Colony-Level Effects of Amygdalin on Honeybees and Their Microbes.

Amygdalin, a cyanogenic glycoside, is found in the nectar and pollen of almond trees, as well as in a variety of other crops, such as cherries, nectarines, apples and others. It is inevitable that western honeybees (Apis mellifera) consistently consume amygdalin during almond pollination season because almond crops are almost exclusively pollinated by honeybees. This study tests the effects of a field-relevant concentration of amygdalin on honeybee microbes and the activities of key honeybee genes. We executed a two-month field trial providing sucrose solutions with or without amygdalin ad libitum to free-flying honeybee colonies. We collected adult worker bees at four time points and used RNA sequencing technology and our HoloBee database to assess global changes in microbes and honeybee transcripts. Our hypothesis was that amygdalin will negatively affect bee microbes and possibly immune gene regulation. Using a log2 fold-change cutoff at two and intraday comparisons, we show no large change of bacterial counts, fungal counts or key bee immune gene transcripts, due to amygdalin treatment in relation to the control. However, relatively large titer decreases in the amygdalin treatment relative to the control were found for several viruses. Chronic bee paralysis virus levels had a sharp decrease (-14.4) with titers then remaining less than the control, Black queen cell virus titers were lower at three time points (<-2) and Deformed wing virus titers were lower at two time points (<-6) in amygdalin-fed compared to sucrose-fed colonies. Titers of Lotmaria passim were lower in the treatment group at three of the four dates (<-4). In contrast, Sacbrood virus had two dates with relative increases in its titers (>2). Overall, viral titers appeared to fluctuate more so than bacteria, as observed by highly inconstant patterns between treatment and control and throughout the season. Our results suggest that amygdalin consumption may reduce several honeybee viruses without affecting other microbes or colony-level expression of immune genes.

Metatranscriptomic analyses of honey bee colonies.

Honey bees face numerous biotic threats from viruses to bacteria, fungi, protists, and mites. Here we describe a thorough analysis of microbes harbored by worker honey bees collected from field colonies in geographically distinct regions of Turkey. Turkey is one of the World's most important centers of apiculture, harboring five subspecies of Apis mellifera L., approximately 20% of the honey bee subspecies in the world. We use deep ILLUMINA-based RNA sequencing to capture RNA species for the honey bee and a sampling of all non-endogenous species carried by bees. After trimming and mapping these reads to the honey bee genome, approximately 10% of the sequences (9-10 million reads per library) remained. These were then mapped to a curated set of public sequences containing ca. Sixty megabase-pairs of sequence representing known microbial species associated with honey bees. Levels of key honey bee pathogens were confirmed using quantitative PCR screens. We contrast microbial matches across different sites in Turkey, showing new country recordings of Lake Sinai virus, two Spiroplasma bacterium species, symbionts Candidatus Schmidhempelia bombi, Frischella perrara, Snodgrassella alvi, Gilliamella apicola, Lactobacillus spp.), neogregarines, and a trypanosome species. By using metagenomic analysis, this study also reveals deep molecular evidence for the presence of bacterial pathogens (Melissococcus plutonius, Paenibacillus larvae), Varroa destructor-1 virus, Sacbrood virus, and fungi. Despite this effort we did not detect KBV, SBPV, Tobacco ringspot virus, VdMLV (Varroa Macula like virus), Acarapis spp., Tropilaeleps spp. and Apocephalus (phorid fly). We discuss possible impacts of management practices and honey bee subspecies on microbial retinues. The described workflow and curated microbial database will be generally useful for microbial surveys of healthy and declining honey bees.