10.4. Concluding remarks
Based on the literature (see Huvenne and Smagghe, 2012 for review) five aspects seem to be most important to conduct successful RNAi knockdown experiments in honey bees.
- Concentration of dsRNA: For every target gene the most effective concentration of RNAi molecules has to be determined. It does not follow the general rule: The more the better. Nunes and Simões (2009) for example report on the removal of 2nd instar larvae, which were fed with 3 and 5 µg dsRNA. In contrast, larvae fed with just 0.5 µg dsRNA did not show a significant higher removal rate than the control group, and moreover exhibited an mRNA silencing effect of about 90 %.
- Nucleotide sequence: Sequences of the RNAi effective molecules have to be carefully designed and tested in order to avoid off-target effects.
- Length of the dsRNA fragment: When not using siRNAs the length of the dsRNA fragments may be crucial for uptake and efficient silencing. Most experiments used dsRNA ranging from 300 to 520 bp (see Huvenne and Smagghe, 2012 for review). Moreover, a minimal length of 211 bp is suggested in S2 cells (Saleh et al., 2006).
- Honey bee life stage: Although adults are easier to handle, literature of other insects suggest a larger silencing effect in younger life stages. E.g. in fall armyworms (Spodoptera frugiperda) the silencing effects after RNAi treatment were reported to be less effective than in S. frugiperda larvae (Griebler et al., 2006). Thus the usage of larvae rather than adults where feasible may be advisable in honey bees as well.
- Application method: The two application methods presented here both have pros and cons. The feeding regimes lead to an individually different consumption of food and therefore to the ingestion of different dosages of dsRNA. But in contrast to injections protocols, feeding is much easier in handling and moreover, it causes less stress in the target animals. Moreover, studies suggest, that the composition of the target tissue may have some influence on the accessibility of dsRNA when choosing injection as application method (Jarosch and Moritz, 2011).