Circular RNA ame_circ_000115 regulates expression of genes in larval gusts of Apis mellifera ligustica stressed by Ascosphaera apis / 生物工程学报

Circular RNAs (circRNAs) are a new class of non-coding RNAs, which have been confirmed to regulate insect gene expression and immune response through multiple manners such as competing endogenous RNA (ceRNA) regulatory network. Currently, function of circRNA in honey bee immune response remains unclear. In this study, PCR and Sanger sequencing were performed to validate the back splicing (BS) site of ame_circ_000115 (in short ac115). RT-qPCR was used to detect the expression profile of ac115 in larval guts of Apis mellifera ligustica stressed by Ascosphaera apis. Dual-luciferase reporter gene assay was conducted to verify the binding relationship between ac115 and ame-miR-13b. Interference of ac115 in larval guts was carried out by feeding specific siRNA, followed by determination of the effect of ac115 interference on expression of six genes relevant to host immune response. The results confirmed the existence of BS site within ac115. Compared with the un-inoculated group, the expression of ac115 in 4-day-old larval gut of the A. apis-inoculated group was up-regulated with extreme significance (P < 0.000 1), while that in 5- and 6-day-old larval guts were significantly up-regulated (P < 0.05). The brightness of specific band for ac115 in 4-, 5- and 6-day-old larval guts of the siRNA-circ_000115-fed group gradually became weak, whereas that of the siRNA-scrambl-fed group was pretty high without obvious variation. Compared with that of the siRNA-scramble-fed group, the expression of ac115 in 4-day-old larval gut of the siRNA-circ_000115-fed group was significantly down-regulated (P < 0.05), whereas that of the 5- and 6-day-old larval guts were down-regulated with extreme significance (P < 0.001). Ame-miR-13b was truly existed and expressed in A. m. ligustica larval guts, and there was true binding relationship between ac115 and ame-miR-13b. Compared with that of the siRNA-scramble-fed group, the expression of antimicrobial peptide genes hymenoptaecin and abaecin in 6-day-old larval gut of the siRNA-circ_000115-fed group was significantly up-regulated (P < 0.05), while that of ecdysone receptor (Ecr) was down-regulated with extreme significance (P < 0.01). These results indicate that ac115 is truly expressed in A. m. ligustica larval guts, BS site truly exists within ac115, and effective interference of ac115 in A. m. ligustica larval guts can be achieved via feeding siRNA. Moreover, ac115 potentially regulates Ecr expression through adsorption of ame-miR-13b and expression of hymenoptaecin and abaecin using a non-ceRNA manner, further participating in host stress-response.

Evaluation of physic contamiants and contamination with coliforms, molds and yeasts of honey from the Northern Brazil / Avaliação de contaminantes físicos e contaminação com coliformes, bolores e leveduras de mel do Norte do Brasil

The Mercosur Technical Regulation for honey determines microbiological parameters for total coliforms and molds and/ yeasts, and absence of physical contaminants, of any nature. The purpose of this study was to verify the biological and physical contamin of Apis mellifera ligustica and Melipona fasciculata, commercialized in the northeastern region of the State of Pará, Brazil. The fourteen samples from informal trade of the municipalities of Bragança, Capanema, Nova Timboteua, São João de Pirabas, Salinas and Tracauateua were analyzed for the Most Probable Number of colifroms at 35ºC, Fungus and Yeast counts, besides the detection of dirt by Macro and Microscopy techniques. The results obtained from the microbiological analyzes were compared to the standard required by MERCOSUR, for coliforms. 57.14% of the samples were non-standard and ranged from <3.0 NMP/g to 20 NMP/g. The molds and/ yeast counts ranged from 1x10¹ CFU/g to 8.6x10² CFU/g, with atotal of 21.42% of the samples being in disagreement with the legislation. From the macroscopic analysis, it was observed that, although 50% of the samples did not present any apparent dirt, microscopically it was possible to observe dirt in 100% of the samples of honey analyzed, which is out of the standard required by the Brazilian legislation. This shows the low sanitary quality of honey commercialized in this region of the state of Pará.

O Regulamento Técnico do Mercosul para mel, determina parâmetros microbiológicos para coliformes totais e fungos e/leveduras, e ausência de contaminantes físicos, de qualquer natureza. A proposta do presente estudo foi verificar a contaminação biológica e física em méis de abelha das espécies Apis mellifera ligustica e Melipona fasciculata, comercializados na região Nordeste do Estado do Pará, Brasil. As amostras provenientes do comercio informal dos de municípios de, Bragança, Capanema, Nova Timboteua, São João de Pirabas, Salinas e Tracauateua, foram analisadas quanto a determinação do Número Mais Provável de coliformes a 35ºC, Contagem de Fungos e/leveduras, além da detecção de sujidades pelas técnicas de Macro e Microscopia. Os resultados obtidos a partir das análises microbiológicas foram comparadas ao padrão exigido pelo MERCOSUL, para coliformes, 57,14% das amostras encontravam-se fora do padrão variaram de <3,0 NMP/g a 20NMP/g. Já a contagem de fungos e/leveduras variou de 1x10¹ UFC/g a 8,6x10² UFC/g, no total de 21,42 % das amostras apresentaram-se em desacordo com a legislação. A partir da análise macroscópica observou-se que, embora 50% das amostras não apresentassem sujidades aparentes, porém microscopicamente foi possível observar a presença sujidades em 100% das amostras de méis analisadas encontrando-se fora do padrão exigido pela legislação Brasileira. Demostrando a baixa qualidade sanitária do mel comercializado nessa região do estado do Pará.