The authors would like to thank Erin Fennern for assisting experiment. This work was funded by the research Council of Norway (180504, 185306 and 191699), PEW Charitable Trust, and the National Institute on Aging (NIA P01 AG22500).

Part 1: RNAi-mediated double gene knockdown
1. dsRNA Synthesis
<ol>
	<li>Design primers of dsRNAs targeting vg, usp and a control gene encoding green fluorescence protein (GFP) which is not in the honey bee genome: primers were designed using online free software Primer3 (<a href="http://frodo.wi.mit.edu/" target="_blank">http://frodo.wi.mit.edu/</a>).</li>
	<li>dsRNA synthesis for vg, usp and GFP: use RiboMax T7 RNA production system from Promega for in ...

<ol>
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E., Zhu, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-miRNA+hairpin+method+that+improves+gene+knockdown+efficiency+and+provides+linked+multi-gene+knockdown.">Multi-miRNA hairpin method that improves gene knockdown efficiency and provides linked multi-gene knockdown.</a> Biotech. 41, 59-63 (2006).</li><li>Ament, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+transcription+factor+ultraspiracle+influences+honey+bee+social+behavior+and+behavior-related+gene+expression.">The transcription factor ultraspiracle influences honey bee social behavior and behavior-related gene expression.</a> PLoS Genet. 8, e1002596 (2012).</li><li>Amdam, G. V., Omholt, S. 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R., Skowronek, W., Guynn, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+changes+in+worker+honey+bees+(Apis+mellifera)+induced+by+injections+of+a+juvenile+hormone+mimic.">Behavioral changes in worker honey bees (Apis mellifera) induced by injections of a juvenile hormone mimic.</a> Ann. Entomol. Soc. Am. 67, 529-534 (1974).</li><li>Scheiner, R., Pluckhahn, S., Oney, B., Blenau, W., Erber, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioural+pharmacology+of+octopamine,+tyramine+and+dopamine+in+honey+bees.">Behavioural pharmacology of octopamine, tyramine and dopamine in honey bees.</a> Behav. Brain Res. 136, 545-553 (2002).</li><li>Robinson, G. 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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+scan+for+cis-regulatory+DNA+motifs+associated+with+social+behavior+in+honey+bees.">Genome scan for cis-regulatory DNA motifs associated with social behavior in honey bees.</a> Proc. Natl. Acad. Sci. U.S.A. 103, 16352-16357 (2006).</li><li>Amdam, G. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hormonal+control+of+the+yolk+precursor+vitellogenin+regulates+immune+function+and+longevity+in+honeybees.">Hormonal control of the yolk precursor vitellogenin regulates immune function and longevity in honeybees.</a> Exp. Gerontol. 39, 767-773 (2004).</li><li>Wang, Q. C., Nie, Q. H., Feng, Z. 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We have nothing to disclose.

Our study is the first effort to simultaneously knock down two genes in adult insects. Our results show that the two strategies of dsRNA injections (single injection and two-day injection) are effective for the double gene suppression, and simultaneous gene silencing of vg and usp can be tested 6 days after the dsRNA injections into bees 8,9. However, gene knockdown efficacy varies in different insect species depending on transcript level of target gene, protein turnover rates and dsRNA uptak...

RNA interference (RNAi) is RNA based post-transcriptional gene silencing, which occurs in a wide variety of eukaryotic organisms. The process of RNAi is triggered by endogenous or exogenous double-stranded RNA (dsRNA) precursors. The dsRNA activates the ribonuclease protein Dicer which binds and cleaves the dsRNA to small fragments (20-25 bp). Then the small fragments of the dsRNA guide a recognition and cleavage of complementary mRNAs by argonaute proteins, a catalytic component of RNA-induced silencing complex (RISC)1. In mammals, dsRNAs longer than 30 nt, activate an antiviral response (interferon response, IFN) which leads to nonspecific degradation of RNA transcripts 2. However, long dsRNAs have proven to be effective and specific in insects since there is a lack of this IFN 3.
Long dsRNAs have been used for downregulation of target genes in different insect species. Honey bees are one of the pioneer insect organisms in which functions of many important genes in the development and behavior have been revealed by using dsRNA 4,5. Several dsRNA delivery methods have been performed in honey bees: dsRNA feeding efficiently downregulates target gene expression in honey bee larvae 6, whereas dsRNA injection is an effective approach for gene knockdown in honey bee embryos 4 and adult bees 7,8.
Gene knockdown effects exhibited by applying dsRNA to insects are transient and localized. Studies have shown that both abdominal dsRNA injections and thoracic dsRNA injections effectively suppress target gene expression in abdominal fat body cells of insects 9,10. DsRNA is injected into abdominal and thoracic cavities and fat body cells are able to take up the dsRNA from the hemolymph where the cells are bathed 10. However, genes in other organs, such as ovaries and brains, cannot be targeted by either abdominal or thoracic injections. In order to target genes in honey bee brain, brain injection of dsRNA has also been performed, which effectively influences target gene expression in local brain areas 11. Here, we only document abdominal dsRNA injection which is more commonly used in adult honey bees.
RNAi has been primarily used to target a single gene and has been a powerful tool to reveal the gene function. However, any gene is not isolated from others; it is in complex regulatory networks. A key to understand a biological process is to dissect how genes interact with each other, which requires simultaneous manipulations of multiple genes rather than a single gene knockdown. In mammalian cell lines, scientists have succeeded in simultaneously inhibiting two or three genes by using delivery systems 12 or multi-microRNA (miRNA) hairpin designs 13. But in insects, multiple gene knockdowns are still untested. Here, we present different injection strategies which can achieve a double gene knockdown. We target two genes: vitellogenin (vg) which encodes a yolk protein precursor, and ultraspiracle (usp) which encodes a putative receptor for juvenile hormone (JH) and may serve as a transcription factor mediating responses to JH 14 in honey bees. Vg and JH regulate each other in a feedback loop 15 and are involved in honey bee behavioral regulation 9. Using the double gene knockdown, we perturb both Vg and JH pathways, and discover how they jointly affect honey bee behavior and physiology and how vg, usp and JH interact 9.
Gustatory perception is a behavioral predictor for honey bee social behavior 16. In terms of behavioral development, nest bees with high gustatory perception behaviorally mature fast, and usually forage early in life and prefer to collect pollen 16,17. Although regulatory mechanisms underlying gustatory perception are still unclear, studies have shown that gustatory perception is linked to internal energy metabolisms 9, hormonal secretion 18,19 and biogenetic amine pathways 20. Both Vg and JH are important hormonal regulators modulating gustatory perception 7,21. In the laboratory, a variation of gustatory perception in honey bees can be evaluated by testing the proboscis extension response (PER) to different sucrose solutions. Each bee is tested by touching both her antennae with a droplet of water followed by an ascending concentration series of 0.1, 0.3, 1, 3, 10, 30% sucrose. A positive response is noted if a bee fully extends her proboscis when a droplet of water or sucrose is touched to each antenna. Based on the number of positive responses to the solutions, the gustatory perception level of each individual can be determined 16. However, the application of the PER is not limited to measuring gustatory perception. The PER is also an effective method to test the metabolic state of bees such as satiation vs. hunger. The bees with greater responses to sucrose are hungrier in general (Wang and Amdam, unpublished data). Furthermore, the PER paradigm can also be used in associative learning and memory in honey bees. In this case bees will be trained to associate the presence of sucrose water with an odor. When the bees learn the association, only the presence of the odor can evoke a positive proboscis response without rewarding them with the sucrose 22,23. In this video, we show how to perform PER to evaluate gustatory perception which has been connected with vg and usp double knockdown in a previous study 9.

<table border="1">
		<tbody>
		 <tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		 </tr>
		 <tr>
			<td>GE Healthcare PCR beads in 96 well plate</td>
			<td>GE Healthcare</td>
			<td>27-9557-01</td>
			<td></td>
		 </tr>
		 <tr>
			<td>QIAquick PCR purification kit</td>
			<td>Qiagen</td>
			<td>28104</td>
			<td></td>
		 </tr>
		 <tr>
			<td>RiboMax T7 RNA production system</td>
			<td>Promega</td>
			<td>P1300</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Trizol LS</td>
			<td>Invitrogen</td>
			<td>10296028</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Chloroform:Isoamyl alcohol 24:1</td>
			<td>Sigma-Aldrich</td>
			<td>C0549</td>
			<td></td>
		 </tr>
		 <tr>
			<td>isopropyl alcohol</td>
			<td>Sigma-Aldrich</td>
			<td>I9516</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Hamilton micro syringe</td>
			<td>Hamilton</td>
			<td>80301</td>
			<td></td>
		 </tr>
		 <tr>
			<td>30G BD disposable needles</td>
			<td>BD Biosciences</td>
			<td>305106</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>84097</td>
			<td></td>
		 </tr>
		 <tr>
			<td>1 ml Syringe</td>
			<td>BD Biosciences</td>
			<td>30960</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Stereo dissection microscope</td>
			<td>Leica Microsystems</td>
			<td>S6D</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Insect pins</td>
			<td>Fine Science Tools</td>
			<td>26000-25</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Wax plate</td>
			<td></td>
			<td></td>
			<td></td>
		 </tr>
		</tbody>
 </table>

rnai-mediated double gene knockdown and gustatory perception measurement in honey bees (apis mellifera)

This video demonstrates novel techniques of RNA interference (RNAi) which downregulate two genes simultaneously in honey bees using double-stranded RNA (dsRNA) injections. It also presents a protocol of proboscis extension response (PER) assay for measuring gustatory perception.	
	RNAi-mediated gene knockdown is an effective technique downregulating target gene expression. This technique is usually used for single gene manipulation, but it has limitations to detect interactions and joint effects between genes. In the first part of this video, we present two strategies to simultaneously knock down two genes (called double gene knockdown). We show both strategies are able to effectively suppress two genes, vitellogenin (vg) and ultraspiracle (usp), which are in a regulatory feedback loop. This double gene knockdown approach can be used to dissect interrelationships between genes and can be readily applied in different insect species.	
	The second part of this video is a demonstration of proboscis extension response (PER) assay in honey bees after the treatment of double gene knockdown. The PER assay is a standard test for measuring gustatory perception in honey bees, which is a key predictor for how fast a honey bee's behavioral maturation is. Greater gustatory perception of nest bees indicates increased behavioral development which is often associated with an earlier age at onset of foraging and foraging specialization in pollen. In addition, PER assay can be applied to identify metabolic states of satiation or hunger in honey bees. Finally, PER assay combined with pairing different odor stimuli for conditioning the bees is also widely used for learning and memory studies in honey bees.

Both the single-injection and two-day-injection strategies significantly reduced vg (One-way ANOVA, p &lt; 0.001) (Figure 2A) and usp (One-way ANOVA, p &lt; 0.001) (Figure 2B) transcript levels in honey bees six days after the dsRNA was injected. The suppression of usp transcript by using the single injection with dsRNA mixture and the two-day injection with vg first and usp second (vg/usp) was significantly lower than the tw...

Watch this Scientific Journal Video about RNAi-mediated Double Gene Knockdown and Gustatory Perception Measurement in Honey Bees Apis mellifera at JoVE.com

RNAi-mediated Double Gene Knockdown and Gustatory Perception Measurement in Honey Bees Apis mellifera

In this protocol, we describe two strategies that simultaneously suppress two genes (double gene knockdown) in honey bees. Then we present how to use the proboscis extension response (PER) assay to study the effect of double gene knockdown on honey bee gustatory perception.

RNAi-mediated Double Gene Knockdown and Gustatory Perception Measurement in Honey Bees (Apis mellifera)

This video demonstrates novel techniques of RNA  interference (RNAi) which downregulate two genes simultaneously in honey bees  using double-stranded RNA ...

rnai-mediated-double-gene-knockdown-gustatory-perception-measurement

Research

JoVE Journal

Neuroscience

14.8K Görüntüleme.  Arizona State University. Bu protokol, biz aynı anda bal arıları iki gen (çift gen demonte) bastırmak iki strateji açıklar. Sonra bal arısı tat algısı çift gen demonte etkisini incelemek için burnumun uzantısı yanıt (PER) testi nasıl kullanılacağı mevcut.