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>
	<li>Okamura, K., Ishizuka, A., Siomi, H., Siomi, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+roles+for+Argonaute+proteins+in+small+RNA-directed+RNA+cleavage+pathways.">Distinct roles for Argonaute proteins in small RNA-directed RNA cleavage pathways.</a> Genes Dev. 18, 1655-1666 (2004).</li><li>Shi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammalian+RNAi+for+the+masses.">Mammalian RNAi for the masses.</a> Trends Genet. 19, 9-12 (2003).</li><li>Huvenne, H., Smagghe, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+dsRNA+uptake+in+insects+and+potential+of+RNAi+for+pest+control:+a+review.">Mechanisms of dsRNA uptake in insects and potential of RNAi for pest control: a review.</a> J. Insect Physiol. 56, 227-235 (2010).</li><li>Beye, M., Hartel, S., Hagen, A., Hasselmann, M., Omholt, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+developmental+gene+silencing+in+the+honey+bee+using+a+homeobox+motif.">Specific developmental gene silencing in the honey bee using a homeobox motif.</a> Insect Mol. Biol. 11, 527-532 (2002).</li><li>Amdam, G. V., Simoes, Z. L., Guidugli, K. R., Norberg, K., Omholt, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disruption+of+vitellogenin+gene+function+in+adult+honeybees+by+intra-abdominal+injection+of+double-stranded+RNA.">Disruption of vitellogenin gene function in adult honeybees by intra-abdominal injection of double-stranded RNA.</a> BMC Biotechnol. 3, 1 (2003).</li><li>Patel, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+making+of+a+queen:+TOR+pathway+governs+diphenic+caste+development.">The making of a queen: TOR pathway governs diphenic caste development.</a> PLoS ONE. 6, e509 (2007).</li><li>Amdam, G. V., Norberg, K., Page, R. E., Erber, J., Scheiner, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Downregulation+of+vitellogenin+gene+activity+increases+the+gustatory+responsiveness+of+honey+bee+workers+(Apis+mellifera).">Downregulation of vitellogenin gene activity increases the gustatory responsiveness of honey bee workers (Apis mellifera).</a> Behav. Brain Res. 169, 201-205 (2006).</li><li>Wang, Y., 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=Down-regulation+of+honey+bee+IRS+gene+biases+behavior+toward+food+rich+in+protein.">Down-regulation of honey bee IRS gene biases behavior toward food rich in protein.</a> PLoS Genet. 6, e1000896 (2010).</li><li>Wang, Y., Brent, C. S., Fennern, E., Amdam, G. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gustatory+perception+and+fat+body+energy+metabolism+are+jointly+affected+by+vitellogenin+and+juvenile+hormone+in+honey+bees.">Gustatory perception and fat body energy metabolism are jointly affected by vitellogenin and juvenile hormone in honey bees.</a> PLoS Genet. 8, e1002779 (2012).</li><li>Luna, B. M., Juhn, J., James, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Injection+of+dsRNA+into+Female+A.+aegypti+Mosquitos.">Injection of dsRNA into Female A. aegypti Mosquitos.</a> J. Vis. Exp. (5), e215 (2007).</li><li>Mussig, L., 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=Acute+disruption+of+the+NMDA+receptor+subunit+NR1+in+the+honeybee+brain+selectively+impairs+memory+formation.">Acute disruption of the NMDA receptor subunit NR1 in the honeybee brain selectively impairs memory formation.</a> J. Neurosci. 30, 7817-7825 (2010).</li><li>Stove, V., Smits, K., Naessens, E., Plum, J., Verhasselt, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+gene+knock-down+by+a+single+lentiviral+vector+expressing+an+array+of+short+hairpin+RNAs.">Multiple gene knock-down by a single lentiviral vector expressing an array of short hairpin RNAs.</a> Electron J. Biotechnol. 19, 13 (2006).</li><li>Sun, D., Melegari, M., Sridhar, S., Rogler, C. 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. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+hive+bee+to+forager+transition+in+honeybee+colonies:+the+double+repressor+hypothesis.">The hive bee to forager transition in honeybee colonies: the double repressor hypothesis.</a> J. Theor. Biol. 223, 451-464 (2003).</li><li>Pankiw, T., Page, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Response+thresholds+to+sucrose+predict+foraging+division+of+labor+in+honeybees.">Response thresholds to sucrose predict foraging division of labor in honeybees.</a> Behav. Ecol. Sociobiol. 47, 265-267 (2000).</li><li>Pankiw, T., Nelson, M., Page, R. E., Fondrk, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+communal+crop:+modulation+of+sucrose+response+thresholds+of+pre-foraging+honey+bees+with+incoming+nectar+quality.">The communal crop: modulation of sucrose response thresholds of pre-foraging honey bees with incoming nectar quality.</a> Behav. Ecol. Sociobiol. 55, 286-292 (2004).</li><li>Jaycox, E. R. <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+L.)+after+Injection+with+Synthetic+Juvenile+Hormone+(Hymenoptera:+Apidae).">Behavioral Changes in Worker Honey Bees (Apis mellifera L.) after Injection with Synthetic Juvenile Hormone (Hymenoptera: Apidae).</a> J. Kan. Entomol. Soc. 49, 165-170 (1976).</li><li>Jaycox, E. 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. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+a+juvenile+hormone+analogue+on+honey+bee+foraging+behaviour+and+alarm+pheromone+production.">Effects of a juvenile hormone analogue on honey bee foraging behaviour and alarm pheromone production.</a> J. Insect. Physiol. , 277-282 (1985).</li><li>Giurfa, M., Malun, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Associative+mechanosensory+conditioning+of+the+proboscis+extension+reflex+in+honeybees.">Associative mechanosensory conditioning of the proboscis extension reflex in honeybees.</a> Lear Memory. 11, 294-302 (2004).</li><li>Scheiner, R., Page, R. E., 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=The+effects+of+genotype,+foraging+role,+and+sucrose+responsiveness+on+the+tactile+learning+performance+of+honey+bees+(Apis+mellifera+L.).">The effects of genotype, foraging role, and sucrose responsiveness on the tactile learning performance of honey bees (Apis mellifera L.).</a> Neurobiol. Learn Mem. 76, 138-150 (2001).</li><li>Wang, Y., 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=PDK1+and+HR46+gene+homologs+tie+social+behavior+to+ovary+signals.">PDK1 and HR46 gene homologs tie social behavior to ovary signals.</a> PLoS ONE. 4, e4899 (2009).</li><li>Wang, Y., 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=Regulation+of+behaviorally+associated+gene+networks+in+worker+honey+bee+ovaries.">Regulation of behaviorally associated gene networks in worker honey bee ovaries.</a> J. Exp. Biology. 215, 124-134 (2012).</li><li>Amdam, G. V., Csondes, A., Fondrk, M. K., Page, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complex+social+behaviour+derived+from+maternal+reproductive+traits.">Complex social behaviour derived from maternal reproductive traits.</a> Nature. 439, 76-78 (2006).</li><li>Amdam, G. V., Ihle, K. E., Page, R. E., Pfaff, D., 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=.">.</a> Hormones, Brain and Behavio. , (2009).</li><li>Guidugli, K. R., 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=Vitellogenin+regulates+hormonal+dynamics+in+the+worker+caste+of+a+eusocial+insect.">Vitellogenin regulates hormonal dynamics in the worker caste of a eusocial insect.</a> FEBS Letters. 579, 4961-4965 (2005).</li><li>Corona, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=juvenile+hormone,+insulin+signaling,+and+queen+honey+bee+longevity.">juvenile hormone, insulin signaling, and queen honey bee longevity.</a> Proc. Natl. Acad. Sci. U.S.A. 104, 7128-7133 (2007).</li><li>Sinha, S., Ling, X., Whitfield, C. W., Zhai, C., Robinson, G. 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. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+interference:+antiviral+weapon+and+beyond.">RNA interference: antiviral weapon and beyond.</a> World J. Gastroenterol. 9, 1657-1661 (2003).</li><li>Carmell, M. A., Xuan, Z., Zhang, M. Q., Hannon, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Argonaute+family:+tentacles+that+reach+into+RNAi,+developmental+control,+stem+cell+maintenance,+and+tumorigenesis.">The Argonaute family: tentacles that reach into RNAi, developmental control, stem cell maintenance, and tumorigenesis.</a> Genes Dev. 16, 2733-2742 (2002).</li><li>Hunt, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+genomics+of+honeybee+foraging+and+nest+defense.">Behavioral genomics of honeybee foraging and nest defense.</a> Naturwissenschaften. 94, 247-267 (2007).</li><li>Pankiw, T., Waddington, K. D., Page, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+sucrose+response+thresholds+in+honey+bees+(Apis+mellifera+L.):+influence+of+genotype,+feeding,+and+foraging+experience.">Modulation of sucrose response thresholds in honey bees (Apis mellifera L.): influence of genotype, feeding, and foraging experience.</a> J. Comp. Physiol. A. 187, 293-301 (2001).</li><li>Pankiw, T., Page, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+pheromones,+hormones,+and+handling+on+sucrose+response+thresholds+of+honey+bees+(Apis+mellifera+L.).">Effect of pheromones, hormones, and handling on sucrose response thresholds of honey bees (Apis mellifera L.).</a> J. Comp. Physiol. A. Neuroethol. Sens. Neural. Behav. Physiol. 189, 675-684 (2003).</li></ol>

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 ビュー.  Arizona State University. このプロトコルでは、我々は同時に、ミツバチの二つの遺伝子（二重遺伝子ノックダウン）を抑制の2つの戦略について説明します。その後私たちはミツバチ味覚知覚に二重遺伝子ノックダウンの効果を研究するために口先拡張応答（PER）アッセイを使用する方法を提示します。

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

Double Fluorescence in situ Hybridization in Fresh Brain Sections

Here we describe a modified version of a double fluorescence in situ hybridization (dFISH) method optimized for detecting two mRNAs of interest in fresh frozen brain sections. Our group has successfully used this approach to study gene co-regulation. More specifically, we have used this dFISH method to explore the anatomical organization, neurochemical properties, and the impact of sensory experience in central sensory circuits, at single cell resolution. This protocol has been validated in brain tissue from mice, rats and songbirds but is expected to be easily adaptable to other vertebrate species, as well as to an array of non-neural tissues. In this film we provide a detailed demonstration of the main steps of this procedure.

Double Fluorescence in situ Hybridization in Fresh Brain Sections

This protocol involves a non-radioactive in-situ hybridization procedure that enables the simultaneous identification of two transcript species, at a single cell resolution, in thin sections of the vertebrate brain.

Here we describe a modified version of a double fluorescence in situ hybridization (dFISH) method optimized for detecting two mRNAs of interest in fresh ...

Behavioural Pharmacology in Classical Conditioning of the Proboscis Extension Response in Honeybees (Apis mellifera)

Honeybees (Apis mellifera) are well known for their communication and orientation skills and for their impressive learning capability1,2. Because the survival of a honeybee colony depends on the exploitation of food sources, forager bees learn and memorize variable flower sites as well as their profitability. Forager bees can be easily trained in natural settings where they forage at a feeding site and learn the related signals such as odor or color. Appetitive associative learning can also be studied under controlled conditions in the laboratory by conditioning the proboscis extension response (PER) of individually harnessed honeybees3,4. This learning paradigm enables the study of the neuronal and molecular mechanisms that underlie learning and memory formation in a simple and highly reliable way5-12. A behavioral pharmacology approach is used to study molecular mechanisms. Drugs are injected systemically to interfere with the function of specific molecules during or after learning and memory formation13-16.
Here we demonstrate how to train harnessed honeybees in PER conditioning and how to apply drugs systemically by injection into the bee flight muscle.

Behavioural Pharmacology in Classical Conditioning of the Proboscis Extension Response in Honeybees Apis mellifera

We demonstrate how to implement a behavioral pharmacology method in an appetitive olfactory conditioning paradigm in honeybees (Apis mellifera) by systemic application of drugs. This method allows investigation of the mechanisms underlying learning and memory formation in a simple and reliable way.

Honeybees (Apis mellifera) are well known for their communication and orientation skills and for their impressive learning capability1,2. Because the ...

Creating Objects and Object Categories for Studying Perception and Perceptual Learning

In order to quantitatively study object perception, be it perception by biological systems or by machines, one needs to create objects and object categories with precisely definable, preferably naturalistic, properties1. Furthermore, for studies on perceptual learning, it is useful to create novel objects and object categories (or object classes) with such properties2.
Many innovative and useful methods currently exist for creating novel objects and object categories3-6 (also see refs. 7,8). However, generally speaking, the existing methods have three broad types of shortcomings.
First, shape variations are generally imposed by the experimenter5,9,10, and may therefore be different from the variability in natural categories, and optimized for a particular recognition algorithm. It would be desirable to have the variations arise independently of the externally imposed constraints.
Second, the existing methods have difficulty capturing the shape complexity of natural objects11-13. If the goal is to study natural object perception, it is desirable for objects and object categories to be naturalistic, so as to avoid possible confounds and special cases.
Third, it is generally hard to quantitatively measure the available information in the stimuli created by conventional methods. It would be desirable to create objects and object categories where the available information can be precisely measured and, where necessary, systematically manipulated (or 'tuned'). This allows one to formulate the underlying object recognition tasks in quantitative terms.
Here we describe a set of algorithms, or methods, that meet all three of the above criteria. Virtual morphogenesis (VM) creates novel, naturalistic virtual 3-D objects called 'digital embryos' by simulating the biological process of embryogenesis14. Virtual phylogenesis (VP) creates novel, naturalistic object categories by simulating the evolutionary process of natural selection9,12,13. Objects and object categories created by these simulations can be further manipulated by various morphing methods to generate systematic variations of shape characteristics15,16. The VP and morphing methods can also be applied, in principle, to novel virtual objects other than digital embryos, or to virtual versions of real-world objects9,13. Virtual objects created in this fashion can be rendered as visual images using a conventional graphical toolkit, with desired manipulations of surface texture, illumination, size, viewpoint and background. The virtual objects can also be 'printed' as haptic objects using a conventional 3-D prototyper.
We also describe some implementations of these computational algorithms to help illustrate the potential utility of the algorithms. It is important to distinguish the algorithms from their implementations. The implementations are demonstrations offered solely as a 'proof of principle' of the underlying algorithms. It is important to note that, in general, an implementation of a computational algorithm often has limitations that the algorithm itself does not have.
Together, these methods represent a set of powerful and flexible tools for studying object recognition and perceptual learning by biological and computational systems alike. With appropriate extensions, these methods may also prove useful in the study of morphogenesis and phylogenesis.

We describe a novel methodology for creating naturalistic 3-D objects and object categories with precisely defined feature variations. We use simulations of the biological processes of morphogenesis and phylogenesis to create novel, naturalistic virtual 3-D objects and object categories that can then be rendered as visual images or haptic objects.

In order to quantitatively study object perception, be it perception by biological systems or by machines, one needs to create objects and object ...

Using Affordable LED Arrays for Photo-Stimulation of Neurons

Standard slice electrophysiology has allowed researchers to probe individual components of neural circuitry by recording electrical responses of single cells in response to electrical or pharmacological manipulations1,2. With the invention of methods to optically control genetically targeted neurons (optogenetics), researchers now have an unprecedented level of control over specific groups of neurons in the standard slice preparation. In particular, photosensitive channelrhodopsin-2 (ChR2) allows researchers to activate neurons with light3,4. By combining careful calibration of LED-based photostimulation of ChR2 with standard slice electrophysiology, we are able to probe with greater detail the role of adult-born interneurons in the olfactory bulb, the first central relay of the olfactory system. Using viral expression of ChR2-YFP specifically in adult-born neurons, we can selectively control young adult-born neurons in a milieu of older and mature neurons. Our optical control uses a simple and inexpensive LED system, and we show how this system can be calibrated to understand how much light is needed to evoke spiking activity in single neurons. Hence, brief flashes of blue light can remotely control the firing pattern of ChR2-transduced newborn cells.

Adult-born neurons expressing ChR2 can be manipulated in slice electrophysiological preparations in order to examine their contribution towards the function of olfactory neural circuits.

Standard slice electrophysiology has allowed researchers to probe individual components of neural circuitry by recording electrical responses of single ...

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents1-3. In this process, called adult neurogenesis, neural stem cells in the walls of the lateral ventricle give rise to neuroblasts that migrate for several millimeters along the rostral migratory stream (RMS) to reach and incorporate into the olfactory bulb. To study the different steps and the impact of adult-born neuron integration into preexisting olfactory circuits, it is necessary to selectively label and manipulate the activity of this specific population of neurons. The recent development of optogenetic technologies offers the opportunity to use light to precisely activate this specific cohort of neurons without affecting surrounding neurons4,5. Here, we present a series of procedures to virally express Channelrhodopsin2(ChR2)-YFP in a temporally restricted cohort of neuroblasts in the RMS before they reach the olfactory bulb and become adult-born neurons. In addition, we show how to implant and calibrate a miniature LED for chronic in vivo stimulation of ChR2-expressing neurons.

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Adult-born neurons of the olfactory bulb can be optogenetically controlled using Channelrhodopsin2-expressing lentiviral injection in the rostral migratory stream and chronic photostimulation with an implanted miniature LED.

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents1-3. In this process, called adult neurogenesis, neural stem cells ...

MPI CyberMotion Simulator: Implementation of a Novel Motion Simulator to Investigate Multisensory Path Integration in Three Dimensions

Path integration is a process in which self-motion is integrated over time to obtain an estimate of one's current position relative to a starting point 1. Humans can do path integration based exclusively on visual 2-3, auditory 4, or inertial cues 5. However, with multiple cues present, inertial cues - particularly kinaesthetic - seem to dominate 6-7. In the absence of vision, humans tend to overestimate short distances (&lt;5 m) and turning angles (&lt;30&deg;), but underestimate longer ones 5. Movement through physical space therefore does not seem to be accurately represented by the brain.
Extensive work has been done on evaluating path integration in the horizontal plane, but little is known about vertical movement (see 3 for virtual movement from vision alone). One reason for this is that traditional motion simulators have a small range of motion restricted mainly to the horizontal plane. Here we take advantage of a motion simulator 8-9 with a large range of motion to assess whether path integration is similar between horizontal and vertical planes. The relative contributions of inertial and visual cues for path navigation were also assessed.
16 observers sat upright in a seat mounted to the flange of a modified KUKA anthropomorphic robot arm. Sensory information was manipulated by providing visual (optic flow, limited lifetime star field), vestibular-kinaesthetic (passive self motion with eyes closed), or visual and vestibular-kinaesthetic motion cues. Movement trajectories in the horizontal, sagittal and frontal planes consisted of two segment lengths (1st: 0.4 m, 2nd: 1 m; &plusmn;0.24 m/s2 peak acceleration). The angle of the two segments was either 45&deg; or 90&deg;. Observers pointed back to their origin by moving an arrow that was superimposed on an avatar presented on the screen.
Observers were more likely to underestimate angle size for movement in the horizontal plane compared to the vertical planes. In the frontal plane observers were more likely to overestimate angle size while there was no such bias in the sagittal plane. Finally, observers responded slower when answering based on vestibular-kinaesthetic information alone. Human path integration based on vestibular-kinaesthetic information alone thus takes longer than when visual information is present. That pointing is consistent with underestimating and overestimating the angle one has moved through in the horizontal and vertical planes respectively, suggests that the neural representation of self-motion through space is non-symmetrical which may relate to the fact that humans experience movement mostly within the horizontal plane.

An efficient way to gain insight into how humans navigate themselves in three dimensions is described. The method takes advantage of a motion simulator capable of moving observers in ways unattainable by traditional simulators. Results confirm that movement in the horizontal plane is underestimated, while vertical movement is overestimated.

Path integration is a process in which self-motion is integrated over time to obtain an estimate of one's current position relative to a starting point 1. ...

Tactile Conditioning And Movement Analysis Of Antennal Sampling Strategies In Honey Bees (Apis mellifera L.)

Honey bees (Apis mellifera L.) are eusocial insects and well known for their complex division of labor and associative learning capability1, 2. The worker bees spend the first half of their life inside the dark hive, where they are nursing the larvae or building the regular hexagonal combs for food (e.g. pollen or nectar) and brood3. The antennae are extraordinary multisensory feelers and play a pivotal role in various tactile mediated tasks4, including hive building5 and pattern recognition6. Later in life, each single bee leaves the hive to forage for food. Then a bee has to learn to discriminate profitable food sources, memorize their location, and communicate it to its nest mates7. Bees use different floral signals like colors or odors7, 8, but also tactile cues from the petal surface9 to form multisensory memories of the food source. Under laboratory conditions, bees can be trained in an appetitive learning paradigm to discriminate tactile object features, such as edges or grooves with their antennae10, 11, 12, 13. This learning paradigm is closely related to the classical olfactory conditioning of the proboscis extension response (PER) in harnessed bees14. The advantage of the tactile learning paradigm in the laboratory is the possibility of combining behavioral experiments on learning with various physiological measurements, including the analysis of the antennal movement pattern.

Tactile Conditioning And Movement Analysis Of Antennal Sampling Strategies In Honey Bees Apis mellifera L.

In this protocol we show how to condition harnessed honey bees to tactile stimuli and introduce a 2D motion capture technique for analyzing the kinematics of fine-scale antennal sampling pattern.

Honey bees (Apis mellifera L.) are eusocial insects and well known for their complex division of labor and associative learning capability1, 2. The worker ...

Double-barreled and Concentric Microelectrodes for Measurement of Extracellular Ion Signals in Brain Tissue

Electrical activity in the brain is accompanied by significant ion fluxes across membranes, resulting in complex changes in the extracellular concentration of all major ions. As these ion shifts bear significant functional consequences, their quantitative determination is often required to understand the function and dysfunction of neural networks under physiological and pathophysiological conditions. In the present study, we demonstrate the fabrication and calibration of double-barreled ion-selective microelectrodes, which have proven to be excellent tools for such measurements in brain tissue. Moreover, so-called &#8220;concentric&#8221; ion-selective microelectrodes are also described, which, based on their different design, offer a far better temporal resolution of fast ion changes. We then show how these electrodes can be employed in acute brain slice preparations of the mouse hippocampus. Using double-barreled, potassium-selective microelectrodes, changes in the extracellular potassium concentration ([K+]o) in response to exogenous application of glutamate receptor agonists or during epileptiform activity are demonstrated. Furthermore, we illustrate the response characteristics of sodium-sensitive, double-barreled and concentric electrodes and compare their detection of changes in the extracellular sodium concentration ([Na+]o) evoked by bath or pressure application of drugs. These measurements show that while response amplitudes are similar, the concentric sodium microelectrodes display a superior signal-to-noise ratio and response time as compared to the double-barreled design. Generally, the demonstrated procedures will be easily transferable to measurement of other ions species, including pH or calcium, and will also be applicable to other preparations.

We demonstrate the fabrication, calibration and properties of two types of ion-selective microelectrodes (double-barreled and concentric) for measurement of ion concentrations in brain tissue. These are then used in the mouse hippocampal slice preparation to show that excitatory activity changes both extracellular potassium and sodium concentrations.

Electrical activity in the brain is accompanied by significant ion fluxes across membranes, resulting in complex changes in the extracellular ...

A Novel Behavioral Assay to Investigate Gustatory Responses of Individual, Freely-moving Bumble Bees (Bombus terrestris)

Generalist pollinators like the buff-tailed bumble bee, Bombus terrestris, encounter both nutrients and toxins in the floral nectar they collect from flowering plants. Only a few studies have described the gustatory responses of bees toward toxins in food, and these experiments have mainly used the proboscis extension response on restrained honey bees. Here, a new behavioral assay is presented for measuring the feeding responses of freely-moving, individual worker bumble bees to nutrients and toxins. This assay measures the amount of solution ingested by each bumble bee and identifies how tastants in food influence the microstructure of the feeding behavior.
The solutions are presented in a microcapillary tube to individual bumble bees that have been previously starved for 2-4 hr. The behavior is captured on digital video. The fine structure of the feeding behavior is analyzed by continuously scoring the position of the proboscis (mouthparts) from video recordings using event logging software. The position of the proboscis is defined by three different behavioral categories: (1) proboscis is extended and in contact with the solution, (2) proboscis is extended but not in contact with the solution and (3) proboscis is stowed under the head. Furthermore the speed of the proboscis retracting away from the solution is also estimated.
In the present assay the volume of solution consumed, the number of feeding bouts, the duration of the feeding bouts and the speed of the proboscis retraction after the first contact is used to evaluate the phagostimulatory or the deterrent activity of the compounds tested.
This new taste assay will allow researchers to measure how compounds found in nectar influence the feeding behavior of bees and will also be useful to pollination biologists, toxicologists and neuroethologists studying the bumble bee&#39;s taste system.

A Novel Behavioral Assay to Investigate Gustatory Responses of Individual, Freely-moving Bumble Bees Bombus terrestris

A novel behavioral assay is described for investigating the short term gustatory responses of the mouthparts of freely-moving bumble bees (Bombus terrestris) toward nutrients and toxins in solution.

Generalist pollinators like the buff-tailed bumble bee, Bombus terrestris, encounter both nutrients and toxins in the floral nectar they collect from ...

Neuropharmacological Manipulation of Restrained and Free-flying Honey Bees, Apis mellifera

Honey bees demonstrate astonishing learning abilities and advanced social behavior and communication. In addition, their brain is small, easy to visualize and to study. Therefore, bees have long been a favored model amongst neurobiologists and neuroethologists for studying the neural basis of social and natural behavior. It is important, however, that the experimental techniques used to study bees do not interfere with the behaviors being studied. Because of this, it has been necessary to develop a range of techniques for pharmacological manipulation of honey bees. In this paper we demonstrate methods for treating restrained or free-flying honey bees with a wide range of pharmacological agents. These include both noninvasive methods such as oral and topical treatments, as well as more invasive methods that allow for precise drug delivery in either systemic or localized fashion. Finally, we discuss the advantages and disadvantages of each method and&#160;describe common hurdles and how to best overcome them. We conclude with a discussion on the importance of adapting the experimental method to the biological questions rather than the other way around.

Neuropharmacological Manipulation of Restrained and Free-flying Honey Bees, Apis mellifera

This manuscript describes several protocols for administering pharmacological agents to honey bees, including simple noninvasive methods for free-flying bees, as well as more invasive variants that allow precise localized treatment of restrained bees.

Honey bees demonstrate astonishing learning abilities and advanced social behavior and communication. In addition, their brain is small, easy to visualize ...