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7. Functional genomics and xenobiotic treatment

7.3. RNA interference

RNA interference (RNAi) is a molecular tool used for transient knock-down (reduction) of gene expression (Brutscher & Flenniken, 2015; Wilson & Doudna, 2013). This technique takes advantage of endogenous molecular machinery that normally helps protect the host against RNA viruses, which replicate their genome via a double-stranded (ds)RNA intermediate. The presence of dsRNA within host cells thus signals viral infection and triggers a cascade of events leading to viral inactivation by the RNA-induced silencing complex (RISC).

Although RNAi evolved as an immune defense against viruses, the dsRNA that leads to the antiviral response does not necessarily need to be a viral sequence. RNAi against nearly any gene is theoretically possible and can be achieved simply through the introduction of short interfering RNA (siRNA; 20-25 bp) or long dsRNA (several hundred bp) to the organism - a technique that has proven to be especially feasible in insects (Huvenne & Smagghe, 2010). Here we present current methods for conducting RNAi experiments in honey bees.

7.3.1. RNAi considerations

  • A common method of introducing RNAi constructs into bees is via injection; however, this is highly invasive and creates a wound on the bee. This might be useful if modeling parasitization by mites, but in most cases, the wound creates undesirable trauma. The methods described here are less invasive.

  • Feeding dsRNA to larvae or adults is also a non-invasive method. It has been covered already in Evans et al. (2013).

  • Non-specific effects associated with RNAi are well-documented (Flenniken & Andino, 2013; Jarosch & Moritz, 2012; Nunes et al., 2013) and probably unavoidable, but the likelihood of observing severe off-target phenotypes can be reduced by cross-referencing the siRNA/dsRNA sequence to the honey bee genome. Only sequences unique to the target should be pursued. Moreover, negative control groups utilizing GFP dsRNA or scrambled dsRNA sequences are essential to distinguish gene-specific phenotypes from non-specific effects.

  • Consider labeling the siRNA construct, either with a fluorescent tag (GFP or RFP) or digoxigenin (DIG) to visualize localization in tissue after treatment and check that the siRNA is correctly introduced to the target tissue.

  • An alternative strategy for RNAi using symbiont-mediation is also documented (Lariviere et al., 2023)

7.3.2. Methods for nanoparticle-mediated RNAi

Honey bees breathe through spiracles, which are small holes in the abdomen connected to a tracheal network. In addition to feeding and injection, one way to introduce a foreign substance into a bee is through inhalation. Spiracles are approximately 200 µm wide; therefore, nebulized nanoparticles carrying RNAi constructs can enter the bee, assuming the nanoparticle-containing droplets or aerosolized particles are sufficiently small. Here we describe the general procedure for using perfluorocarbon (PFC) nanoparticles for delivering siRNA to bees, adapted from methods described in (Li-Byarlay et al., 2013).

7.3.2.1. Materials

  • Large equipment: humidity-controlled incubator

  • Medical nebulizer (purchased from a medical health supply store)

  • Collecting chamber with end cap (Bioquip, Catalog #2820GA: 2820D) attached to the nebulizer. Bioquip is now closed permanently. Equivalent needs to be searched

  • Nanoparticles that can carry small interfere RNAs (PFCs purchased from Thermo Fisher, for example)

  • siRNA (follow methods outlined in Evans et al. (2013) for siRNA design, production, and purification, or purchase from a vendor)

  • Acrylic or plexiglass cages for maintaining live bees in the laboratory and which are suitable for exposure to nebulized nanoparticles

7.3.2.2. Procedure

  1. To coat nanoparticles with siRNA, mix siRNA and add nanoparticles to create a solution with a ratio of 1 μM siRNA to 200 pM nanoparticles.

    TIP: Higher ratios may be used, but will not necessarily be more efficacious.

  2. Select bees and maintain them in laboratory cages according to methods outlined in Williams et al. (2013).

  3. Add the siRNA and nanoparticle solution to a nebulizer compressor machine. Live bees to be treated are housed in the collecting chamber. The solution is then sprayed on bees for ~5 min.

  4. Allow bees to recover in a dark incubator held at 32 °C and at least 40% humidity for 96 h. Provide pollen paste (50% w/v honey, 50% w/v pollen) and sugar water (50% w/v sucrose in water) ad libitum.