Table_1_Omega-6:3 Ratio More Than Absolute Lipid Level in Diet Affects Associative Learning in Honey Bees.DOCX

<p>Floral pollen is a major source of honey bee nutrition that provides them with micro- and macro-nutrients, including proteins, fatty acids, vitamins, and minerals. Different pollens vary in composition, including in the essential fatty acids, alpha-linolenic acid (omega-3) and linoleic acid (omega-6). Monocultures, prevalent in modern agriculture, may expose honey bee colonies to unbalanced omega-6:3 diets. The importance of omega-3 in the diet for adequate learning and cognitive function, with a focus on suitable omega-6:3 ratio, is well documented in mammals. We have recently shown, for the first time in invertebrates, the importance of omega-3 in diets for associative learning ability in honey bees. In the current work, we examine the effect of the absolute amount of omega-3 in diet compared to the omega-6:3 ratio on honey bee associative learning. We fed newly emerged bees for 1 week on different artificial diets, which had lipid concentration of 1, 2, 4, or 8%, with omega-6:3 ratios of 0.3, 1, or 5, respectively. We then tested the bees in a proboscis-extension response olfactory conditioning assay. We found that both omega-6:3 ratio and total lipid concentration affected learning. The most detrimental diet for learning was that with a high omega-6:3 ratio of 5, regardless of the absolute amount of omega-3 in the diet. Bees fed an omega-6:3 ratio of 1, with 4% total lipid concentration achieved the best performance. Our results with honey bees are consistent with those found in mammals. Best cognitive performance is achieved by a diet that is sufficiently rich in essential fatty acids, but as long as the omega-6:3 ratio is not high.</p

Bidirectional Transfer of RNAi between Honey Bee and <em>Varroa destructor</em>: <em>Varroa</em> Gene Silencing Reduces <em>Varroa</em> Population

<div><p>The mite <em>Varroa destructor</em> is an obligatory ectoparasite of the honey bee (<em>Apis mellifera</em>) and is one of the major threats to apiculture worldwide. We previously reported that honey bees fed on double-stranded RNA (dsRNA) with a sequence homologous to that of the Israeli acute paralysis virus are protected from the viral disease. Here we show that dsRNA ingested by bees is transferred to the <em>Varroa</em> mite and from mite on to a parasitized bee. This cross-species, reciprocal exchange of dsRNA between bee and <em>Varroa</em> engendered targeted gene silencing in the latter, and resulted in an over 60% decrease in the mite population. Thus, transfer of gene-silencing-triggering molecules between this invertebrate host and its ectoparasite could lead to a conceptually novel approach to <em>Varroa</em> control.</p> </div

List of <i>Varroa</i> dsRNA sequences used in <i>Varroa</i> gene silencing.

<p>This table lists the <i>Varroa</i> dsRNA sequences numbers, genes function, and the mixtures they are contained in. The full sequences are located in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003035#ppat.1003035.s003" target="_blank">Table S1</a>.</p

Mean (+ SE) number of <i>Varroa</i> mites per bee in four treatments.

<p>Different letters above columns indicate significant differences between treatments (<i>P</i><0.05).</p

Demonstration of dsRNA transmission from adult bee to <i>Varroa</i> via the bee hemolymph.

<p>Northern blot assay was performed on RNA extracted from a bee that had ingested dsRNA-GFP (B+) and from an untreated bee (B−), pooled RNA extracted from hemolymph collected from bees that had ingested dsRNA-GFP (H+) and from untreated bees (H−), and pooled RNA extracted from <i>Varroa</i> mites parasitizing dsRNA-GFP-treated bees (V+) and untreated bees (V−). C = positive control (GFP-carrying plasmid).</p

Mean (+ SE) total number of bees (capped brood and adults) in four treatments.

<p>Treatments did not differ significantly.</p

Demonstration of dsRNA transfer from bee to <i>Varroa</i> and from <i>Varroa</i> to bee.

<p>(A) Indirect dsRNA transmission from bee to <i>Varroa</i> parasitizing bee brood. RT-PCR of <i>Varroa</i>-extracted RNA. Numbers represent days from the beginning of dsRNA feeding. M = size markers. C = positive control (GFP-carrying plasmid). + indicates samples collected from dsRNA-GFP-treated hives. – indicates samples collected from untreated, control hives. (B) DsRNA transmission from <i>Varroa</i> to bee. RT-PCR was performed on RNA extracted from a dsRNA-GFP-carrying <i>Varroa</i> (V+) and from <i>Varroa</i> devoid of dsRNA-GFP (V−). B+ represents amplification of RNA from bees infested with dsRNA-GFP-carrying <i>Varroa</i> and B– represents amplification from bees infested with dsRNA-GFP-devoid <i>Varroa</i>. M and C: as in legend for A.</p

Schematic representation of the honey bee feeding regimen.

<p>The experiment lasted for 60 days. Top: schematic representation of a honey bee's life cycle. Bottom: experimental procedures on a timescale.</p

Silencing of <i>Varroa</i> gene expression following horizontal transfer of dsRNA from bee to <i>Varroa</i>.

<p>(A–C) <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003035#s2" target="_blank">Results</a> (mean ± SE) of real-time RT-PCR of <i>Varroa</i> genes 4, 14 and 9, respectively (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003035#ppat.1003035.s003" target="_blank">Table S1</a>). RNA from <i>Varroa</i> infesting untreated bees, which did not ingest dsRNA, or those that ingested the physiologically inert dsRNA-GFP served as controls. Note that treatment I is devoid of sequence 9. Different letters above columns indicate significant differences between treatments (<i>P</i><0.05). Details of the RT-PCR assays and statistical analysis are described in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003035#s4" target="_blank">Materials and Methods</a>. (D–E) Semi-quantitative RT-PCR for expression of the apoptosis inhibitor FAS gene (sequence 12, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003035#ppat.1003035.s003" target="_blank">Table S1</a>). RNA was extracted from <i>Varroa</i> fed on bees that had ingested Mixture I or II (D). RNA was extracted from <i>Varroa</i> fed on dsRNA-untreated bees, and on bees that had ingested dsRNA-GFP (E). (F) Amplification of actin as a standardizing internal control. Reactions devoid of reverse transcriptase (RT) served as controls for the absence of DNA contamination. Number of PCR cycles is indicated at the top of each electropherogram. SM = size markers.</p