-Aethina tumida (small hive beetle, SHB) was first detected in September 2014 in Calabria region, southern Italy, and in a single apiary in Sicily in November 2014. In September 2015, SHB was again recorded in Calabria, and in 2016, only sentinel honey bee nucleus colonies were found to be infested. Its phylogenetic relationship and possible origin were investigated comparing the cox1 sequences with the corresponding region available in the GenBank database. The neighbour-joining method revealed that the first Italian specimen belonged to a group also containing an African specimen from Cameroon. The Italian specimens differ from the SHBs spread worldwide and are split into two different groups: group B1 includes the AfricCam3 sequence and the first SHB identified in Calabria; group B2 includes specimens from Calabria and the only one from Sicily which share identical cox1 sequences. SHB in Italy appears to have been introduced from Africa and includes independent or contemporary incursions in the two concerned regions. The most likely scenario is that SHB was introduced into Calabria followed by man-mediated migration to Sicily.Aethina tumida / Apis mellifera / cox1 gene / exotic bee infestation / southern Italy
Prognosis and therapeutic management of dogs with cutaneous mast cell tumors (MCTs) depend on clinical stage and histological grade. However, the prognostic value of this latter is still questionable. In the present study, MCT transcriptome was analyzed to identify a set of candidate genes potentially useful for predicting the biological behavior of MCTs. Fifty-one canine MCT biopsies were analyzed. Isolated and purified total RNAs were individually hybridized to the Agilent Canine V2 4x44k DNA microarray. The comparison of reference differentiated and undifferentiated MCT transcriptome revealed a total of 597 differentially expressed genes (147 down-regulated and 450 up-regulated). The functional analysis of this set of genes provided evidence that they were mainly involved in cell cycle, DNA replication, p53 signaling pathway, nucleotide excision repair and pyrimidine metabolism. Class prediction analysis identified 13 transcripts providing the greatest accuracy of class prediction and divided samples into two categories (differentiated and undifferentiated), harboring a different prognosis. The Principal Component Analysis of all samples, made by using the selected 13 markers, confirmed MCT classification. The first three components accounted for 99.924% of the total variance. This molecular classification significantly correlated with survival time (p = 0.0026). Furthermore, among all marker genes, a significant association was found between mRNA expression and MCT-related mortality for FOXM1, GSN, FEN1 and KPNA2 (p<0.05). Finally, marker genes mRNA expression was evaluated in a cohort of 22 independent samples. Data obtained enabled to identify MCT cases with different prognosis. Overall, the molecular characterization of canine MCT transcriptome allowed the identification of a set of 13 transcripts that clearly separated differentiated from undifferentiated MCTs, thus predicting outcome regardless of the histological grade. These results may have clinical relevance and warrant future validation in a prospective study.
Formalin-fixed, paraffin-embedded (FFPE) tissues represent a unique source of archived biological material, but obtaining suitable DNA and RNA for retrospective "-omic" investigations is still challenging. In the current study, canine tumor FFPE blocks were used to 1) compare common commercial DNA and RNA extraction kits; 2) compare target gene expression measured in FFPE blocks and biopsies stored in a commercial storage reagent; 3) assess the impact of fixation time; and 4) perform biomolecular investigations on archival samples chosen according to formalin fixation times. Nucleic acids yield and quality were determined by spectrophotometer and capillary electrophoresis, respectively. Quantitative real-time polymerase chain reaction assays for the following genes: BCL-2-associated X protein, B-cell lymphoma extra large, antigen identified by monoclonal antibody Ki-67, proto-oncogene c-KIT (c-kit). Two internal control genes (Golgin A1 and canine transmembrane BAX inhibitor motif containing 4), together with direct sequencing of c-kit exons 8, 9, 11, and 17, were used as end points. Differences in DNA/RNA yield and purity were noticed among the commercial kits. Nucleic acids (particularly RNA) extracted from paraffin blocks were degraded, even at lower fixation times. Compared to samples held in the commercial storage reagent, archived tissues showed a poorer amplification. Therefore, a gold standard protocol for DNA/RNA isolation from canine tumor FFPE blocks for molecular investigations is still troublesome. More standardized storage conditions, including time between sample acquisition and fixation, fixation time, and sample thickness, are needed to guarantee the preservation of nucleic acids and, then, their possible use in retrospective transcriptomic analysis.
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