos were highly toxic (LD50 100 μg a.i./bee) to M. quadrifasciata and A. mellifera, except for abamectin, which was moderately toxic to A. mellifera (LD50 1-10 μg a.i./bee). The contact toxicity of abamectin and deltamethrin was moderate (LD50 1-10 μg a.i./bee) and methamidophos was found to be slightly toxic (LD50 10-100 μg a.i./bee) for M. quadrifasciata. In contrast, deltamethrin exhibited moderate toxicity to workers of A. mellifera (LD50 1-10 μg a.i./bee), abamectin was slightly toxic (LD50 10-100 μg a.i./bee) and methamidophos virtually non toxic (LD50 >100 μg a.i./bee). A Melipona quadrifasciata (Lepeletier, 1836) é considerada um potencial polinizador da cultura do tomateiro em ambiente protegido. No entanto, esse polinizador pode ser afetado diretamente por compostos tóxicos pulverizados na cultura para o controle de pragas e/ou doenças. O objetivo foi avaliar os efeitos letais (DL50) dos inseticidas abamectina, deltametrina, espinosade e metamidofós para a abelha-sem- ferrão M. quadrifasciata (Lepeletier, 1836) e para Apis mellifera (Linnaeus, 1758) africanizada (Hymenoptera: Apidae), utilizando metodologias adaptadas de ensaios com A. mellifera por diferentes vias de exposição. Nos testes de exposição por via oral, as formulações os inseticidas abamectina, deltametrina, espinosade e metamidofós se mostraram altamente tóxicos (DL50 < 1,0 μg i.a./abelha) para operárias da M. quadrifasciata. Assim como para a A. mellifera, com exceção do metamidofós foi moderadamente tóxico (DL50 1-10 μg i.a./abelha). Na exposição tópica os ingredientes ativos abamectina, deltametrina e metamidofós foram considerados virtualmente atóxicos (DL50 >100 μg i.a./abelha) para M. quadrifasciata. Assim como para a A. mellifera, com exceção da abamectina foi moderadamente tóxica (DL50 1-10 μg i.a./abelha). Na aplicação por contato, abamectina e deltametrina apresentaram toxicidade moderada (DL50 1-10 μg i.a./abelha) e o metamidofós mostrou-se ligeiramente tóxico (DL50 10-100 μg i.a./abelha) para M. quadrifasciata. Porém, para operárias de A. mellifera a deltametrina apresentou toxicidade moderada (DL50 1-10 μg i.a./abelha), abamectina foi ligeiramente tóxica (DL50 10-100 μg i.a./abelha) e o metamidofós virtualmente atóxico (DL50 >100 μg i.a./abelha).

Pollination is an important factor in agricultural systems, especially in growing fruits and seed production, which depend greatly on bee visiting during blossom season; highly successful gains within these activities varies between 80 and nearly 100 per cent, owing to the bees. The assessment of bee diversity in commercial orchards of guava may contribute to a more desirable strategic design and consequent improvement of production. The aim of the study was identify the diversity of visiting bees to guava flowers (Psidium guajava) in a commercial orchard in Salinas, State of Minas Gerais, Brazil. The work was carried during blossom season of May - 2005. Field works occurred between 6:00 am to 6:00 pm, counting with 44 hours of collection, when 705 bees were collected. The richness observed was of 17 species, the most frequent and dominant being Trigona spinipes. Among the collection there were some considered accessory species: Apis mellifera, Melipona quadrifasciata and Tetragonisca angustula. Most of individual bees have been captured during early periods of morning, between 6:00 and 10:00 am.

Queens and workers of social insects differ in the rates of egg laying. Using genomic information we determined the sequence of vasa, a highly conserved gene specific to the germ line of metazoans, for the honey bee and four stingless bees. The vasa sequence of social bees differed from that of other insects in two motifs. By RT-PCR we confirmed the germ line specificity of Amvasa expression in honey bees. In situ hybridization on ovarioles showed that Amvasa is expressed throughout the germarium, except for the transition zone beneath the terminal filament. A diffuse vasa signal was also seen in terminal filaments suggesting the presence of germ line cells. Oocytes showed elevated levels of Amvasa transcripts in the lower germarium and after follicles became segregated. In previtellogenic follicles, Amvasa transcription was detected in the trophocytes, which appear to supply its mRNA to the growing oocyte. A similar picture was obtained for ovarioles of the stingless bee Melipona quadrifasciata, except that Amvasa expression was higher in the oocytes of previtellogenic follicles. The social bees differ in this respect from Drosophila, the model system for insect oogenesis, suggesting that changes in the sequence and expression pattern of vasa may have occurred during social evolution.

Currently, the identification of pollinators is a critical necessity of conservation programs. After it was found that features extracted from patterns of wing venation are sufficient to discriminate among insect species, various studies have focused on this structure. We examined wing venation patterns of males and workers of five stingless bee species in order to determine if there are differences between sexes and if these differences are greater within than between species. Geometric morphometric analyses were made of the forewings of males and workers of Nannotrigona testaceicornis, Melipona quadrifasciata, Frieseomelitta varia, and Scaptotrigona aff. depilis and Plebeia remota. The patterns of males and workers from the same species were more similar than the patterns of individuals of the same sex from different species, and the patterns of both males and workers, when analyzed alone, were sufficiently different to distinguish among these five species. This demonstrates that we can use this kind of analysis for the identification of stingless bee species and that the sex of the individual does not impede identification. Computer-assisted morphometric analysis of bee wing images can be a useful tool for biodiversity studies and conservation programs.

The possible significance of nestmate recognition in prevention of robbing and parasitism in three species of stingless bees was assessed. Nestmate discrimination abilities vary among them; Melipona quadrifasciataworkers attacked 74% of nonnestmate conspecifics that were encountered, while M. scutellarisand M. rufiventriswere less discriminating, attacking only 14 and 60% of non-nestmates, respectively. In tests of interspecific interactions, M. quadrifasciataand M. scutellariswere the least mutually tolerant of all species pairs tested. Tests with Apis melliferashowed a high degree of intolerance by two of the three Meliponaspecies.

Conselho Nacional de Desenvolvimento Científico e Tecnológico Melipona quadrifasciata, regionally known as mandaçaia , presents two distinct subspecies: M. quadrifasciata anthidioides and M. quadrifasciata quadrifasciata. They differ in the yellow tergal stripes, which are continuous in M. q. quadrifasciata and discontinuous in M. q. anthidioides. The correlation between geographic distribution and metasomal coloration was investigated and characterized the restriction sites in the mtDNA of both morphotypes of M. quadrifasciata, as determined by the tergal stripes patterns. Specimens from 198 localities were examined, and the variation observed in the pattern of tergal stripes was grouped into four distinct classes. The distribution pattern of M. quadrifasciata found in the present work agrees with the previously reported pattern for this species, i.e., M. q. quadrifasciata, a form characterized by continuous tergal stripes, inhabits the southern portion of the distribution, from Rio Grande do Sul to southern São Paulo, including Misiones, in Argentina, and southeastern Paraguay to the west, whereas M. q. anthidioides, a form with interrupted stripes, occurs from northeastern São Paulo to the northern portion of Diamantina Plateau, in Bahia, and westwards to the western tip of Minas Gerais and central portion of the Goiás State. The data of RFLP showed two restriction patterns, one present in M. q. quadrifasciata, and another in M. q. anthidioides and in populations with continuous tergal stripes from northern Minas Gerais and northeastern Bahia and Sergipe. The phylogeography was evaluated across its range of M. quadrifasciata populations. Using 852 bp of mtDNA COI gene of 145 individuals from 56 locates of RS RS, SC, PR, SP, RJ, MG, ES, BA and SE states in Brazil. Fifty haplotypes were identified 50 with a nucleotide diversity (π) of 0,0055 and a haplotypic diversity (Hd) of 0,957. The phylogenetic analysis showed the presence of two clades: the south clade comprising the subspecies M. q. quadrifasciata, and the north clade formed by the subspecies M. q. anthidioides and the populations with continuous tergal stripes from northern Minas Gerais and northeastern Bahia and Sergipe. AMOVA showed a percentage of variation between the clades of 68,56% and a ΦST 0,905. The barrier of gene flow, estimated with SAMOVA, was located close to Ribeira do Iguape River Valley in the south of the State of São Paulo. The unimodal mismatch distribution suggested bottleneck events affecting both clades. Coalescence analysis estimated time of divergence estimated between 490.000 and 390.000 years B.P. Other species of Melipona of the Atlantic forest, M. bicolor and M. marginata, also present subspecies that display morphological differences along north-south ranges that match the distribution of M. q. quadrifasciata and M. q. anthidioides. Concordant phylogeographyc patterns have been reported for other animal groups, as birds (Xiphorhynchus fuscus) and serpents (Bothrops jararaca). A abelha Melipona quadrifasciata, conhecida popularmente como mandaçaia, apresenta duas subespécies: M. q. anthidioides e M. q. quadrifasciata. A principal diferença entre as subespécies são as faixas tergais amarelas, que são contínuas em M. q. quadrifasciata e descontínuas em M. q. anthidioides. A correlação entre a distribuição geográfica e a coloração metassomal e os sítios de restrição do DNA mitocondrial foi caracterizado de ambos morfotipos de M. quadrifasciata, como determinado pelos padrões de faixas tergais. Foram examinados exemplares provenientes de um total de 198 localidades no Brasil, e com base na variação observada os exemplares foram distribuidos em quatro classes. A distribuição geográfica de M. quadrifasciata observada mostra M. q. quadrifasciata, uma forma com faixas tergais contínuas, ocupando a porção sul da distribuição, do Rio Grande do Sul até a metade sul de São Paulo, indo a oeste até a província de Misiones, na Argentina e porção sudeste do Paraguai. A forma com faixas interrompidas, M. q. anthidioides, distribui-se da metade nordeste de São Paulo até o extremo norte da região da Chapada Diamantina, na Bahia, estendendo-se a oeste pelo Triângulo Mineiro e região central do estado de Goiás. Dois padrões RFLP foram identificados, sendo um presente em M. q. quadrifasciata, e o outro em M. q. anthidioides e nas populações com padrão de faixas tergais continuo do norte de Minas Gerais e Sergipe e nordeste da Bahia. Avaliou-se também o padrão filogeográfico das populações de M. quadrifasciata ao longo de sua área de distribuição. Para análise filogeográfica foram utilizados 852 pb referentes a parte do gene COI do mtDNA de 145 indivíduos de M. quadrifasciata provenientes de 56 localidades dos estados do RS, SC, PR, SP, RJ, MG, ES, BA e SE. Foram identificados 50 haplótipos, com uma diversidade nucleotídica (π) de 0,0055 e uma diversidade haplotípica (Hd) de 0,957. Foi evidenciado, na inferência filogenética e na rede de haplótipos, a presença de dois clados: o clado sul compreendendo à subespécie M. q. quadrifasciata, e o clado norte formado pela subespécie M. q. anthidioides e as populações de faixa tergal contínua do norte de Minas Gerais e do Sergipe e nordeste da Bahia. A AMOVA mostrou um percentual de variação entre os clados de 68,56% e um ΦST de 0,905. A barreira de fluxo gênico, estimada pela SAMOVA, foi localizada próxima ao Vale do Ribeira do Iguape no sul do estado de São Paulo, concordando também com a separação dos clados. A mismatch distribution evidenciou gargalos populacionais em ambos clados por meio de distribuição unimodal. O tempo de divergência estimado pela análise de coalescência foi entre 490.000 e 390.000 anos A.P. Outras espécies de abelhas, M. bicolor e M. marginata, exibem diferenciação morfológica que recebe o status de subespécies, apresentando zona de separação entre as subespécies concordante com M. quadrifasciata. Padrões filogeográficos com vicariância semelhante foram reportados para pássaros (Xiphorhynchus fuscus) e serpentes (Bothrops jararaca).

The forid fly P. kerteszi is a pest that mainly attacks the brood of social bees. The consequences of this attack range from weakening to total extermination of bee colonies. The objective of the present study was to develop a methodology that would eradicate this pest when P. kerteszi is parasitizing colonies of Melipona bees. Three M. quadrifasciata colonies parasitized with the forid fly were treated with a solution of meliponid honey or vinegar in combination with the oktrine fly poison. A flask containing this solution was placed between the nest and a garbage tray separated by a mesh net that would permit the passage of forid flies but not of bees. Dead forid flies inside or close to the solution were found daily for a period of three to seven days. O forideo P. kerteszi é uma praga que ataca principalmente as crias de abelhas sociais. As conseqüências do ataque vão desde o enfraquecimento até o extermínio total da colônia de abelhas. O objetivo deste trabalho foi a criação de uma metodologia que permita a erradicação deste parasita quando estiver atacando colônias de abelhas melipona. Três colônias de M. quadrifasciata parasitadas com o forídeo foram tratadas com solução de mel de meliponídeos ou vinagre associada ao veneno de mosca oktrine. Um frasco contendo essa solução foi colocado entre o ninho e a lixeira em colmeias do modelo Uberlândia. Uma tela de malha que não permitisse a passagem das abelhas foi colocada entre o ninho e a lixeira. Por um período de três a sete dias foram encontrados forídeos mortos dentro ou próximo à solução.

Learning in insects has been extensively studied using different experimental approaches. One of them, the proboscis extension response (PER) paradigm, is particularly well suited for quantitative studies of cognitive abilities of honeybees under controlled conditions. The goal of this study was to analyze the capability of three eusocial bee species to be olfactory conditioned in the PER paradigm. We worked with two Brazilian stingless bees species, Melipona quadrifasciata and Scaptotrigona aff. depilis, and with the invasive Africanized honeybee, Apis mellifera. These three species present very different recruitment strategies, which could be related with different odor-learning abilities. We evaluated their gustatory responsiveness and learning capability to discriminate floral odors. Gustatory responsiveness was similar for the three species, although S. aff. depilis workers showed fluctuations along the experimental period. Results for the learning assays revealed that M. quadrifasciata workers can be conditioned to discriminate floral odors in a classical differential conditioning protocol and that this discrimination is maintained 15 min after training. During conditioning, Africanized honeybees presented the highest discrimination, for M. quadrifasciata it was intermediate, and S. aff. depilis bees presented no discrimination. The differences found are discussed considering the putative different learning abilities and procedure effect for each species.

Background: The stingless bee genus Melipona has been divided into two groups, based on their heterochromatin content. Melipona quadrifasciata and Melipona rufiventris have low and high levels of heterochromatin, respectively. Since condensed chromatin may be rich in methylated DNA sequences, M. quadrifasciata and M. rufiventris nuclei may contain different amounts of methylated CpG. These differences could be assessed by comparing Feulgen-DNA values obtained by image analysis of cells treated with the restriction enzymes Msp I and Hpa II that distinguish between methylated and unmethylated DNA. Msp I and Hpa II cleave the sequence -CCGG-, but there is no cleavage by Hpa II if the cytosine of the central CG dinucleotide is methylated. Methods: Malpighian tubules of M. quadrifasciata and M. rufiventris were treated with Msp I and Hpa II prior to the Feulgen reaction, and analyzed by automatic scanning microspectrophotometry. Results: The Feulgen-DNA values for the heterochromatin of M. rufiventris and for the small heterochromatin and some euchromatin domains of M. quadrifasciata mostly decreased after treatment with Msp I, but were unchanged after treatment with Hpa II. Conclusion: CpG methylation, although detected in diverse chromatin compartments in different bee species, may induce silencing effects required for the same cell physiology. © 2006 International Society for Analytical Cytology

Conselho Nacional de Desenvolvimento Científico e Tecnológico In this work it was aimed at a) to investigate the influence of the workers' bee behavioral status of different origins in the recognition process and defensive behavior; b) the influence of the age of the queens and of the workers' bee behavioral status in the acceptance process of virgin queens; c) influence of the workers' bee behavioral status on the survival and preoviposition period of queens of M. quadrifasciata in laboratory conditions. In the first chapter three trials were accomplished (aggression initiative, number of fights with the time and occurrence of injuries), where workers bee of different origins groups and behavioral status were confronted in a neutral arena. In the controls situation, no happened any aggressive interaction among the confronted groups. The results verified for the experimental group they indicate that the behavioral status and the degree of the workers' bee experience modulate the type of aggressive response the conspecific non-nestmates ones. Encounters between groups of forager workers and of foragers with nurse workers, they resulted in mutual aggressiveness and high number of injured individuals. Result contrary to the found for newly-emerged workers, that, although they have begun some confrontations with forager or nurse workers, these never resulted in counterattacks, and consequently, in injured workers. Though, confronts between groups of nurse workers and nurses with newly-emerged workers, haven t result in perceptible physical damages. In the second chapter of this thesis, virgin queens of zero to three days were introduced in mini-colonies formed by populations of nurse or foraging bees. It was verified that the rejection of queens by foraging bees increases with the age of the queens, being the recently-emerged queens more tolerated. However in the encounters with nurse bees, most of the virgin queens they were accepted independently of the age, indicating that foraging bees and nurse bees of M. quadrifasciata react in different ways to the presence of virgin queens. In the third chapter of this thesis, copulated queens in laboratory conditions were introduced in mini-colonies constituted by foraging bees, nurse bees, recently-emerged bees and mixed population. We verified that the worker bees age influences the time of pre-oviposition and survival of queens of M. quadrifasciata in laboratory conditions. The results indicate that, in colonies constituted by nurse bees the mortality was low, the number of queens in posture was elevated and the pre-oviposition period was reduced. In colonies with mixed population, the pre-oviposition period was similar to the colonies with nurse bees, however, the survival of the queens was shorter than in the treatment previously referred. However, in colonies constituted by recently-emerged bees, there was elevated mortality, delayed posture and reduced percentage of queens in posture. In colonies formed by forager workers no posture happened and all the queens died. No presente trabalho objetivou-se, a) investigar a influência do status comportamental de operárias de diferentes origens no processo de reconhecimento e comportamento defensivo; b) a influência da idade das rainhas e do status comportamental das operárias no processo de aceitação de rainhas virgens; c) influência do status comportamental das operárias sobre a sobrevivência e período de préoviposição de rainhas de M. quadrifasciata em condições de laboratório. No primeiro capítulo foram realizados três ensaios (iniciativa de agressão, número de lutas com o tempo e ocorrência de injúrias), onde grupos de operárias de diferentes origens e status comportamental foram confrontados numa arena neutra. Na situação controle, não ocorreu qualquer interação agressiva entre os grupos confrontados. Os resultados verificados para o grupo experimental indicam que o status comportamental e o grau de experiência das operárias modulam o tipo de resposta agressiva a co-específicos não familiares. Encontros realizados entre grupos de operárias forrageiras com operárias forrageiras e de operárias forrageiras com nutridoras, resultaram em agressividade mútua e elevado número de indivíduos injuriados. Resultado contrário ao encontrado para operárias recém-emergidas, que, embora tenham iniciado alguns confrontos com operárias forrageiras ou nutridoras, estes nunca resultaram em contra-ataques, e conseqüentemente, em operárias injuriadas. Encontros entre grupos de operárias nutridoras com nutridoras e de operárias nutridoras com recém-emergidas não resultaram em manifestações agressivas perceptíveis. No segundo capítulo desta tese, rainhas virgens de zero a três dias de idade foram marcadas e introduzidas em minicolônias formadas por populações de operárias nutridoras ou forrageiras. Verificou-se que a rejeição de rainhas por operárias forrageiras aumenta com a idade das rainhas, sendo as rainhas recém-emergidas de zero dia de idade as mais toleradas. Já nos encontros com operárias nutridoras, a maioria das rainhas virgens foi aceita independentemente da idade. Estes resultados indicam que operárias forrageiras e nutridoras de M. quadrifasciata reagem de forma diferenciada à presença de rainhas virgens. No terceiro capítulo, rainhas copuladas em condições de laboratório foram introduzidas em mini-colônias constituídas por operárias forrageiras, nutridoras, recém-emergidas e população mista. Verificou-se que a idade das operárias influencia o tempo de pré-oviposição e sobrevivência de rainhas de M. quadrifasciata em condições de laboratório. Os resultados indicam que, em colônias constituídas por operárias nutridoras ou população mista, a mortalidade foi baixa, o número de rainhas em postura foi elevado e o período de pré-oviposição foi reduzido. Em colônias constituídas por operárias recém-emergidas, houve elevada mortalidade, postura tardia e reduzida porcentagem de rainhas em postura. Em colônias formadas por operárias forrageiras não ocorreu postura e todas as rainhas morreram.

To evaluate caste-biased gene expression in Melipona quadrifasciata, a stingless bee, we generated 1278 ESTs using Representational Difference Analysis. Most annotated sequences were similar to honey bee genes of unknown function. Only few queen-biased sequences had their putative function assigned by sequence comparison, contrasting with the worker-biased ESTs. The expression of six annotated genes connected to caste specificity was validated by real time PCR. Interestingly, queens that were developmentally induced by treatment with a juvenile hormone analogue displayed an expression profile clearly different from natural queens for this set of genes. In summary, this study represents an important first step in applying a comparative genomic approach to queen/worker polyphenism in the bee.

This study describes an optimized protocol for the generation of Amplified Fragment Length Polymorphism (AFLP) markers in a stingless bee. Essential modifications to standard protocols are a re- striction enzyme digestion (EcoRI and Tru1I) in a two-step procedure, combined with a touchdown program in the selective PCR amplification step and product labelling by incorporation of α( 33 P)dATP. In an analysis of 75 workers collected from three colonies of Melipona quadrifasciata we obtained 719 markers. Analy- sis of genetic variability revealed that on average 32% of the markers were polymorphic within a colony. Compared to the overall percentage of polymorphism (44% of the markers detected in our bee samples), the observed rates of within-colony polymorphism are remarkably high, considering that the workers of each colony were all offspring of a singly mated queen. amplified fragment length polymorphism / genotyping / stingless bee / genetic polymorphism / Melipona / Apidae

The endemic range of the stingless bee Melipona quadrifasciata Lepeletier extends from the southern most state in Brazil, Rio Grande do Sul, up to the state of Paraiba, where it originally inhabited the Atlantic Rain Forest. Two subspecies have been morphologically recognized based on the tergal band pattern (yellow stripes across the abdomen), M. q. quadrifasciata and M. q. anthidioides. The objective of the present study was to characterize restriction site variation in the mitochondrial DNA of each subspecies. Four colonies of each subspecies were sampled. One individual per colony was used for DNA extraction and further analysis through amplification of nine fragments of the mitochondrial genome and digestion with 13 restriction enzymes (PCR+RFLP). Three enzymes showed restriction pattern differences between the subspecies. Those differences will be useful to determine the maternal origin of hybrids colonies, which may occur naturally or due to colony transportation among breeders.

Conselho Nacional de Desenvolvimento Científico e Tecnológico Foi avaliado o potencial da abelha-sem-ferrão Melipona quadrifasciata Lepeletier (Hymenoptera: Apidae) como polinizador de tomateiros (híbrido longa-vida; variedade Rodas) sob cultivo hidropônico e conceitos orgânicos em casa-de- vegetação em Minas Gerais, Brasil. O efeito do tratamento polinização pelas abelhas mais polinização manual não diferiu do efeito do tratamento polinização das abelhas ou manual. O (1) diâmetro máximo, a (2) altura e a circunferência do fruto (quociente entre 1 e 2), não diferiram entre os tratamentos. Entretanto, os frutos resultantes das flores visitadas somente por M. quadrifasciata, apresentaram 10,8% menos sementes (massa seca) do que os produzidos por polinização manual. Na variedade testada, as flores protegidas com sacos de poliéster (não polinizadas) não produziram frutos. A aparente baixa eficiência da polinização feita por M. quadrifasciata poderia ser atribuída à sobreposição de apenas 30 minutos entre o pico máximo de forrageamento das abelhas e o horário de maior receptividade do estigma das flores. Concluiu-se que é possível usar M. quadrifasciata como polinizador de tomateiros cultivados em ambiente protegido devido ao (i) aumento na qualidade dos frutos, considerando-se a diminuição das injúrias mecânicas quando comparadas à polinização manual tradicional, (ii) a não- diminuição do tamanho do fruto e (iii) ao alto preço do produto no mercado. Todavia, para o uso sustentável de M. quadrifasciata como polinizador em técnicas de criação e manejo em ambientes protegidos, cativeiro precisam ser desenvolvidas e difundidas, o que poderia prevenir sérios declínios das populações naturais desta abelha. Isto poderia ocorrer caso o único modo de aquisição de novas colônias para uso em programas de polinização fosse a retirada delas de seu ambiente natural. The neotropical stingless bee Melipona quadrifasciata Lepeletier was evaluated for pollinating tomatoes (long-life hybrid variety Rodas) grew in a plastic house with hydroponic system and organic concepts in Minas Gerais State, Brazil. Flowers not pollinated did not set any fruit. Pollination by bees plus manual pollination did not differ from either bee or manual pollination. (1) Maximum diameter, (2) fruit height and roundness (quotient between 1 and 2) was not significantly different between treatments, but flowers visited by M. quadrifasciata, set fruits with 10.8% less seeds (dry mass) than ones set from manual pollination. This apparently low efficiency of M. quadrifasciata as pollinator might be attributed to the overlap of only 30 minutes between highest bee foraging activity and highest flower stigma receptivity. Yet it was concluded M. quadrifasciata may be a feasible pollinator of greenhouse tomatoes because of (i) the observed increase in fruit quality due lower mechanical injury compared to traditional manual pollination, (ii) no significant decrease in fruit size, and (iii) high price of such product in the market. Some considerations for sustainable use of M. quadrifasciata as greenhouse pollinator are presented. Since while techniques for keeping captive colonies for M. quadrifasciata are currently available, the current method for acquiring new ones is removing them from the forest and, if colony demand was created for commercial use, techniques for captive colonies growing and distribution for pollination use must be developed to prevent serious declines in wild populations.

Summary The origin of the peritrophic membrane (PM) in bees is still a matter of debate. It is either of type I (synthesized by the entire midgut epithelium) or of type II (released from the anterior midgut end). The present study identified secretory sites of peritrophin-55 kDa, a PM protein in larvae of Melipona quadrifasciata anthidioides . Peritrophin-55 was isolated from PMs and was used for the production of a polyclonal antibody. Our study demonstrates the presence of peritrophin-55 in vesicles and on microvilli of digestive cells and in the PM. It suggests that the PM is of type-I, being specific for the larval phase of this stingless bee.

Species of plants used by Melipona quadrifasciata Lepeletier for pollen and nectar gathering in an urban forest fragment were recorded in Belo Horizonte, Minas Gerais, Brazil. Melipona quadrifasciata visited 22 out of 103 flowering plant species. The plant species belonged mainly to Myrtaceae, Asteraceae, and Convolvulaceae (64% of the visits). Melipona quadrifasciata tended to collect pollen or nectar each time, except for Myrtaceae species, from which both pollen and nectar were collected. Bee abundance at flowers did not significantly correlate to food availability (expressed by flowering plant richness). We found a relatively high similarity (50%) between plant species used by M. quadrifasciata, which was also found in studies carried out in São Paulo State. However, low similarity (17%) was found between the results of this study and those of another done in Bahia State, Brazil.

Conselho Nacional de Desenvolvimento Científico e Tecnológico O tubo digestivo dos insetos pode ser dividido em intestino anterior, intestino médio ou ventrículo e intestino posterior, sendo o ventrículo de origem endodérmica e os intestinos anterior e posterior de origem ectodérmica. No ventrículo ocorre a secreção de enzimas digestivas e a absorção dos nutrientes. Os principais tipos celulares encontrados no ventrículo são as células digestivas, que secretam enzimas e absorvem nutrientes e as células regenerativas, que se diferenciam e originam os demais tipos celulares do epitélio do ventrículo. Durante a metamorfose das abelhas, o epitélio do ventrículo é substituído, e esta substituição ocorre às custas da diferenciação das células regenerativas larvais. O presente trabalho busca investigar as modificações ocorridas no epitélio do ventrículo da abelha Melipona quadrifasciata anthidioides durante o desenvolvimento pós-embrionário e se existe proliferação das células regenerativas durante a metamorfose do ventrículo, utilizando-se técnicas anatômicas, de imunofluorescência e imunoperoxidase com anticorpo anti BrdU. Foram analisadas larvas de diferentes idades, pré-pupas, pupas de operárias em diferentes fases de desenvolvimento: pupas de olhos brancos, olhos marrons, olhos pretos e operárias adultas. Os resultados revelaram que na larva ocorre proliferação de células regenerativas, o que pode ser comprovado pela observação de metáfases, culminando com o aumento do número de células regenerativas por ninho. Com o envelhecimento da larva o número e o tamanho dos ninhos de células regenerativas aumentam. A substituição do epitélio larval pelo epitélio do adulto ocorre a partir do segundo dia após o inicio da defecação. Neste período os ninhos de células regenerativas estão em contato, formando uma espécie de rede, lembrando um sincício pelo fato de não ter sido observado o limite celular. Concomintante à degeneração das células digestivas larvais ocorre a diferenciação das células regenerativas, formando as células digestivas do adulto. Células regenerativas BrdU positivas são observadas somente na fase de pré-pupa, 12 h após a injeção de BrdU, indicando que é durante esta fase que a população de células regenerativas aumenta. Pode-se entender a proliferação das células regenerativas como um processo compensatório, que repõe as células regenerativas que se diferenciam durante a metamorfose do ventrículo. Parece que a existência de proliferação de células regenerativas culmina no aumento da população de células regenerativas e não na formação de novas células digestivas. A hipótese de que células digestivas dos adultos se originam através da proliferação e posterior diferenciação das células regenerativas durante a metamorfose do ventrículo de M. quadrifasciata anthidioides pode ser rejeitada porque a proliferação das células regenerativas durante a pupação não seria suficiente para restabelecer os ninhos de células regenerativas e ao mesmo tempo formar novas células digestivas e pelo fato de que a população de células regenerativas existente durante o processo de defecação parece ser suficiente para promover a renovação do epitélio do ventrículo durante a metamorfose. The digestive tract of insects can be divided into foregut, midgut and hindgut. Fore and hindgut have an ectodermic origin, while midgut is endodermic. Midgut is responsible for the enzyme secretion and nutrient absorption. The midgut epithelium is formed basically by the digestive cells, responsible for the enzyme secretion and nutrient absorption and for the small regenerative cells that are placed in nests scattered among the digestive cells. During midgut metamorphosis, the epithelium is substituted to attend adult's nutritional needs, and this substitution occurs due to larval regenerative cells differentiation. The present work focused on the midgut epithelial modifications during the post- embryonic development of the stingless bee Melipona quadrifasciata anthidioides. The presence of regenerative cells proliferation during midgut metamorphosis was also investigated, using anatomical and imunofluorescence techniques with antibody against BrdU. Were analyzed specimens at the following developmental stage: larvae of different ages, prepupae, White eyed, brown eyed and black eyed pupae and adult workers. Regenerative cell proliferation is easily detected during larval period by the presence of metaphase, which results in the regenerative cell number increasing. During larval aging the number and the size of the nests of regenerative cells also increases. Larval epithelium degeneration starts in the second day after defecation process had initiated. In this period the nests of regenerative cells are in contact and cell limit were not visualized, resembling a sincicium. The degeneration of the larval digestive cells and the regenerative cells differentiation into adult's digestive cells occurs at the same time. The BrdU positive regenerative cells are only observed in the prepupae period, 12 h after BrdU injection indicating that regenerative cell population increase during this period. Regenerative cell proliferation can be understood as a compensatory process, responsible for the regenerative cell replacing that are consumed during midgut epithelial renewing during metamorphosis. It probably assures the maintenance of the adult midgut epithelium. It seems that the regenerative cell proliferation culminates in the increase of the regenerative cells population and not in the formation of new digestive cells because the proliferation of regenerative cells would not be enough to reestablish the nests of regenerative cells and at the same time form new adult digestive cells. In this sense the hypothesis that digestive adult cells are originated through the regenerative cell proliferation and posterior differentiation during metamorphosis can be rejected. M. quadrifasciata anthidioides midgut

Conselho Nacional de Desenvolvimento Científico e Tecnológico O objetivo deste trabalho foi estudar os efeitos do aumento na abundância de Apis mellifera, espécie exótica, sobre a coleta de pólen por Melipona quadrifasciata, espécie nativa, em Viçosa, MG. Para isso, foi estudado o padrão diário de atividade de coleta de pólen por M. quadrifasciata em áreas de pastagem abandonada e em áreas de fragmentos de mata secundária, com a finalidade de testar a hipótese de que a abundância aumentada de A. mellifera causa alterações neste padrão. Também foram identificadas as espécies de plantas que são fontes de pólen para ambas as espécies nas mesmas áreas, a fim de testar a hipótese de que a abundância aumentada de A. mellifera causa alterações na quantidade e na qualidade do pólen coletado por M. quadrifasciata. A atividade de coleta de pólen por M. quadrifasciata se iniciava ainda durante o nascer do sol e se concentrava nas primeiras horas da manhã. Cerca de 69.40% de todas as cargas de pólen foram coletadas até as 10 horas da manhã. A abundância aumentada de A. mellifera não causou alterações sobre o padrão diário de coleta de pólen por M. quadrifasciata. Para a coleta de pólen, M. quadrifasciata visitou, nas áreas de pastagem abandonada, 23 espécies de plantas pertencentes a 9 famílias de Angiospermae e, nos fragmentos de mata, foram coletadas cargas de pólen de 20 espécies pertencentes a 10 famílias. M. quadrifasciata visitou, para a coleta de pólen, principalmente plantas de porte arbóreo, com floração massal, e flores que oferecem o pólen como recurso. Embora seja generalista, M. quadrifasciata é mais restritiva que outras abelhas eussociais para a coleta de pólen e apresenta uma oligolecia temporal. As abelhas dos ninhos de A. mellifera instalados nas áreas de pastagem visitaram 25 espécies de plantas, pertencentes a 16 famílias. Nos ninhos instalados na mata, foram encontradas 33 espécies pertencentes a 21 famílias. A. mellifera também é generalista e utilizou plantas com floradas abundantes, embora não tenha utilizado apenas plantas de porte arbóreo para a coleta de pólen. Embora houvesse uma sobreposição de nicho relativamente alta, a abundância aumentada de A. mellifera não apresentou efeitos sobre a largura de nicho e sobre a quantidade e a qualidade do pólen coletado por M. quadrifasciata. A não detecção de efeitos da abundância aumentada de A. mellifera pode acontecer em decorrência do fato de que pólen não é um fator limitante ou do fato de que o sistema já está estável, moldado à presença de A. mellifera. É possível, também, que a não detecção de efeitos seja decorrente do número de colônias de A. mellifera utilizadas em cada experimento não ser suficiente para significativamente aumentar a abundância nas áreas estudadas, embora seja útil para se medir efeitos da introdução de ninhos de abelhas africanizadas para a produção apícola. Embora os dados desse trabalho apontem para uma ausência de efeitos sobre as abelhas eussociais, a presença da A. mellifera nas regiões Neotropicais ainda tem muitos aspectos a serem estudados. This work’s goal is to study the effects of the increased abundance of Apis mellifera, an exotic species, in the pollen collection of Melipona quadrifasciata, a native species. In order to do this the daily pattern of pollen collection by M. quadrifasciata was studied, to test the hypothesis that the increased abundance of A. mellifera resulted in changes in this pattern. The plant species used as pollen sources by both species were also studied, to test the hypothesis that the increased abundance of A. mellifera resulted in quantitative and qualitative changes in pollen collection by M. quadrifasciata. Pollen collection by M. quadrifasciata began during sunrise and was concentrated in the first hours of the morning. About 69.40% of all the pollen loads were collected until 10 o’clock in the morning. The increased abundance of A. mellifera didn’t change the daily pattern of pollen collection by M. quadrifasciata. Twenty-three plant species belonging to 9 families of Angiosperms were visited by M. quadrifasciata for pollen collection in the abandoned grazing areas while in the areas of forest fragments, it visited 20 plant species from 10 families. M. quadrifasciata used mainly trees with mass flowering as pollen sources and flowers that offer pollen as reward. Though it is generalist, M. quadrifasciata is more restrictive than other eusocial bees and shows a temporal oligolecy. The bees from the A. mellifera nests visited 25 plants species from 16 families in the grazing abandoned areas and 33 plant species from 21 families in the areas of forest fragments. A. mellifera is also a generalist and used plants with abundant flowering, though it didn’t used only trees. Even though there was a relatively high niche overlap, the increased abundance of A. mellifera didn’t change M. quadrifasciata’s niche breadth and the quality or quantity of pollen collected. Not detecting effects of the increased abundance of can happen due to the fact that pollen is not a limiting factor or that the system is stable, adapted to the presence of A. mellifera. Or maybe it is due to the fact that the number of colonies of A. mellifera used wasn’t high enough to significantly increase its abundance; it is useful, however, to measure the effects of introduction of africanized honey bee nests for beekeeping. Even though data from this work show that there are no effects on eusocial bees, the presence of A. mellifera in the Neotropics still has many aspects to be studied.

The yeast communities associated with the stingless bees Tetragonisca angustula, Melipona quadrifasciata and Frieseomelitta varia were studied. The bees T. angustula and F. varia showed a strong association with the yeast Starmerella meliponinorum. M. quadrifasciata more frequently carried a species related to Candida apicola, but also vectored low numbers of S. meliponinorum. Some of the yeasts isolated from adult bees were typical of species known to occur in flowers. Other yeast species found in adult bees were more typical of those found in the phylloplane. S. meliponinorum and the species in the C. apicola complex, also part of the Starmerella clade, may have a mutualistic relationship with the bees studied. Many yeasts in that group are often found in bees or substrates visited by bees, suggesting that a mutually beneficial interaction exists between them.

Neste estudo descrevemos a ultra-estrutura das células enteroendócrinas observadas no intestino médio de M. quadrifasciata anthidioides, abelha na qual ocorrem dois tipos de células enteroendócrinas, concentradas na região posterior do intestino médio. As células do tipo fechado são menores e possuem grânulos de secreção menos eletrodensos e menos regulares do que as células do tipo aberto. As células do tipo aberto possuem ainda longas mitocôndrias concentradas principalmente na região basal, onde também se concentra a maior parte dos grânulos de secreção. Além dos grânulos de secreção e das mitocôndrias, as células endócrinas nessa espécie possuem grandes vacúolos autofágicos e elementos do complexo de Golgi bem desenvolvidos. In this study we describe the ultrastructure of the endocrine cells observed in the midgut of M. quadrifasciata anthidioides. This bee has two types of endocrine cells, which are numerous on the posterior midgut region. Cells of the closed type are smaller and have irregular secretory granules with lower electrondensity than those of the open cell type. The open cell type has elongated mitochondria mainly on the basal area, where most of the secretory granules are also found. Besides the secretion granules and mitochondria, endocrine cells in this species have well-developed autophagic vacuoles and Golgi complex elements.

Thirty-two strains of the novel species Starmerella meliponinorum sp. nov. were isolated from various substrates associated with three stingless bee species (tribe Meliponini) in Brazil and one in Costa Rica. The strains were found in garbage pellets (faecal material, discarded pollen or food, etc.), pollen provisions, adult bees, honey and propolis of Tetragonisca angustula, in honey from Melipona quadrifasciata and in adults of Melipona rufiventris and Trigona fulviventris. The sequence of the D1/D2 domains of the large-subunit rDNA showed that the novel species belongs to the Starmerella clade and is most closely related to Candida etchellsii, although the two differ in their sequences by 7 % base substitutions. S. meliponinorum is homothallic and assimilates few carbon sources. Nitrate is utilized as a sole nitrogen source. The type strain of S. meliponinorum is strain UFMG-01-J26.1T (=CBS 9117T).

Testes length in diploid and haploid larvae drones of Melipona quadrifasciata were investigated to ascertain whether it is a good character to distinguish haploid from diploid drones. Two groups of haploid and two groups of diploid drones were compared. The results show that diploid males have shorter testes than haploid males. Another interesting observation was that whereas diploid males from different groups had testes of similar sizes, haploid males produced by different queens had testes which differed significantly in size. It is suggested that measurements of the testes length could be used to determine ploidy and contribute to the understanding of the population biology of this and other related Hymenoptera species. Melipona quadrifasciata / stingless bee / diploid drone / testicule

We analyzed patterns of heterochromatic bands in the Neotropical stingless bee genus Melipona (Hymenoptera, Meliponini). Group I species (Melipona bicolor bicolor, Melipona quadrifasciata, Melipona asilvae, Melipona marginata, Melipona subnitida) were characterized by low heterochromatic content. Group II species (Melipona capixaba, Melipona compressipes, Melipona crinita, Melipona seminigra fuscopilosa e Melipona scutellaris) had high heterochromatic content. All species had 2n = 18 and n = 9. In species of Group I heterochromatin was pericentromeric and located on the short arm of acrocentric chromosomes, while in Group II species heterochromatin was distributed along most of the chromosome length. The most effective sequential staining was quinacrine mustard (QM)/distamycin (DA)/chromomycin A3(CMA3)/4-6-diamidino-2-phenylindole (DAPI). Heterochromatic and euchromatic bands varied extensively within Group I. In Group II species euchromatin was restricted to the chromosome tips and it was uniformly GC+. Patterns of restriction enzymes (EcoRI, DraI, HindIII) showed that heterochromatin was heterogeneous. In all species the first pair of homologues was of unequal size and showed heteromorphism of a GC+ pericentromeric heterochromatin. In M. asilvae (Group I) this pair bore NOR and in M. compressipes (Group II) it hybridized with a rDNA FISH probe. As for Group I species the second pair was AT+ in M. subnitida and neutral for AT and GC in the remaining species of this group. Outgroup comparison indicates that high levels of heterochromatin represent a derived condition within Melipona. The pattern of karyotypic evolution sets Melipona in an isolated position within the Meliponini.

Them aim scope of this study is to characterize the enzymatic polymorphism found in the Melipona quadrifasciata Lepeletier, 1936 populations from Ribeirao Preto, Sao Paulo and Espirito Santo, Brazil and its hybrids. Samples from each colony (about 52) were prepared for starch gel electrophoresis in order to investigate the genetic variation of the following enzimes: esterase (EST), isocitrate dehydrogenase (IDH), malic enzyme (ME), phosphoglucomutase (PGM), superoxide desmutase (SOD), α-glycerophosphate dehydrogenase (αPGD), malate dehydrogenase (MDH), leucine aminopeptidase (LAP), hexokinase (HK) and phosphoglucose isomerase (PGI). The analysis showed that LAP and HK did not show enzymatic activity and EST showed two alleles(est-sand and est-f) while all the others were shown to be monomorphic. The allele EST-S showed a frequency of 82,6%.

International audience; To find out what foragers of the stingless bees Melipona scutellaris and M. quadrifasciata actually do while recruiting nestmates to a food source we videotaped their behavior in the nest and simultaneously recorded the sounds and vibrations produced by them when returning from a rich food source. Neither temporal nor spectral characteristics of the sounds and vibrations correlated significantly with distance or direction to the food. Foragers motivated recruits to search for food at random by a "jostling run". The number of jostles by a forager correlates with the number of collecting bees. There was no correlation between the movements of a returning forager and either distance or direction to the feeder. "Zigzag flights", guiding flights and scent marking of foragers were excluded as a way to communicate the location of the food source. Aside from the indication of the mere existence of a food source the mechanisms by which foragers communicate its location are still obscure.; Comportement de recrutement chez les abeilles sans aiguillon, Melipona scutellaris et Melipona quadrifasciata. II. Mécanismes éventuels de communication. Afin d'élucider ce que font réellement les abeilles sans aiguillon Melipona scutellaris et Melipona quadrifascita (Hymenoptera, Apidae) lorsqu'elles recrutent des congénères du nid à une source de nourriture, nous avons dressé trois abeilles à venir visiter un nourrisseur situé à diverses distances de la ruche et dans diverses directions. Les butineuses qui rentraient déchargeaient leur nourriture à l'intérieur d'une boîte d'enregistrement installée entre l'entrée du nid et le nid lui-même (Fig. 1). Là, nous avons enregistré en vidéo le comportement des butineuses et leurs interactions avec les abeilles de la ruche et avec les recrues et enregistré simultanément les sons et les vibrations qu'elles produisaient de retour d'un nourrisseur avec un sirop très concentré (1,5 mol.l$^{-1}$). L'analyse des sons et des vibrations a montré que tous les paramètres mesurés (durée du signal, nombre et durée des impulsions, durée des intervalles entre les impulsions et fréquence principale d'un signal ; Figs. 9 et 10) variaient largement (Fig. 11). Contrairement aux résultats de Esch [2] pour M. quadrifasciata et M. merillae et de Nieh et Roubik [11] pour M. panamica, nous n'avons trouvé aucune corrélation entre les durées des impulsions et la distance à une source de nourriture (Fig. 13). Ni la répartition dans le temps ni les caractéristiques spectrales des signaux ne sont corrélées significativement avec la distance ou la direction du nourrisseur (Tab. III). Outre l'indication par le son et les vibrations de l'existence d'une source de nourriture bien approvisionnée, les butineuses stimulent les recrues à rechercher de la nourriture au hasard à l'aide d'une " course-bousculade "(Fig. 3). Le nombre de bousculades est corrélé au nombre d'abeilles qui s'envolent (Fig. 12). Lorsqu'elles courent à travers la ruche, les butineuses tournent et changent de direction fréquemment. Pourtant il n'y a pas de "danse "indiquant la localisation de la source de nourriture et aucune corrélation n'a été trouvée entre ces mouvements et soit la distance, soit la direction de la source (Fig. 5 ; Tabs. I et II). On peut exclure les vols en zig-zag et les vols d'accompagnement des mécanismes qui indiquent la localisation d'une source de nourriture. Les butineuses et les recrues ne quittaient la ruche ensemble que dans 8 % des observations. Les futures recrues ne quittaient jamais la ruche plus d'une fois ($0,7 \pm 0,2$) avant de s'envoler pour une source de nourriture, où elles étaient capturées. De même la fréquence du marquage olfactif était bien trop faible dans nos expériences pour être utilisé comme moyen de communication de la localisation de la source de nourriture. Les futures recrues n'entrent en contact avec les butineuses ou avec la nourriture que celles-ci rapportent qu'environ une heure avant leur propre envol. Nos résultats suggèrent que les recrues obtiennent d'abord une information sur la qualité de la nourriture (nombre maximum de contacts alimentaires 20 minutes avant leur capture), puis une information sur la localisation de la source (nombre maximum de contacts directs avec les butineuses environ 10 minutes avant leur capture, Fig. 6). Lorsque les futures recrues de M. scutellaris et M. quadrifasciata établissent le contact avec une butineuse qui rapporte de la nourriture, c'est de préférence dans la région thorax-abdomen (Fig. 7). Nous avons donc montré comment l'existence d'une source de nourriture bien approvisionnée était indiquée ; par contre les mécanismes par lesquels les butineuses communiquent la distance et la direction restent toujours obscurs.

International audience; The two stingless bee species Melipona scutellaris and M. quadrifasciata recruit nestmates to a rich foraging site. We tested this with feeders up to 140 m away from the hive. Foragers of M. scutellaris communicated direction (up to 140 m) more accurately than distance (up to 30 m) whereas those of M. quadrifasciata communicated direction only up to 30 m and distance up to 40 m. Our data indicate that in both species recruitment is divided into two temporal phases. Whereas in an initial phase alarmed nestmates search for food at random, bees leaving the hive in the following phase are obviously provided with information about its specific location. As a consequence after 35 minutes (M. scutellaris) and 85 minutes (M. quadrifasciata), respectively, significantly more newcomers arrive at the feeder than at an identical control feeder. The differences found in the recruitment success of M. scutellaris and M. quadrifasciata are discussed in regard to the different demands of their natural habitats.; Comportement de recrutement des abeilles sans aiguillon, Melipona scutellaris et Melipona quadrifasciata. I. Butinage à des sources de nourriture situées dans des directions et à des distances variables. Cherchant principalement à étudier les mécanismes sensoriels impliqués dans le comportement de recrutement des abeilles sans aiguillon (voir l'article associé [5]), nous avons d'abord voulu établir de façon certaine si les deux espèces étudiées Melipona scutellaris et Melipona quadrifasciata recrutaient ou non des butineuses pour une source de nourriture donnée. Pour chaque expérience nous avons dressé cinq butineuses marquées à venir visiter un nourrisseur distant de 140 m au maximum de la ruche (fig. 1). Durant trois heures nous avons prélevé toutes les nouvelles venues sur le nourrisseur test et sur le nourrisseur témoin, qui différait l'un de l'autre soit par la distance, soit par la direction. A l'aide du test $\chi^2$ (niveau de signification $p \le 0,05$) nous avons testé la répartition des abeilles sur les deux nourrisseurs par rapport au hasard. Les deux espèces recrutent bien des congénères du nid pour une source de nourriture bien approvisionnée. Chez M. scutellaris la direction (jusqu'à 140 m, fig. 2a) est communiquée plus précisément que la distance (jusqu'à 30 m, fig. 3a), alors que chez M. quadrifasciata la direction n'est communiquée que jusqu'à 30 m (fig. 2b) et la distance jusqu'à 40 m (fig. 3b). Chez les deux espèces le recrutement comporte deux phases : dans la phase initiale les congénères mises en alerte cherchent la nourriture au hasard ; dans la seconde phase, les abeilles qui quittent la ruche possèdent visiblement l'information sur la localisation spécifique de la nourriture. En conséquence, au bout de 35 min pour M. scutellaris et 85 min pour M. quadrifasciata, les nouvelles venues arrivent en plus grand nombre sur le nourrisseur test (fig. 4). Ce résultat suggère la succession suivante d'événements : 1) quand une butineuse découvre une source de nourriture bien approvisionnée, elle communique d'abord son existence, son odeur et sa qualité à l'intérieur de la ruche ; 2) les congénères alertées sortent et cherchent au hasard une source de nourriture équivalente. Ceci peut constituer une stratégie fructueuse dans des habitats, tels que la forêt tropicale, où les plantes fleurissent en masse ; 3) les abeilles, dont la recherche au hasard a été infructueuse retournent à la ruche, et obtiennent, des butineuses, qui rentrent chargées, l'information sur la localisation de la source. Les différences entre les deux espèces peuvent peut-être s'expliquer par la différence de leurs milieux naturels : M. quadrifasciata vit dans la végétation dense de la forêt pluviale atlantique du Brésil, où les arbres de floraison massive peuvent être plus abondants ou plus faciles à rencontrer que dans une forêt ouverte, qui est l'habitat de M. scutellaris. Une recherche faite plus ou moins au hasard peut suffire à M. quadrifasciata.

Scent marking of profitable food sources improves foraging efficiency and helps social bees meet colony demands for unpredictable floral resources (Giurfa and Nunez, 1992). Sophisticated marking has evolved in the stingless bees (Apidae, Meliponini); several genera recruit nestmates to profitable food sources with scent signals (reviewed in Nieh, 2004). Scent cues such as “footprints” (Wilms and Eltz, 2008) can also facilitate navigation and orientation to rewarding parts of food patches. Scent marks may improve colonies’ competitive ability, especially in the tropics, where social bee abundance and diversity are high. We assessed scent mark use by the stingless bee Melipona quadrifasciata. This species poorly communicates to nestmates the location of rich food sources (Jarau et al., 2000), thus could benefit from within-patch orientation information provided by attractive odor marks. However, its ability to deposit and use fieldbased information is not known. We studied four Melipona quadrifasciata anthidioides colonies at the Universidade de Sao Paulo, Ribeirao Preto in July 2007, and July– August 2008, using feeder choice tests (see, for example, Hrncir et al., 2004). To begin a trial, we allowed foragers trained to feed on 1.5 M sucrose solution to recruit 15 nestmates not previously used in the experiment. The 15 bees freely fed at a “visited” feeder for 10 min, during which we counted the number of visits. Next, we aspirated all bees and replaced the original apparatus with the visited and clean feeders on clean tripods, separated by 30 cm.

Three methods of division of Melipona quadrifasciata Lep. colonies were evaluated: fisogastric queen naturally mated; orphans colonies, and queen mated in laboratory. Out of six colonies, five had grade 7 in 221 days, reaching 83,3% of success, in the 1st method. Out of four, 100% were formed using the 2nd method, in 221 days, and 71,4% out of seven colonies were formed with grade 7 in 195 days using the 3rd method. All colonies were treated with Syrup-A; the same management and same system of evaluation. Colonies obtained were described performance by the following mathematic models: Grade1 = 1.202 + 0.262 x reading; Grade2 = 1.474 + 0.250 x reading and Grade3 = 1.406 + 0.287 x reading. The 3rd method was the best considering time of the colonies development; however, it consumed more labor and laboratory equipments.

Observation colonies containing only young workers from 10 matrix colonies were set up to investigate the genetic aspects involved in task division in Melipona quadrifasciata. Wide variation among origins was observed for all behaviors analyzed, but these differences were significant only for brood cell construction and propolis preparation Para o estudo dos aspectos genéticos envolvidos na divisão de trabalho em Melipona quadrifasciata foram construídas colônias de observação contendo apenas operárias jovens originadas de 10 colônias. Em todos comportamentos analisados houve uma grande variação entre as origens, mas somente os comportamentos de construção de células de cria e propolisação apresentaram diferenças significativas entre as origens

The objective of the present study was to test three different procedures for DNA extraction of Melipona quadrifasciata based on existing methods for DNA extraction of Apis, plants and fungi. These methods differ in the concentrations of specific substances in the extraction buffer. The results demonstrate that the method used for Apis is not adequate for DNA extraction from M. quadrifasciata. On the other hand, with minor modifications this method and the methods for plants and fungi were adequate for DNA extraction of this stingless bee, both for adults and larvae O objetivo deste trabalho foi o de testar três protocolos de extração de DNA utilizados para Apis, plantas e fungos, visando determinar um que seja eficiente para extração de DNA de Melipona quadrifasciata. Esses métodos diferem nas concentrações de componentes específicos do tampão de extração. Os resultados obtidos mostraram que a metodologia recomendada para extração de DNA de Apis não é adequada para a extração de DNA de M. quadrifasciata. Entretanto, com pequenas modificações, este método, bem como aquele utilizado para a extração de DNA de plantas e fungos, mostrou-se eficiente para a extração de DNA desta abelha sem ferrão, tanto de indivíduos adultos como de larvas