Scientific note on the first molecular detection of the acute bee paralysis virus in Brazilian stingless bees

Abstract

Meliponiculture is a traditional activity in northern and northeastern Brazil, where it represents a complementary source of income (Ueira-Vieira et al. 2013). Indigenous Brazilian people have domesticated some species of stingless bees, such as the Melipona genus, due to their heartiness and honey production. Rational rearing for commercialization of the Melipona scutellaris bee is common in northeastern Brazil. Their hive structure is different from honeybee mainly for lower number of workers than Apis mellifera . The honey and pollen are stored in a pot. The larval food is provisioned by workers, and after the queen lays eggs, the cell is totally closed by the worker. Therefore, there is no contact between the workers and larvae like in the honeybee. The caste determination mechanism in the Melipona genus is based on the interaction between environmental and genetic components, which is different from other bees (Kerr and Nielsen 1966). Recently, our research group has received a large amount of information from beekeepers about a decrease in the stingless bee population as well as an increase in the number of dead stingless bees. Therefore, we performed reverse transcription polymerase chain reaction (RT-PCR) screening of seven viruses in M. scutellaris sampled from two meliponaries in Brazil. Workers of M. scutellaris were collected from the meliponary at the Federal University of Uberlandia, Minas Gerais State (S 180 55′/W 450 17′) (n=10 hives) and a meliponary at Igarassu City, Pernambuco State (07° 50′ 03′′ S 34° 54′ 23′′W) (n=5 hives). Only forager bees were used for molecular analysis, and they were collected after arriving back to the hive. The total RNAwas extracted from five stingless bees per hive using the TRIzol reagent (Invitrogen), according to manufacturer’s recommendations. The total RNA was treated with DNAse enzyme (Promega) and used for reverse transcription using M-MLV reverse transcriptase (USB), according to the manufacturer’s recommendations. The rp49 gene was used to test the quality of complementary DNA (cDNA). Seven RNA viruses were analyzed using specific primers synthesized for each type of virus (see supplementary material, Table S1). DNA amplification of the different viruses was performed using Taq polymerase Platinum (Invitrogen) according to the manufacturer’s instructions. For in silico analysis, the CAP3 sequence assembly software was used to form a contig among sequencing repetitions (three replicate sequences for each amplicon). Phylogenetic analysis was performed using the Maximum Parsimony Method Software Mega 6 with the statistical analysis based on 1000 bootstrap replications. After observing stingless bee death in our meliponary (Minas Gerais State in Brazil), we analyzed whether viruses were present inM. scutellaris stingless bees. Among the seven viruses screened by RT-PCR, positive results were only obtained for the acute bee paralysis virus (ABPV), and surprisingly, the infection was present in all 10 of the hives tested (Figure 1). The hives were observed weekly and the population was Electronic supplementary material The online version of this article (doi:10.1007/s13592-015-0353-2) contains supplementary material, which is available to authorized users.