Divergência genética interpopulacional em Tetragonisca angustula Latreille, 1811 (Hymenoptera, Apidae, Meliponidae)

Trabalho de Conclusão de Curso (Graduação)Tetragonisca angustula (jataí) apresenta uma das mais amplas distribuições entre os Meliponíneos, abrangendo praticamente toda a America do Sul, Central e México.» A técnica de RAPD (Random 'Amp/ifiedPo—lymorphic DNA)-tem sido utilizada na caracterização de populações, detecção de polimorfismos genéticos, análises filogenéticas e, inclusive, confirmação de taxonomia baseada — em dados morfológicos. Esse trabalho objetivou-se estimar a diversidade genética entre populações de T. angustula de três regiões (Triângulo Mineiro/MGz-Uberlândia 'l, 2, 3, 4 e 5, Ituiutaba 1, 2 e 3 e Uberaba 1 e 2; Sul de Minas/MG: São Miguel do Anta 1, 2, 3, 4a ti;-Argentina: Aristóbulo-delValle-e Posadas 1 e 2), usando Tetragoma clavipes como outgroup. Foram feitos bu/ks com 5 individuos por local de amostragem, utilizando-se »15 primers (13 curtos e 2 longos) «com as seguintes condições de reação: 10ng de DNA, 10pmoles de primer, 2mM de MgClz, 1.5U de Taq DNA polimerase, vloopM de cada dNTP, 50mM de KCl e iOmM de Tris—HCl pH 8.0 .Os produtos das amplificações foram separados em gel de agarose 12% e fotografados. Os resultados foram analisadospelo método de- porcentagem de desacordo e análise de cluster por UPGMA. Ao nível de 162%, as amostras de jataí foram divididas em duas clades: uma correspondente à região do Triângulo Mineiro e outra para as amostras da Argentina e Sul de Minas que, por sua vez, se divergiram em 2 grupos -a 14.2—%.- As amostras »do Triângulo Mineiro apresentaram divergência máxima de 6,5%, com 3 genótipos (dois de Uberlândia e um de ituiutaba) que não apresentaram?dissimilaridade genética. As amostras do- Sul de Minas apresentaram divergência genética máxima de 4.4% e as da Argentina de 55%. Tetragona clavipes divergiu em 69%das amostras de T. angustula. Os valores encontrados para T. angustula foram similares aos encontrados na literatura dentro de uma mesma região - (Triangulo Mineiro); No entanto,- para regiões geograficamente distantes (Sul de Minas e Triângulo Mineiro), os valores encontrados foram superiores. isto pode ser devido ao uso de DNA proveniente de bulks de cinco indivíduos, diferenças na Taq DNA polimerase e nos protocolos de extração. Novas- estimativas de divergência para esta espécie usando—se bulks-odm maior número de indivíduos e maior número de primers forneceriam dados conclusivos sobre o número mínimo “ amostral- por localidade e taxa- de dissimilaridade genética padrão para a espécie

Leucine-aminopeptidase A (LAP-A) Encoding Gene in Apoidea: from Genomic Identification to Functional Insights Based on Gene Expression

Aminopeptidases are enzymes that cleave the N-terminal region of proteins and show structural conservation in prokaryotes and eukaryotes. We aimed to identify leucine-aminopeptidase A (LAP-A) orthologs in the genome of bee species with diff erent levels of social organization, and to explore the putative roles of this enzyme based on gene expression data. We identified a single gene for LAP-A on chromosome 15 of Apis mellifera L. and predicted orthologs in genomes of 11 bee species. We found evidence of LAP-A expression in more than 50 bee species. In honeybee and other bees, LAP-A transcripts were expressed in diverse tissues, including: brains, fat bodies, ovaries, testicles, integuments, and glands, on diff erent developmental stages that spanned from embryogenesis to adult life. Our fi ndings on the transcriptional activity of LAP-A are consistent with previously published data on enzymatic activity of LAP-A in bees throughout the development in different tissues and in both sexes. The presence of LAP-A gene in the Apoidea genomes and its ubiquitous expression support housekeeping roles of this enzyme and broad-spectrum functions in bees, independente of their life styles

Double-stranded RNA ingested by Apis mellifera larvae promotes phisiological disturbs in caste development

Abelhas adultas produzem vitelogenina, a principal proteína da hemolinfa. Ela está envolvida na reprodução, comportamento, imunidade, longevidade e regulação da organização social. A interferência por RNA interference é a mais promissora ferramenta para estudos de função gênica, baseada na introdução de duplex de RNA (dsRNA) que induz a degradação de transcritos alvo-específicos. Injeção de dsRNA altera a transcrição de vitelogenina, mas evidências apontam que a ativação do sistema imune em abelhas seja um efeito colateral destaa manipulação. Desenvolvemos um método para o silenciamento do gene codificador de vitelogenina no desenvolvimento pós-embrionário, que minimiza os efeitos da manipulação, onde 0,5 ?g de dsRNA de vitelogenina (dsVg) ou de GFP (controle exógeno, dsGFP) foi oferecido na dieta natural de larvas de segundo estágio, as quais foram mantidas na colônia. Nosso enfoque principal foi a compreensão dos efeitos do silenciamento pós-transcricional de rainhas e operárias de A. mellifera, em especial na fase larval. Operárias adultas reconhecem larvas tratadas e as remove. Mantemos certa distância entre as células de cria que recebiam o tratamento e a remoção de larvas tratadas diminuiu consideravelmente. A expressão de transcritos de vitelogenina em indivíduos sem tratamento e tratados foi analisada no quinto estágio larval de ambas as castas, bem como em operárias adultas de 7 dias e rainhas recémnascidas, utilizando-se PCR em tempo real e a expressão do gene codificador de actina como controle endógeno. Em adultos, controles sem tratamento e dsGFP expressaram quantidades similares de transcritos de vitelogenina. Os grupos alimentados com dsVg tiveram expressão reduzida de vitelogenina, a saber: quinto estágio larval de operárias (91%) e de rainhas (71%), operárias de 7 dias (88%) e rainhas recém-nascidas (70%). O silenciamento da vitelogenina não afetou a morfologia dos adultos, mas sim a fisiologia de larvas de ambas as castas, como nos títulos de hormônio juvenil e concentração de proteínas circulantes na hemolinfa. Concluímos que a ingestão de dsRNA é um método não-invasivo que induz silenciamento gênico e, assim, uma ferramenta eficiente para estudos funcionais pós-genoma. Os mecanismos regulatórios do gene codificador de vitelogenina e seu papel na diferenciação de castas estão em discussão.Adult bees produce vitellogenin (Vg), the main protein in hemolymph; it is involved in honey bee (Apis mellifera) reproduction, behavior, immunity, longevity and regulation of social organization. Genetic interference mediated by injection of double-stranded RNA (dsRNA) is a powerful tool for the analysis of gene function in Apis mellifera. Injection of dsRNA effectively alters vitellogenin transcription; however, evidence has been found of immune system activation in treated bees, which could be a collateral effect of treatment. Consequently, we developed a non-invasive protocol for disruption of the A. mellifera genes exemplified by vitellogenin mRNA silencing, to understand it, mainly, in the female larval context. Second instar larvae were treated as follows: the treatment group received 0,5 ?g of double-stranded vitellogenin RNA (dsVg) mixed with larval food deposited in the worker brood cells; control group 1 was left to develop without treatment, while control group 2 received dsGFP (Green Fluorescent Protein), as an exogenous control. Treated and control larvae were maintained in the colony until adult emergence. Workers recognized dsRNAtreated larvae and frequently removed them. To circumvent this problem we increased the distance between the treatment groups. Vg gene expression were determined for fifth instar larvae of both castes and for 7 day-old workers and newly-emerged queens, evaluated by quantitative real time PCR, using actin as an endogenous control. For adults, we found that controls, dsGFP- and non-treated bees expressed similar amounts of Vg transcripts. The dsVg-fed groups had significantly reduced Vg gene expression in fifth instar larvae of workers (91%) and queens (71%) and, also, in 7 day-old workers (88%) and newly-emerged queens (70%). Disruption of the Vg gene did not affect adults morphology but physiological larval traits of both castes, as juvenile hormone titre and protein concentration. We conclude that dsRNA ingestion is an effective non-invasive method for inducing knockdown and an efficient approach for post-genome functional studies. The regulatory mechanisms of vitellogenin gene and its rules during caste differentiation are discussed

A non-invasive method for silencing gene transcription in honeybees maintained under natural conditions

in the Apis mellifera post-genomic era, RNAi protocols have been used in functional approaches. However, sample manipulation and invasive methods such as injection of double-stranded RNA (dsRNA) can compromise physiology and survival. To circumvent these problems, we developed a non-invasive method for honeybee gene knockdown, using a well-established vitellogenin RNAi system as a model. Second instar larvae received dsRNA for vitellogenin (dsVg-RNA) in their natural diet. For exogenous control, larvae received dsRNA for GFP (dsGFP-RNA). Untreated larvae formed another control group. Around 60% of the treated larvae naturally developed until adult emergence when 0.5 mu g of dsVg-RNA or dsGFP-RNA was offered while no larvae that received 3.0 mu g of dsRNA reached pupal stages. Diet dilution did not affect the removal rates. Viability depends not only on the delivered doses but also on the internal conditions of colonies. The weight of treated and untreated groups showed no statistical differences. This showed that RNAi ingestion did not elicit drastic collateral effects. Approximately 90% of vitellogenin transcripts from 7-day-old workers were silenced compared to controls. A large number of samples are handled in a relatively short time and smaller quantities of RNAi molecules are used compared to invasive methods. These advantages culminate in a versatile and a cost-effective approach. (c) 2008 Elsevier Ltd. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

A honeybee storage protein gene, hex 70a, expressed in developing gonads and nutritionally regulated in adult fat body

In preparing for metamorphosis, insect larvae store a huge amount of proteins in hemolymph, mainly hexamerins. Out of the four hexamerins present in the honeybee larvae, one, HEX 70a, exhibited a distinct developmental pattern, especially since it is also present in adults. Here, we report sequence data and experimental evidence suggesting alternative functions for HEX 70a, besides its well-known role as an amino acid resource during metamorphosis. The hex 70a gene consists of 6 exons and encodes a 684 amino acid chain containing the conserved hemocyanin N, M, and C domains. HEX 70a classifies as an arylphorin since it contains more than 15% of aromatic amino acids. In the fat body of adult workers, hex 70a expression turned out to be a nutrient-limited process. However, the fat body is not the only site for hex 70a expression. Both, transcript and protein subunits were also detected in developing gonads from workers, queens and drones, suggesting a role in ovary differentiation and testes maturation and functioning. In its putative reproductive role, HEX 70a however differs from the yolk protein, vitellogenin, since it was not detected in eggs or embryos. (C) 2008 Elsevier Ltd. All rights reserved

Genetic divergence in Tetragonisca angustula Latreille, 1811 (Hymenoptera, Meliponinae, Trigonini) based on rapd markers

One of the commonest neotropical stingless bees is Tetragonisca angustula (Latreille, 1811), popularly known in Portuguese as jataí, which occurs in variable nesting sites from Mexico to Argentina. We used 18 primers to generate 218 RAPD markers which we used to determined the genetic distance between T. angustula populations from 25 localities in three different Latin America countries, using Tetragonisca weyrauchi from the Brazilian state of Acre and the common honey bee (Apis mellifera) as outgroups. Genetic distance, calculated as the Percentage of Dissimilarity (14%), based on all markers divided the T. angustula population into eastern (group 1) and western (group 2) groups. However, we were able to separate the two groups by using only two primers that have generated five specific molecular markers. The eastern group consists of T. angustula angustula which occurs from Panama to the Brazilian states of Maranhão and northern Minas Gerais and has spread through the Brazilian Atlantic Forest as far as the southern Brazilian state of Santa Catarina. Group 2 is made up of T. angustula fiebrigi which has a more southerly and western distribution, occurring only in the western Brazilian states of Mato Grosso and Mato Grosso do Sul as well as the west of some other Brazilian states (Goiás, Minas Gerais, São Paulo, Paraná and Santa Catarina) and northeastern Argentina