Os autores gostariam de agradecer a Erin Fennern para auxiliar experimento. Este trabalho foi financiado pelo Conselho de Pesquisa da Noruega (180504, 185306 e 191699), PEW Charitable Trust, eo Instituto Nacional do Envelhecimento (NIA P01 AG22500).

Parte 1: RNAi mediado pelo gene knockdown dupla 1. dsRNA Synthesis <ol><li> Iniciadores do projeto de dsRNAs segmentação vg, usp e um controle de gene que codifica a proteína verde fluorescente (GFP), que não está no genoma da abelha do mel: primers foram desenhados utilizando o software online gratuitamente Primer3 ( <a href="http://frodo.wi.mit.edu/" target="_blank">http://frodo.wi.mit.edu/</a> ) . </li><li> síntese de dsRNA para vg, usp e GFP: use RiboMax T7 sistema de produção de RNA da Promega para transcrição in vitro. </li><li> dsRNA purificação: <ol><li> Desnaturação e renaturação: aqueça o dsRNA a 85 ° C por 5 minutos e deixe esfriar em temperatura ambiente por 1 hora. </li><li> DNase tratamento I: adicione 1 mL DNase I em cada reação dsRNA e misturá-los sacudindo os tubos. Incubar durante 15 min a 37 ° C. </li><li> Adicionar 150 mL de água livre de nuclease e 750 mL TRIZOL - LS em cada reação, e misture-as delicadamente por inverting do tubo. Incubar as amostras durante 5 min a 30 ° C. </li><li> Adicionar 200 ul de clorofórmio para cada amostra. Misturar vigorosamente durante 20 segundos. Centrifugar as amostras durante 15 min a 12.000 xg, a 4 ° C. </li><li> Transferir a fase sobrenadante de cada tubo novo e adicionar 500 mL de álcool isopropílico para cada tubo. Misture invertendo o tubo. Incube-se a mistura durante 20 min a -20 ° C. Centrifugar durante 10 min a 12.000 xg, a 4 ° C. </li><li> Remover o sobrenadante e utilizar 1.000 ul de 75% de etanol para lavar o sedimento. Após centrifugação o tubo durante 5 min a 7500 xg, a 4 ° C, o ar seco do ARNdc pelete, e usar água livre de nuclease para dissolver o sedimento. A fim de assegurar a eficácia do gene da sub-regulação, a concentração deve ser cerca de ARNdc 9 -10 ug / uL. </li></ol></li></ol> 2. Injeção de dsRNA Abdominal Para gene knockdown dupla, existem duas estratégias: 1) injeção única: mix dsRNA de dois genes e inject-se a mistura, 2) dois dias de injecção: injectar o primeiro dsRNA alvo um gene no primeiro dia, e injectar a segunda ARNdc nos mesmos abelhas no segundo dia. <ol><li> Frio recentemente surgiram abelhas em um 4 ° C geladeira por 1-2 min. Quando as abelhas são imobilizadas, montar 3-4 abelhas em paralelo em placas de Petri cheias de cera sólida, com pinos de insetos cruzaram entre seus abdomens e thoraces. </li><li> Relaxar as abelhas novamente em um 4 ° C geladeira. Certifique-se de que as abelhas são completamente imóvel. Demora cerca de 1-2 min. As abelhas devem olhar solto. Se as abelhas se enrolado ou contorcidos, que significa que eles são arrefecidas muito tempo que deve ser evitado. Tempo Livre demais provoca uma elevada mortalidade, mas imobilização completa é importante porque qualquer movimento aumenta o tamanho da ferida e mortalidade. </li><li> Coloque uma agulha descartável 30 G (BD) em um micro seringa Hamilton. Take 3 dsRNA mL e verifique se não há nenhuma bolha de ar na seringa. A agulha é inserida para um lado do abdómen para evitar damaging órgãos internos. Pressione o êmbolo da seringa, lentamente para permitir que o ARNdc ser absorvida. Uma vez que o dsRNA é muito viscosa, que leva 2-3 segundos para ser completamente expelido da seringa. Após completamente empurrando o êmbolo para baixo, deixando a agulha na ferida para 4-5 seg. Observe as abelhas por 3-5 seg após as injeções. Se uma gota de hemolinfa vazamentos da ferida, descartar a abelha. </li><li> Use cores diferentes para marcar seus thoraces correspondentes a diferentes tratamentos. Neste ponto, se a estratégia de injeção única foi utilizada, as abelhas podem ser colocados de volta em uma colônia depois de 1 hora de observação. No entanto, se a estratégia de injecção de dois dias foi utilizado, após a injecção com o primeiro dsRNA, as abelhas devem ser colocados numa gaiola de malha de cilindro com mel fornecido na parte lateral e ser mantido numa incubadora a 34 ° C e 80% de humidade . </li><li> No segundo dia, siga os passos 2.2 e 2.3 para relaxar as abelhas nas gaiolas de malha e montá-los em placas de cera. Realizar as injecções com a segunda ARNdc como é descrileito na etapa 2.4. </li><li> Deixe que as abelhas se recuperar em temperatura ambiente por 1 hora e apresentá-los em uma colônia. </li></ol> Nota: Os efeitos Knockdown pode ser detectado por RT-PCR quantitativo (qRT-PCR) e transferência western. Em geral, a eficácia knockdown varia e depende de muitos fatores. Em nosso estudo, o gene knockdown dupla pode ser detectado de 7 dias após as injeções individuais e injeções de dois dias. Parte 2: narigudo extensão resposta ensaio (PER) 1. Coleta de abelhas da colmeia No dia 6 após as injeções, coleta de abelhas tratadas da colônia sem o uso de um fumante. Coletar as abelhas em gaiolas cilindro de malha de metal. Mantenha três abelhas em cada gaiola. 2. Abelhas de montagem para PER Frio as abelhas a 4 ° C até serem completamente imobilizado. Monte cada abelha verticalmente em um tubo de plástico especialmente projetado para PER. Mantenha o abdômen dentro do tubo por using dois pequenos pedaços de fita, e manter a cabeça para fora do tubo. Coloque os tubos em um rack com duas fileiras de colunas pequenas de plástico, e atribuir a cada tubo (abelha) com um número de identificação. Em seguida, coloque as abelhas tratadas nas prateleiras em uma incubadora (34 ° C e 80% de umidade) e mantê-los na incubadora por 2 horas. 3. Preparando uma série de sacarose Water Solutions Prepare a 0%, 0,1%, 0,3%, 1%, 3%, 10% e 30% de água, soluções de sacarose e carregar as soluções para as seringas com agulhas de 15-20. 4. Por ensaio Toque a antena da abelha com uma gota de solução a 0% (água), seguido de 0,1%, 0,3%, 1%, 3%, 10% e 30% de água, soluções de sacarose. Testar todas as abelhas com água primeiro e permitir que pelo menos dois minutos, como o intervalo entre cada solução é utilizada. Por isso, costumamos ter pelo menos 20 abelhas para cada tentativa. Se uma abelha estende totalmente seus tromba, uma resposta positiva é contado. 5.Gerenciamento de Dados Soma-se o total de respostas positivas para cada indivíduo para a pontuação resposta gustativa (GRS). Realizar uma análise estatística adequada sobre os dados. Parte 3. Validação knockdown Gene Corpos gordos são dissecados a partir de um outro conjunto de abelhas tratadas idade de 7 dias. Um procedimento padrão Trizol é utilizado para a extracção de RNA, que é seguido pelo tratamento com DNase I 24. Vg e expressão do gene da USP é analisado por um de dois passos de qRT-PCR. Actina é usado como um gene de referência, uma vez que tem expressões estáveis ​​em tecidos diferentes das abelhas. Publicada em tempo real para os iniciadores de PCR e o gene vg USP são utilizados nesta experiência 24. Os dados são analisados ​​usando o método de delta-delta CT 25.

<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>

We have nothing to disclose.

Nosso estudo é o primeiro esforço para bater simultaneamente até dois genes em insetos adultos. Nossos resultados mostram que as duas estratégias de dsRNA injeção (injeção única e injeção de dois dias) são eficazes para a supressão gene casal e silenciamento gênico simultânea de vg e usp pode ser testado seis dias após as injeções de dsRNA em abelhas 8,9. No entanto, a eficácia knockdown gene varia em diferentes espécies de insetos, dependendo do nível de transcrição d...

Interferência de RNA (RNAi) é baseada em ARN o silenciamento pós-transcricional que ocorre em uma ampla variedade de organismos eucarióticos. O processo de RNAi é desencadeada por RNA (dsRNA) precursores double-stranded endógenos ou exógenos. O dsRNA ativa a proteína ribonuclease Dicer que liga e corta o dsRNA de pequenos fragmentos (20-25 pb). Em seguida, os pequenos fragmentos de ARNdc de um guia de reconhecimento e de clivagem de mRNAs complementares por proteínas Argonaute, um componente catalítico do RNA induzida por complexo silenciamento (RISC) 1. Nos mamíferos, dsRNAs mais de 30 nt, ativar uma resposta antiviral (resposta interferon, IFN), que leva à degradação inespecífica de RNA transcrições 2. No entanto, os longos dsRNAs têm provado ser eficazes e específicos no insectos já que existe uma falta de este IFN 3. Longos dsRNAs têm sido utilizados para regulação negativa dos genes alvo em diferentes espécies de insectos. As abelhas são um dos pionorganismos insectos EER em que as funções de muitos genes importantes no desenvolvimento e comportamento foram revelados usando ARNdc 4,5. Vários métodos de entrega de dsRNA foram realizados em abelhas: Alimentação dsRNA downregulates eficiente expressão do gene alvo em larvas de abelhas 6, enquanto a injeção dsRNA é uma abordagem eficaz para knockdown do gene em embriões de mel de abelha 4 e abelhas adultas 7,8. Efeitos knockdown do gene exibidos pela aplicação de dsRNA de insetos são transitórias e localizadas. Estudos têm demonstrado que ambas as injecções de dsRNA abdominais e torácicas injecções dsRNA suprimir eficazmente a expressão do gene alvo em células de gordura corporal abdominal de insectos 9,10. DsRNA é injetado na cavidade abdominal e torácica e células de gordura corporal são capazes de assumir o dsRNA da hemolinfa, onde as células são banhadas 10. No entanto, os genes de outros órgãos, tais como o cérebro, ovários e não pode ser alvo de qualquerinjeções abdominal ou torácica. A fim de visar genes no cérebro da abelha do mel, a injecção de ARNcd cérebro também tem sido realizada, o que influencia de forma eficaz a expressão do gene alvo em áreas cerebrais locais 11. Aqui, nós só documentar injeção dsRNA abdominal, que é mais comumente usado em abelhas adultas. RNAi tem sido utilizado principalmente para atingir um único gene e tem sido uma ferramenta poderosa para revelar a função do gene. No entanto, qualquer gene não está isolada das outras, é em redes reguladoras complexas. Uma chave para a compreensão de um processo biológico é dissecar como os genes interagem uns com os outros, o que requer manipulações simultâneas de múltiplos genes, em vez de um único gene knockdown. Em linhagens de células de mamíferos, os cientistas conseguiram inibir simultaneamente dois ou três genes por meio de sistemas de entrega de 12 ou multi-microRNA (miRNA) projeta hairpin 13. Mas, em insetos, vários knockdowns genes ainda não foram testados. Aqui, nós present estratégias de injeção diferentes, que podem atingir um gene knockdown dupla. Nós alvo dois genes: vitelogenina (vg), que codifica um precursor da proteína da gema e ultraspiracle (USP), que codifica um receptor putativo para a hormona juvenil (HA), pode servir como um factor de transcrição de mediação de respostas a JH 14 em abelhas de mel. Vg e JH regular o outro em um ciclo de realimentação 15 e estão envolvidos em mel de abelha comportamental regra 9. Usando o gene knockdown dupla, que perturbam tanto Vg e vias de JH, e descobrir como eles afetam conjuntamente o comportamento das abelhas de mel e fisiologia e como vg, usp e JH interagir 9. Percepção gustativa é um preditor comportamental para mel de abelha comportamento social 16. Em termos de desenvolvimento comportamental, abelhas ninho com alta percepção gustativa comportamentalmente maduro rápidos, e geralmente de forragem no início da vida e preferem coletar pólen 16,17. Embora m regulamentarecanismos subjacentes percepção gustativa ainda não estão claros, os estudos mostraram que a percepção gustativa está ligada ao metabolismo de energia interna 9, secreção hormonal 18,19 e biogenética amina caminhos 20. Ambos Vg e JH são importantes reguladores hormonais modulando a percepção gustativa 7,21. No laboratório, a uma variação da percepção gustativa em abelhas de mel pode ser avaliada testando a resposta extensão tromba (PER) para diferentes soluções de sacarose. Cada abelha é testada tocando tanto suas antenas com uma gota de água, seguido por uma série ascendente de concentração de 0,1, 0,3, 1, 3, 10, 30% de sacarose. A resposta positiva é observado se a abelha estende totalmente seus tromba quando uma gota de água ou sacarose é tocado a cada antena. Com base no número de respostas positivas para as soluções, o nível de percepção gustativa de cada indivíduo pode ser determinada 16. No entanto, a aplicação do PER não se limita a medir gpercepção ustatory. O PER é também um método eficaz para testar o estado metabólico de abelhas, tais como a saciedade vs fome. As abelhas com maiores respostas a sacarose são mais faminto em geral (Wang e Amdam, dados não publicados). Além disso, o paradigma PER também pode ser utilizado na aprendizagem e na memória associativa em abelhas de mel. Neste caso, as abelhas serão treinados para associar a presença de água de sacarose, com um odor. Quando as abelhas aprender a associação, apenas a presença do odor pode evocar uma resposta positiva tromba sem premiar os com sacarose a 22,23. Neste vídeo, vamos mostrar como realizar PER para avaliar a percepção gustativa, que tem sido relacionada com vg e usp knockdown casal em um estudo anterior 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)

Este vídeo demonstra novas técnicas de interferência de RNA (RNAi), que downregulate dois genes simultaneamente em abelhas usando RNA (dsRNA) injeções de cadeia dupla. Ele também apresenta um protocolo de resposta extensão probóscide (PER) ensaio para medir a percepção gustativa. Knockdown gene RNAi mediado é uma técnica eficaz diminuindo a expressão de genes-alvo. Esta técnica é geralmente utilizada para a manipulação de um único gene, mas tem limitações para detectar interações e efeitos conjuntas entre genes. Na primeira parte deste vídeo, apresentamos duas estratégias para bater simultaneamente até dois genes (chamados gene knockdown duplo). Mostramos as duas estratégias são capazes de suprimir efetivamente dois genes, vitelogenina (VG) e ultraspiracle (USP), que estão em um ciclo de feedback regulador. Esta abordagem gene knockdown dupla pode ser usada para dissecar inter-relações entre genes e podem ser prontamente aplicadasdiferentes espécies de insetos. A segunda parte deste vídeo é uma demonstração de tromba extensão resposta ensaio (PER), em abelhas produtoras de mel após o tratamento de gene knockdown dupla. O ensaio PER é um teste padrão para medir a percepção gustativa em abelhas produtoras de mel, que é um indicador chave para o quão rápido amadurecimento comportamental de uma abelha de mel é. Maior percepção gustativa de abelhas ninho indica maior desenvolvimento comportamental, que é muitas vezes associada a uma idade mais precoce de início do forrageamento e forrageamento especialização em pólen. Além disso, por ensaio pode ser aplicado para identificar estados metabólicos de saciedade ou a fome em abelhas de mel. Finalmente, por ensaio combinado com diferentes estímulos emparelhamento de odor para o condicionamento as abelhas também é amplamente utilizada para estudos de aprendizagem e memória em abelhas de mel.

Tanto o single-injeção e dois dias de injeção de estratégias reduziu significativamente vg (One-way ANOVA, p &lt;0,001) (Figura 2A) e USP (One-way ANOVA, p &lt;0,001) (Figura 2B) níveis de transcrição em abelhas seis dias depois do ARNdc foi injectada. A supressão da USP transcrição usando a única injecção com uma mistura de ARNdc e ​​a injecção de dois dias com vg primeiro e segundo USP (vg / USP) foi significati...

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

Neste protocolo, descrevemos duas estratégias que suprimem simultaneamente dois genes (gene knockdown double) em abelhas. Em seguida, apresentamos como usar a resposta de extensão probóscide (PER) ensaio para estudar o efeito do gene knockdown duplo em mel de abelha percepção gustativa.

RNAi mediado por Gene Knockdown duplo e Mensuração Percepção gustativa em abelhas do mel (<em&gt; Apis mellifera</em&gt;)

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

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

14.8K visualizações.  Arizona State University. Neste protocolo, descrevemos duas estratégias que suprimem simultaneamente dois genes (gene knockdown double) em abelhas. Em seguida, apresentamos como usar a resposta de extensão probóscide (PER) ensaio para estudar o efeito do gene knockdown duplo em mel de abelha percepção gustativa.

Vídeo: 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.

Fluorescência dupla In situ Hibridação nas Seções cérebro fresco

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

Neurociência

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.

Farmacologia Comportamental no condicionamento clássico da Resposta Extensão Proboscis em Abelhas ( Apis mellifera)

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.

Criação de Objetos e categorias de objetos para o Estudo Percepção e Aprendizagem Perceptual

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.

Acessível usando Arrays LED para Foto Estimulação de Neurônios

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.

Transdução Viral seletiva de Adultos-nascido neurônios olfativos de crônica In vivo Estímulo Optogenetic

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.

MPI Simulator CyberMotion: Implementação de um simulador de movimento Novela Caminho para Investigar Integração multisensorial em Três Dimensões

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.

Condicionado tátil e Análise do Movimento de estratégias de amostragem antenais de abelhas (<em&gt; Apis mellifera</em&gt; 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 ...

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.

Microeletrodos de cano duplo e concêntricos para a medição de Extracelular Ion Signals no tecido cerebral

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.

A Novel Behavioral Ensaio investigar as gustativas de um indivíduo, livre-se deslocam Bumble abelhas (<em&gt; Terrestris do Bombus</em&gt;)

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.

Manipulação neurofarmacologia de contido e vôo livre abelhas do mel,<em&gt; Apis mellifera</em

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