<p class='jove_content'><strong>Mohammed R. Milad</strong> is supported by NIMH grant R01 MH097880 and institutional funds from the Department of Psychiatry at MGH.</p>

<p class="jove_content"> NOTA: Todos os procedimentos descritos neste protocolo ter sido aprovado pela Subcomissão de Investigação Animal Care, que serve como Cuidado e Uso Comitê de animal Institucional (IACUC) para o Hospital Geral de Massachusetts, e estão de acordo com os Institutos Nacionais de diretrizes de saúde. </p><p class="jove_title"> 1. animal Habitação e Procedimento Handling </p><ol><li> Após a chegada, os ratos fêmeas casa adulto Sprague Dawley cerca de 8-10 semanas de idade (pesando 200-225 g) em grupos de caixas de plástico 3-4 polegadas com cama de lascas de madeira. Mantê-los em uma dieta de acesso ad libitum à comida e água e um ciclo claro / escuro de 12 horas de luz. Deixar ratos para se aclimatar às condições de habitação e de animais facilidade para um mínimo de 1 semana. <br /> NOTA: Todos os procedimentos deste manuscrito são realizados durante o ciclo de luz. </li><li> Após este período de aclimatação, lidar com todos os animais durante 5 min cada, para 2-3 dias. Adequadamente pegar a ratos, uma de cada vez (suportado pelo tronco e não pela cauda),mantê-los de forma segura, e acariciá-los durante este tempo. Lidar com os animais que aparecem mais estressado, nervoso ou ansioso por longos períodos de tempo. Faça isso para minimizar a lidar com o estresse, o que pode afetar os seguintes passos neste protocolo. <br /> NOTA: O número de dias e quantidade de tempo lidar com os animais pode variar entre diferentes laboratórios; no entanto, estes devem ser coerentes entre os experimentos dentro do mesmo laboratório. </li></ol><p class="jove_title"> 2. Monitorização do ciclo estral: Vaginal Swab Manchas e Procedimento de coloração </p><ol><li> Adicione solução salina a 0,9% (cloreto de sódio dissolvido em água destilada). Umedeça um aplicador com ponta de algodão nesta solução. Blot a ponta usando uma toalha de papel para evitar a saturação excessiva, o que poderia levar a coleta de amostra insuficiente ou perda de células. Segurando a cauda para cima longe da abertura vaginal, insira suavemente a ponta de algodão dentro do canal vaginal e rolar em torno das paredes para capturar células vaginais soltos. </li><li> Se o animal urina na camaponta ton, substitua-o por um novo, como a contaminação da urina irá tornar mais difícil para a identificação de fase. Tenha muito cuidado para não causar sofrimento ao animal durante este procedimento, porque a exposição ao estresse vai interromper o ciclo estral. Ao recolher amostras, visam alcançar uma inserção rápida e retirada que não é muito profundo como para minimizar o estímulo extra para o colo do útero, o que pode induzir a pseudociese. <br /> NOTA: pseudogestação pode ser identificado como um ciclo estral acíclicos que tipicamente dura cerca de 12 dias, <em>isto é,</em> dias consecutivos de diestro <sup>25.</sup> </li><li> Retire com cuidado a ponta do aplicador e enrolá-lo sobre uma lâmina de microscópio pré-rotulados. Evite pressioná muito difícil como muito líquido no slide pode fazer identificação de fase difícil, uma vez seco. <br /> NOTA: Um slide pode conter vários dias para cada animal, embora isso possa ser feito de acordo com as preferências individuais. </li><li> Certifique-se que as transferências de amostra para o slide. Coleta de amostras para Several dias consecutivos em uma lâmina para ser capaz de melhor acompanhar e identificar fases estral diárias alterações. <br /> NOTA: Se possível, também é ideal se as pessoas que coletam amostras de continuar a ser a mesma para todos os animais. </li><li> Uma vez que a lâmina é seco, comece o protocolo de coloração para a identificação da fase. Usando o kit de coloração (ver Tabela de Materiais), mergulhar slides de 10 vezes no fixador (azul claro), 10 vezes em solução corante (cor de rosa), e 5 vezes em solução counterstain (roxo). Lavar as lâminas com cuidado em água corrente. Vamos lâminas completamente ar seco antes de ver ao microscópio. <br /> NOTA: O longo ciclo estral tipicamente 4-5 dias consiste de (nesta ordem): estro (E), metaestro (M), diestro (D), e proestro <sup>26</sup> (P). O estro é caracterizado por células azuis, cornificado, metaestro por leucócitos e uma combinação de células cornificadas e nucleadas, diestro é semelhante ao metaestro na presença de tipos de células excepto eles são muito escassos, e por agregados proestro roxo-coradascélulas de nucleadas <strong>(Figura 1).</strong> Existem vários procedimentos de coloração, em adição às descritas acima, que são igualmente eficazes para citologia vaginal esfregaço. </li><li> Recolha de amostras para, pelo menos, dois ciclos completos (~ 8-10 dias), a fim de controlar com precisão as fases antes da experimentação. Seja consistente com a hora do dia da colheita de amostras. Coletar amostras de esfregaço vaginal diariamente durante e até o final do experimento. </li></ol><p class="jove_title"> 3. Pré-exposição </p><ol><li> Antes do início do experimento, pré-expor os animais ao condicionamento câmaras que os animais possam se adaptar ao contexto (câmaras de condicionamento) antes de qualquer teste comportamental. <ol><li> Pré-expor as fêmeas para as câmaras de condicionamento (localizados em caixas atenuantes de sons), com as luzes da casa por 20-30 min por dia durante 3 dias antes de qualquer experimentação. Atribuir a caixa eles são pré-expostos a como a sua câmara para o estudo. Limpe bem tele caixas (paredes e bandejas) entre as sessões e animais para remover os odores, pois podem afetar o comportamento e ciclo estral. </li></ol></li></ol><p class="jove_title"> 4. Dia 1: Habituação / Medo Condicionado </p><ol><li> Uma vez que os animais tenham sido verificada na fase do estro dos seus ciclos estrais, começar a fase de habituação / condicionamento da experiência de 3 dias. <ol><li> Tome pré-tom medidas iniciais de congelamento prévio para o estímulo condicionado primeiro tom (CS) apresentações em cada dia do experimento. Faça isso por meio da aquisição pontuações por cento de congelamento (calculado dividindo o segundos gastos imóvel pela duração do julgamento, multiplicado por 100) durante o intervalo de tempo de estímulo ausente no início do primeiro julgamento CS-alone antes do início CS. <br /> NOTA: Estas medidas podem ser usados ​​para avaliar e comparar os níveis de congelamento da linha de base com aquelas durante habituação, condicionado, extinção, e recall. </li><li> Para realizar o treinamento extinção durante a fase de metaestro,condicionar os ratos no seu último dia de estro. Swab e identificar fase de estro (passo 2) todos os dias antes de iniciar o protocolo experimental (condicionado / extinção / chamada). Como a fase do cio pode durar mais de um dia, o uso história ciclo de prever duração das fases para determinar quando os animais estão prontos para começar a treinar. </li></ol></li><li> Nas câmaras condicionado, ligue o chão grade pé-choque para um gerador de estimulação elétrica. Usar um nível de estimulação de 0,5-0,6 mA como o estímulo não condicionado (US). Use um kHz 4, 80 dB tom como o estímulo condicionado (CS). <br /> NOTA: Estes parâmetros podem variar entre laboratórios e podem ser modificados para o condicionamento ideal. <ol><li> Se o programa de análise de congelação requer sinais visuais (em oposição a temporal), co-activar uma luz LED com o tom. Posicionar a luz do lado de fora da câmara operante, dentro do quadro de vídeo. No início de cada ensaio, activar o diodo emissor de luz iluminada e mantê-lo até que a extremidade do the julgamento. <br /> NOTA: Esta luz não é visível pelo animal e serve apenas como um marcador para o início e final do julgamento dentro dos arquivos de vídeo gravados (a ser usado para medir o comportamento de congelamento). </li></ol></li><li> Coloque os animais para dentro das câmaras de condicionamento para 5 ensaios CS-alone de habituação. Imediatamente após a sessão de habituação, executar 7 ensaios CS-americanas de medo condicionado emparelhados. Certifique-se de que o tom CS tem a duração de 30 segundos e coterminates com o choque sec US 0,5. Ensaios são executados em todas as sessões (habituação, condicionado, extinção, e de recall), com um intervalo entre variável média de 3 minutos. </li><li> No fim da sessão de condicionamento, os animais regressar às suas gaiolas de alojamento e à instalação para animais até ao dia seguinte. </li></ol><p class="jove_title"> 5. Dia 2: Estradiol Preparação </p><ol><li> Swab e identificar a fase de estro para todos os animais que foram submetidos Dia 1 do presente protocolo. Prepare o estradiol (também conhecida como E2, 17beta-estradiol, oubeta-estradiol) para a 15 ug / kg de dose de injecção subcutânea utilizando óleo de sésamo ou de 0,9% solução salina como veículo. </li><li> Para calcular a quantidade de estradiol para se preparar para injeções, use os pesos corporais de todos os animais, que foram medidos e registados quando eles foram condicionados no dia 1 deste protocolo. </li><li> Faça os cálculos para padronizar o volume administrado de estradiol <em>(ou seja,</em> 0,2 ml) por rato. Dado que o estradiol é de difícil dissolução, devido à sua insolubilidade em água e requer um grande volume de solvente para a dose pequena, preparar uma solução de reserva por mistura de estradiol em óleo de sésamo sobre o calor até se dissolver. A solução pode então ser passada através de um filtro de 0,22 um, para remover contaminantes. Por exemplo, adicionar um volume calculado de a / 1 ml de solução de óleo de sésamo 3,000 ug de estradiol para o volume total necessário de óleo de sésamo, a fim de atingir um 15 ug / kg num volume de 0,2 ml injecção por animal. <br /> NOTA: Como alternativa, utilizar outras formas de estrogênio tha<html> t são solúveis em água e mais fácil de dissolver. </li></ol><p class="jove_title"> 6. Dia 2: Administração Estradiol / Formação Extinction </p><ol><li> Administrar o estradiol subcutaneamente. Levante a pele solta entre a escápula pelo pescoço em uma pitada suave e inserir a agulha da seringa no triângulo que é formada pelas dobras. </li><li> 30 minutos após as injecções subcutâneas de estradiol, colocar os animais nas câmaras de condicionamento e de novo começar a formação de extinção. Nesta sessão extinção, realizar 20 CS-alone ensaios, com cada ensaio que consiste em um 30 seg, 4 kHz, 80 tom dB, apresentado em um intervalo entre variável média de 3 min. <br /> NOTA: O efeito do estradiol na recordação ocorre quando o estradiol é administrado antes da formação extinção ou imediatamente após a extinção. O efeito não é observado 4 horas após a extinção, por isso é recomendado para injetar próximo da hora de início ou no final da sessão de treinamento <sup>15.</sup> </li></ol>. le &quot;&gt; 7 Day 3: Extinction recall </p><ol><li> 24 horas após o treinamento extinção, pincelar os animais que Dia 1 e 2 do presente protocolo concluídas. </li><li> Coloque os animais em suas câmaras de condicionamento atribuídos (o mesmo que o dia 1 e 2). Inicie a sessão recordação extinção, apresentando 3 CS-alone ensaios (os três ensaios de recordação extinção), que consiste de apenas 3,80 dB CS-alone ensaios com intervalos entre tentativas semelhantes às 20 ensaios administrados para o treinamento extinção como descrito no passo 6.2. </li><li> Após a conclusão da sessão de recall, retornar os animais para suas gaiolas. </li></ol><p class="jove_title"> Análise 8. Dados </p><ol><li> Adquirir dados comportamentais através de vídeo e analisá-los utilizando programas de computador. <br /> NOTA: Congelamento pontuações também podem ser contadas manualmente por um experimentador que é cego para o tratamento medicamentoso cronometrando episódios de congelamento durante a apresentação CS de cada julgamento, expresso em percentagem do tempo gasto congelamento durante o tom. O congelamento édefinido pela completa falta de movimento, com excepção da respiração durante os ensaios 30 sec CS-americanos. Calcule por cento congelamento dividindo os segundos gastos congelamento (imóvel) por 30 segundos (duração do julgamento) multiplicado por 100. </li></ol>

<ol><li>Baron-Cohen, S., Knickmeyer, R. C., Belmonte, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sex+differences+in+the+brain%3A+implications+for+explaining+autism%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Sex differences in the brain: implications for explaining autism.</a> <em>Science</em>. <strong>310</strong> (5749), 819-823 (2005).</li>
<li>Voyer, D., Voyer, S., Bryden, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Magnitude+of+sex+differences+in+spatial+abilities%3A+a+meta-analysis+and+consideration+of+critical+variables%5BTitle%5D)+AND+%22Psychological+Bulletin%22%5BJournal%5D)">Magnitude of sex differences in spatial abilities: a meta-analysis and consideration of critical variables.</a> <em>Psychological Bulletin</em>. <strong>117</strong> (2), 250-270 (1995).</li>
<li>Pinker, S., Spelke, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+Conversation+with+Steven+Pinker+and+Elizabeth+Spelke%5BTitle%5D)+AND+%22The+Science+of+Gender+and+Science%22%5BJournal%5D)">A Conversation with Steven Pinker and Elizabeth Spelke.</a> <em>The Science of Gender and Science</em>. , Harvard University: Mind/Brain/Behavior Initiative. (2005).</li>
<li>Hampson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Estrogen-related+variations+in+human+spatial+and+articulatory-motor+skills%5BTitle%5D)+AND+%22Psychoneuroendocrinology%22%5BJournal%5D)">Estrogen-related variations in human spatial and articulatory-motor skills.</a> <em>Psychoneuroendocrinology</em>. <strong>15</strong>, 97-111 (1990).</li>
<li>Hampson, E., Kimura, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reciprocal+effects+of+hormonal+fluctuations+on+human+motor+and+perceptual-spatial+skills%5BTitle%5D)+AND+%22Behavioral+Neuroscience%22%5BJournal%5D)">Reciprocal effects of hormonal fluctuations on human motor and perceptual-spatial skills.</a> <em>Behavioral Neuroscience</em>. <strong>102</strong>, 456-459 (1988).</li>
<li>Hampson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Variations+in+sex-related+cognitive+abilities+across+the+menstrual+cycle%5BTitle%5D)+AND+%22Brain+and+Cognition%22%5BJournal%5D)">Variations in sex-related cognitive abilities across the menstrual cycle.</a> <em>Brain and Cognition</em>. <strong>14</strong> (1), 26-43 (1990).</li>
<li>Kilpatrick, D. G., Resnick, H. S., Milanak, M. E., Miller, M. W., Keyes, K. M., Friedman, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((National+estimates+of+exposure+to+traumatic+events+and+PTSD+prevalence+using+DSM-IV+and+DSM-5+criteria%5BTitle%5D)+AND+%22Journal+of+Traumatic+Stress%22%5BJournal%5D)">National estimates of exposure to traumatic events and PTSD prevalence using DSM-IV and DSM-5 criteria.</a> <em>Journal of Traumatic Stress</em>. <strong>26</strong> (5), 537-547 (2013).</li>
<li>Breslau, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gender+differences+in+trauma+and+posttraumatic+stress+disorder%5BTitle%5D)+AND+%22Journal+of+Gender-Specific+Medicine%22%5BJournal%5D)">Gender differences in trauma and posttraumatic stress disorder.</a> <em>Journal of Gender-Specific Medicine</em>. <strong>5</strong> (1), 34-40 (2002).</li>
<li>Frans, O., Rimmo, P. A., Aberg, L., Fredrikson, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Trauma+exposure+and+post-traumatic+stress+disorder+in+the+general+population%5BTitle%5D)+AND+%22Acta+Psychiatrica+Scandinavica%22%5BJournal%5D)">Trauma exposure and post-traumatic stress disorder in the general population.</a> <em>Acta Psychiatrica Scandinavica</em>. <strong>111</strong> (4), 291-299 (2005).</li>
<li>Breslau, N., Kessler, R. C., Chilcoat, H. D., Schultz, L. R., Davis, G. C., Andreski, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Trauma+and+posttraumatic+stress+disorder+in+the+community%3A+the+1996+Detroit+Area+Survey+of+Trauma%5BTitle%5D)+AND+%22Archives+of+General+Psychiatry%22%5BJournal%5D)">Trauma and posttraumatic stress disorder in the community: the 1996 Detroit Area Survey of Trauma.</a> <em>Archives of General Psychiatry</em>. <strong>55</strong> (7), 626-632 (1996).</li>
<li>Seedat, S., Stein, D. J., Carey, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Post-traumatic+stress+disorder+in+women%3A+epidemiological+and+treatment+issues%5BTitle%5D)+AND+%22CNS+Drugs%22%5BJournal%5D)">Post-traumatic stress disorder in women: epidemiological and treatment issues.</a> <em>CNS Drugs</em>. <strong>19</strong> (5), 411-427 (2005).</li>
<li>Holbrook, T. L., Hoyt, D. B., Stein, M. B., Sieber, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gender+differences+in+long-term+posttraumatic+stress+disorder+outcomes+after+major+trauma%3A+women+are+at+higher+risk+of+adverse+outcomes+than+men%5BTitle%5D)+AND+%22Journal+of+Trauma%22%5BJournal%5D)">Gender differences in long-term posttraumatic stress disorder outcomes after major trauma: women are at higher risk of adverse outcomes than men.</a> <em>Journal of Trauma</em>. <strong>53</strong> (5), 882-888 (2002).</li>
<li>Labad, J., Menchon, J. M., Alonso, P., Segalas, C., Jimenez, S., Jaurrieta, N., <em>et al.</em> <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gender+differences+in+obsessive-compulsive+symptom+dimensions%5BTitle%5D)+AND+%22Depression+and+Anxiety%22%5BJournal%5D)">Gender differences in obsessive-compulsive symptom dimensions.</a> <em>Depression and Anxiety</em>. <strong>25</strong> (10), 832-838 (2008).</li>
<li>Gupta, R. R., Sen, S., Diepenhorst, L. L., Rudick, C. N., Maren, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Estrogen+modulates+sexually+dimorphic+contextual+fear+conditioning+and+hippocampal+long-term+potentiation+%28LTP%29+in+rats%5BTitle%5D)+AND+%22Brain+Research%22%5BJournal%5D)">Estrogen modulates sexually dimorphic contextual fear conditioning and hippocampal long-term potentiation (LTP) in rats.</a> <em>Brain Research</em>. <strong>888</strong>, 356-365 (2001).</li>
<li>Milad, M. R., Igoe, S. A., Lebron-Milad, K., Novales, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Estrous+cycle+phase+and+gonadal+hormones+influence+conditioned+fear+extinction%5BTitle%5D)+AND+%22Neuroscience%22%5BJournal%5D)">Estrous cycle phase and gonadal hormones influence conditioned fear extinction.</a> <em>Neuroscience</em>. <strong>164</strong> (3), 887-895 (2009).</li>
<li>Zeidan, M., <em>et al.</em> <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Estradiol+modulates+medial+prefrontal+cortex+and+amygdala+activity+during+fear+extinction+in+women+and+female+rats%5BTitle%5D)+AND+%22Biological+Psychiatry%22%5BJournal%5D)">Estradiol modulates medial prefrontal cortex and amygdala activity during fear extinction in women and female rats.</a> <em>Biological Psychiatry</em>. <strong>70</strong> (10), 920-927 (2011).</li>
<li>Lebron-Milad, K., Milad, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sex+differences%2C+gonadal+hormones+and+the+fear+extinction+network%3A+implications+for+anxiety+disorders%5BTitle%5D)+AND+%22Biology+of+Mood+%26+Anxiety+Disorders%22%5BJournal%5D)">Sex differences, gonadal hormones and the fear extinction network: implications for anxiety disorders.</a> <em>Biology of Mood & Anxiety Disorders</em>. <strong>2</strong> (3), (2012).</li>
<li>Rey, C. D., Lipps, J., Shansky, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dopamine+d1+receptor+activation+rescues+extinction+impairments+in+low-estrogen+female+rats+and+induces+cortical+layer-specific+activation+changes+in+prefrontal-amygdala+circuits%5BTitle%5D)+AND+%22Neuropsychopharmacology%22%5BJournal%5D)">Dopamine d1 receptor activation rescues extinction impairments in low-estrogen female rats and induces cortical layer-specific activation changes in prefrontal-amygdala circuits.</a> <em>Neuropsychopharmacology</em>. <strong>39</strong> (5), 1282-1289 (2013).</li>
<li>Ström, J. O., Theodorsson, A., Ingberg, E., Isaksson, I. M., Theodorsson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ovariectomy+and+17%CE%B2-estradiol+Replacement+in+Rats+and+Mice%3A+A+Visual+Demonstration%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Ovariectomy and 17β-estradiol Replacement in Rats and Mice: A Visual Demonstration.</a> <em>Journal of Visualized Experiments</em>. , e4013(2012).</li>
<li>Markham, J. A., Pych, J. C., Juraska, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ovarian+hormone+replacement+to+aged+ovariectomized+female+rats+benefits+acquisition+of+the+morris+water+maze%5BTitle%5D)+AND+%22Hormones+%26+Behavior%22%5BJournal%5D)">Ovarian hormone replacement to aged ovariectomized female rats benefits acquisition of the morris water maze.</a> <em>Hormones & Behavior</em>. <strong>42</strong> (3), 284-293 (2002).</li>
<li>Markowska, A. L., Savonenko, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effectiveness+of+estrogen+replacement+in+restoration+of+cognitive+function+after+long-term+estrogen+withdrawal+in+aging+rats%5BTitle%5D)+AND+%22Journal+of+Neuroscience%22%5BJournal%5D)">Effectiveness of estrogen replacement in restoration of cognitive function after long-term estrogen withdrawal in aging rats.</a> <em>Journal of Neuroscience</em>. <strong>22</strong> (24), 10985-10995 (2002).</li>
<li>Bredemann, T. M., McMahon, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((17%CE%B2+Estradiol+Increases+Resilience+and+Improves+Hippocampal+Synaptic+Function+in+Helpless+Ovariectomized+Rats%5BTitle%5D)+AND+%22Psychoneuroendocrinology%22%5BJournal%5D)">17β Estradiol Increases Resilience and Improves Hippocampal Synaptic Function in Helpless Ovariectomized Rats.</a> <em>Psychoneuroendocrinology</em>. <strong>42</strong>, 77-88 (2014).</li>
<li>Grueso, R., <em>et al.</em> <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Early%2C+but+not+late+onset+estrogen+replacement+therapy+prevents+oxidative+stress+and+metabolic+alterations+caused+by+ovariectomy%5BTitle%5D)+AND+%22Antioxidants+and+Redox+Signaling%22%5BJournal%5D)">Early, but not late onset estrogen replacement therapy prevents oxidative stress and metabolic alterations caused by ovariectomy.</a> <em>Antioxidants and Redox Signaling</em>. <strong>20</strong> (2), 236-246 (2014).</li>
<li>Shughrue, P. J., Bushnell, C. D., Dorsa, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Estrogen+receptor+messenger+ribonucleic+acid+in+female+rat+brain+during+the+estrous+cycle%3A+a+comparison+with+ovariectomized+females+and+intact+males%5BTitle%5D)+AND+%22Endocrinology%22%5BJournal%5D)">Estrogen receptor messenger ribonucleic acid in female rat brain during the estrous cycle: a comparison with ovariectomized females and intact males.</a> <em>Endocrinology</em>. <strong>131</strong> (1), 381-388 (1992).</li>
<li>Frye, C. A., Erskine, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Influence+of+time+of+mating+and+paced+copulation+on+induction+of+pseudopregnancy+in+cyclic+female+rats%5BTitle%5D)+AND+%22Journal+of+Reproduction+and+Fertility%22%5BJournal%5D)">Influence of time of mating and paced copulation on induction of pseudopregnancy in cyclic female rats.</a> <em>Journal of Reproduction and Fertility</em>. <strong>90</strong> (2), 375-385 (1990).</li>
<li>Becker, J. B., <em>et al.</em> <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Strategies+and+methods+for+research+on+sex+differences+in+brain+and+behavior%5BTitle%5D)+AND+%22Endocrinology%22%5BJournal%5D)">Strategies and methods for research on sex differences in brain and behavior.</a> <em>Endocrinology</em>. <strong>146</strong>, 1650-1673 (2005).</li>
<li>Westwood, F. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+female+rat+reproductive+cycle%3A+a+practical+histological+guide+to+staging%5BTitle%5D)+AND+%22Toxicologic+Pathology%22%5BJournal%5D)">The female rat reproductive cycle: a practical histological guide to staging.</a> <em>Toxicologic Pathology</em>. <strong>36</strong> (3), 375-384 (2008).</li>
<li>Hurn, P. D., Macrae, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Estrogen+as+a+Neuroprotectant+in+Stroke%5BTitle%5D)+AND+%22Journal+of+Cerebral+Blood+Flow+%26+Metabolism%22%5BJournal%5D)">Estrogen as a Neuroprotectant in Stroke.</a> <em>Journal of Cerebral Blood Flow & Metabolism</em>. <strong>20</strong>, 631-652 (2000).</li>
<li>Parasuraman, S., Raveendran, R., Kesavan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Blood+sample+collection+in+small+laboratory+animals%5BTitle%5D)+AND+%22Journal+of+Pharmacology+and+Pharmacotherapeutics%22%5BJournal%5D)">Blood sample collection in small laboratory animals.</a> <em>Journal of Pharmacology and Pharmacotherapeutics</em>. <strong>1</strong> (2), 87-93 (2010).</li>
<li>Gillies, G. E., McArthur, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Estrogen+actions+in+the+brain+and+the+basis+for+differential+action+in+men+and+women%3A+a+case+for+sex-specific+medicines%5BTitle%5D)+AND+%22Pharmacological+Reviews%22%5BJournal%5D)">Estrogen actions in the brain and the basis for differential action in men and women: a case for sex-specific medicines.</a> <em>Pharmacological Reviews</em>. <strong>62</strong> (2), 155-198 (2010).</li>
<li>Tolin, D. F., Foa, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sex+differences+in+trauma+and+posttraumatic+stress+disorder%3A+a+quantitative+review+of+25+years+of+research%5BTitle%5D)+AND+%22Psychological+Bulletin%22%5BJournal%5D)">Sex differences in trauma and posttraumatic stress disorder: a quantitative review of 25 years of research.</a> <em>Psychological Bulletin</em>. <strong>132</strong>, 959-992 (2006).</li>
</ol>

<p class="jove_content"> Os autores deste manuscrito não tem concorrentes interesses financeiros ou outros conflitos de interesse a divulgar. </p>

<p class="jove_content"> Extinção do medo tem sido extensivamente estudada em ratos machos com os mecanismos neurais que medeiam extinção medo condicionado identificados e avaliados sob várias manipulações. Relativamente poucos estudos analisaram ratas ou o papel das hormonas sexuais na extinção do medo. Para estudar especificamente os efeitos do estrogénio sobre a extinção do medo, naturalmente ciclagem ratas são submetidos a um paradigma comportamental de três dias. Este procedimento consiste em fases habituação / condicionado...

<p class="jove_content"> Diferenças sexuais inerentes são observados em vários comportamentos cognitivos e paradigmas de aprendizagem em humanos e roedores. Por exemplo, foi relatado que as mulheres geralmente têm habilidades verbais e orientada para o detalhe mais fortes, enquanto os homens têm melhores habilidades espaciais <sup>1-3.</sup> Estas diferenças entre os sexos pode ser em parte devido à influência dos hormônios sexuais. Altos níveis de estradiol melhorar o desempenho em tarefas em que as mulheres são melhores, mas pioram o desempenho em tarefas que os homens geralmente fazem bem <sup>4-6.</sup> Embora esta evidência é convincente, a capacidade limitada de controlar completamente ambientes experimentais em estudos humanos torna difícil determinar se esses efeitos sobre o comportamento pode ser atribuído especificamente aos hormônios. Os estudos em animais, em contraste, permite a circunstâncias totalmente controladas. </p><p class="jove_content"> Embora os mecanismos neurais básicos de extinção do medo, foram identificados e são bem estudada em machos, não é claro seestes sistemas são os mesmos em fêmeas ou como eles mudam ao longo do ciclo estral. O condicionamento do medo e extinção são amplamente utilizados paradigmas comportamentais em roedores e humanos para realizar estudos relacionados a transtornos de ansiedade. Dado que as mulheres têm um maior risco de transtornos de ansiedade, bem como a duração dos sintomas e a gravidade maior <sup>7-13,</sup> é crítico para incluir fêmeas nestes estudos. A sub-representação das mulheres na pesquisa pode ser atribuída aos desafios de monitoramento fase de estro e contábil para os efeitos comportamentais de hormônios sexuais. Laboratórios que fazem examinar fêmeas nesta problemas encontro em conta que muitas vezes passam não declarada ou não estão descritos nos métodos para seus estudos. </p><p class="jove_content"> Emergentes evidência em roedores sugere que as diferenças sexuais em extinção do medo são modulados por hormonas sexuais <sup>14-18.</sup> Com medo condicionado, os animais são treinados para temer um estímulo particular. Depois de uma série de apresentações de unreinforcedo estímulo, os animais aprendem a não temer a sugestão, um processo chamado de extinção. Como bem o animal aprende e consolida a memória de aprender esta tarefa pode ser observado durante um teste de recordação extinção dado algum tempo após o treinamento extinção. Menos expressão medo durante a chamada extinção demonstra boa consolidação da memória extinção. Descobertas recentes de nosso laboratório sugerem que o estrogênio pode modular a consolidação da memória de extinção do medo e melhorar a recordação extinção. Especificamente, os ratos fêmeas que são extintas em proestro, a fase do ciclo estral, durante o qual os níveis circulantes de estrogênio pico, apresentam uma melhor retenção de memória extinção. Em contraste, as mulheres que se extinguem no visor fase metaestro baixo estrogênio relativamente pobre recordação extinção, o que pode ser melhorado com a administração de estradiol exógeno antes ou imediatamente após o treino extinção <sup>15,16.</sup> Os mecanismos neurais da extinção consolidação da memória (including o papel dos hormônios sexuais) em fêmeas não são claras. </p><p class="jove_content"> Em animais de laboratório, o papel dos hormônios pode ser investigado utilizando procedimentos de remoção cirúrgicos invasivos, como a castração e ooforectomia. Após a recuperação da cirurgia, os hormônios sexuais são frequentemente exogenamente manipulada durante a realização de uma tarefa comportamental <sup>19.</sup> Esta abordagem tem fornecido informações importantes sobre os hormônios sexuais e é útil porque permite manipulações bem controlados de hormônios sexuais (em termos de tempo e dose) <sup>20-23.</sup> Esta abordagem, no entanto, não se avaliar a influência das flutuações naturais que ocorrem em todo o ciclo estral nem representam animais &quot;normais&quot;, limitando assim o potencial de translação para estudos em seres humanos. Tem sido bem documentado que os níveis das hormonas sexuais femininas pico e declínio em fases específicas do ciclo estral, e as alterações de expressão de receptor de estrogénio no ciclo, ae <sup>24</sup> após a ovariectomia. Assim, existe a necessidade de realizar estudos sobre as fêmeas com gônadas intactas e refinar modelos experimentais para estudar com segurança os efeitos que os estados altos e baixos de estrogênio pode ter sobre as mulheres ao longo da sua vida útil. </p><p class="jove_content"> Este protocolo incide sobre o efeito do estrogênio sobre os sistemas neurobiológicos envolvidos na extinção do medo. Ele descreve como: 1) monitorar cuidadosamente o ciclo estral, 2) preparar as doses eficazes de estradiol para administração sistêmica, e 3) seguem um paradigma comportamental que inclui o condicionamento do medo, extinção, e recordar na naturalmente ciclismo ratas. Este protocolo pode ser modificado com outras manipulações farmacológicas e ferramentas celulares e moleculares para auxiliar estudos para compreender melhor as diferenças de sexo que são observados no medo condicionado comportamento extinção. Note-se que os procedimentos descritos abaixo são aqueles utilizados no nosso laboratório, e existe uma série de variações destes procedimentos in literatura. </p>

<table><tbody><tr><td>Fear conditioning chamber</td><td>Coulbourn Instruments</td><td></td><td></td></tr><tr><td>Graphic State</td><td>Coulbourn Instruments</td><td></td><td></td></tr><tr><td>Sound-attenuating box</td><td>Med Associates, Inc.</td><td>NIR-022MD</td><td></td></tr><tr><td>Estradiol</td><td>Sigma-Aldrich</td><td>E1024</td><td>In sesame oil for subcutaneous injection</td></tr><tr><td>Sesame oil</td><td>Sigma-Aldrich</td><td>S3547-250ML</td><td></td></tr><tr><td>Freezescan</td><td>Cleversys, Inc.</td><td></td><td></td></tr><tr><td>Dip quick stain</td><td>Jorgensen Laboratories, Inc.</td><td>J0322A1, J0322A2, J0322A3</td><td></td></tr><tr><td>Cotton-tipped applicators</td><td>Fisher Scientific</td><td>23-400-114</td><td>6-inch, sterile</td></tr><tr><td>0.9% saline</td><td>LabChem, Inc.</td><td>LC23460-2</td><td>Sodium chloride w/v</td></tr><tr><td>Selectfrost microscope slides</td><td>Fisher Scientific</td><td>12-550-003</td><td></td></tr><tr><td>Virex II 256</td><td>Diversey, Inc.</td><td>5019317</td><td>Dilute 1:256 with water</td></tr><tr><td>Luer-Lok Tip 1ml Syringes</td><td>Becton Dickinson</td><td>309628</td><td></td></tr><tr><td>Hypodermic disposable needles</td><td>Exelint International, Co.</td><td>26402</td><td>26-gauge</td></tr></tbody></table>

protocol for studying extinction of conditioned fear in naturally cycling female rats

<p class="jove_content"> A extinção do medo condicionado tem sido extensivamente estudada em roedores machos. Recentemente, tem havido um número crescente de estudos que indicam que os mecanismos neurais para certas tarefas comportamentais e comportamentos de resposta são diferentes em fêmeas e machos. Usando fêmeas em estudos de pesquisa pode representar um desafio por causa da variação de hormônios sexuais durante o ciclo estral. Este protocolo descreve procedimentos bem estabelecidos que são úteis para investigar o papel do estrogénio na extinção medo a consolidação da memória em ratos fêmeas. Fase do ciclo estral e administração de estrogênio exógeno antes do treino extinção pode influenciar recordação extinção 24 horas mais tarde. A técnica de swab vaginal para identificação fase de estro descrito aqui ajuda o exame e manipulação de ciclismo naturalmente hormônios sexuais. O uso deste modelo básico roedor pode delimitar melhor os mecanismos pelos quais o estrogênio podem modular memória extinção do medo emfêmeas. </p>

<p class="jove_content"> Neste protocolo recordação extinção do medo, por cento congelamento foi medida como um indicador do medo. Animais que extinguiu bem e retidos a memória da formação extinção exibiu baixa medo no último dia do teste comportamental durante a chamada extinção. Ratos machos e fêmeas não apresentam diferenças significativas na expressão de medo condicionado durante o condicionamento, extinção, e recordar as fases <strong>(Figura 2).</strong> No entanto, a diferença entre os sexos se torna evidente quando os...

Watch this Scientific Journal Video about Protocol for Studying Extinction of Conditioned Fear in Naturally Cycling Female Rats at JoVE.com

Protocol for Studying Extinction of Conditioned Fear in Naturally Cycling Female Rats

<p class='jove_content'>Gonadal hormones such as estrogen modulate memory formation in a number of experimental paradigms including fear extinction memory. This protocol describes a set of methods for investigating the influence of gonadal hormones specifically during extinction in naturally cycling females, including estrous cycle monitoring and exogenous estrogen administration.</p>

Protocolo de Estudos de Extinção do medo condicionado em Naturally Ciclismo ratas

Extinction of conditioned fear has been extensively studied in male rodents. Recently, there have been an increasing number of studies indicating that ...

protocol-for-studying-extinction-conditioned-fear-naturally-cycling

Research

JoVE Journal

Behavior

13.3K visualizações.  Massachusetts General Hospital. Gonadal hormones such as estrogen modulate memory formation in a number of experimental paradigms including fear extinction memory. This protocol describes a set of methods for investigating the influence of gonadal hormones specifically during extinction in naturally cycling females, including estrous cycle monitoring and exogenous estrogen administration.

Vídeo: Protocol for Studying Extinction of Conditioned Fear in Naturally Cycling Female Rats

Scientific Method

<p>The scientific method is used to solve problems and explain phenomena. The development of the scientific method coincided with changes in philosophy underpinning scientific discovery, radically transforming the views of society about nature. During the European Renaissance, individuals such as Francis Bacon, Galileo, and Isaac Newton formalized the concept of the scientific method and put it into practice. Although the scientific method has been revised since its early conceptions, much of the framework and philosophy remains in practice today.</p>
<h4>Step 1: The Observation and Question</h4>
<p>Prior to investigation, a scientist must define the question to be addressed. This crucial first step in the scientific process involves observing some natural phenomena of interest. This observation should then lead to a number of questions about the phenomena. This stage frequently requires background research necessary to understand the subject matter and past work on similar ideas. Reviewing and evaluating previous research allows scientists to refine their questions to more accurately address gaps in scientific knowledge. Defining a research question and understanding relevant prior research will influence how the scientific method is applied, making it an important first step in the research process.</p>
<p>An everyday example: You are trying to get to school or work and your car won&#8217;t start. The thought process that most people go through in that situation clearly mirrors the official scientific method (after you are finished getting upset). First, you make an observation: my car won&#8217;t start! The question that follows: why isn&#8217;t it working?</p>
<h4>Step 2: The Hypothesis</h4>
<p>The next step is making a hypothesis, based on prior knowledge. A hypothesis is an &#8220;uncertain explanation&#8221; or an unproven conjecture that seeks to explain some phenomenon based on knowledge obtained while executing subsequent experiments or observations. Generally, scientists develop multiple hypotheses to address their questions and test them systematically.</p>
<p>All hypotheses must meet certain criteria for the scientific process to work. First, a hypothesis must be testable and falsifiable. This aspect of the hypothesis is critical and of much greater importance than the hypothesis being correct. A testable hypothesis is one that generates testable predictions, addressed through observations or experiments. A falsifiable hypothesis is one that, through observation of conflicting outcomes, can be proven wrong. This allows investigators to gain more confidence over time, not by accumulating evidence showing that a hypothesis is correct, but rather by showing that situations that could establish its falsity do not occur.</p>
<p>Hypotheses come in two forms: null hypotheses and alternative hypotheses. The null hypothesis is tested against the alternative hypothesis and reflects that there will be no observed change in the experiment. The alternative hypothesis is generally the one described in the previous two paragraphs, also referred to as the experimental hypothesis. The alternative hypothesis is the predicted outcome of the experiment. If the null hypothesis is rejected, then this builds evidence for the alternative hypothesis.</p>
<p>An everyday example: Maybe it is freezing outside and therefore it is fairly likely that your car battery is dead. Maybe you know you were low on gas the night before and therefore it is likely that the tank is empty.</p>
<h4>Step 3: Experimentation and Data Collection</h4>
<p>Either way, the next step is to make more observations or to conduct experiments leading to conclusions. Following the formulation of hypotheses, scientists plan and conduct experiments to test their hypotheses. These experiments provide data that will either support or falsify the hypothesis. Data can be collected from quantitative or qualitative observations. Qualitative information refers to observations that can be made simply using one&#39;s senses, be that through sight, sound, taste, smell, or touch. In contrast, quantitative observations are ones in which precise measurements of some type are used to investigate one&#39;s hypothesis.</p>
<p>An experiment is a procedure designed to determine whether observations of the real world agree with or refute the derived predictions in the hypothesis. If evidence from an experiment supports a hypothesis, that gives the hypothesis more credibility. This does not indicate that the hypothesis is true, as future experiments may reveal new information about the original hypothesis. Experimental design is another critical step in the scientific method and can have a great effect on the results and conclusions one draws from an experiment. Careful thought and time should be devoted to experimental design and minimizing possible errors. The experiment should be designed so that every variable or factor that could influence the outcome of the experiment be under control of the researcher. Two types of variables are used to describe the conditions in an experiment: the independent and the dependent, or response, variable. The independent variable is directly manipulated or controlled by the scientist and is generally what one predicts will affect the dependent variable. The dependent, or response, variable thus depends on the value of the independent variable. Experiments are generally designed so that one specific factor is manipulated in the experiment in order to illuminate cause and effect relationships.</p>
<p>An everyday example: Does the car still have all of its parts? Is this the right key? What does the gas gauge say? Does a jump start help?</p>
<p>Another important aspect in experimental design is the role of the control treatment, which represents a non-manipulated treatment condition. The control treatment is kept in the same conditions as the experimental treatment, but the experimental manipulation is not applied to the control. For example, if a researcher were testing the effects of soil salinity on plant growth, the soil in the control treatment would have no added salt. The control provides a baseline of &#8220;normal&#8221; conditions with which to compare the experimental treatments.</p>
<p>Experimental design should also incorporate replicates of each treatment. Repeatability of experimental results is an important part of the scientific method that ensures the validity and accuracy of data. It is quite difficult to control all aspects of an experiment so there is inherent variation in results that cannot be controlled for even under the most carefully designed and controlled experiments. Having replicates enables an investigator to estimate this inherent variation in results. Precise recording and measurement of data is also of great importance for ensuring the accuracy of results and the conclusions one draws from the results.</p>
<h4>Step 4: Results and Data Analysis</h4>
<p>The next step in the scientific method involves determining what the results from the experiment mean. Scientists compare the predictions of their null hypothesis to that of their alternative hypothesis to determine if they are able to reject the null hypothesis. Rejecting the null hypothesis means that there is a significant probability that values of the dependent variable in the control versus experimental treatments are not equal to each other. If significant differences exist, then one can reject the null hypothesis and accept the alternative hypothesis. Conversely, the investigator may fail to reject the null hypothesis, meaning the treatment has no effect on the results. Before scientists can make any claims about their null hypothesis from their experimental data or observations, statistical tests are required to ensure the validity of the data and further interpretation of the data. Statistical tests allow researchers to determine if there are genuine differences between the control and experimental treatments. From there, they can create figures and tables to illustrate their findings.</p>
<h4>Step 5: Conclusions</h4>
<p>The last portion of the scientific method involves providing explanations of the results and the conclusions that can be logically drawn from the results. Generally, this step of the scientific process also requires one to revisit scientific literature and compare their results with other experiments or observations on related topics. This allows researchers to put their experiment in a more general context and elaborate on the significance of particular results. Additionally, it allows them to explain how their work fits into a larger context in their discipline.</p>
<p>The scientific process does not stop here! The scientific process works through time as knowledge on topics in science accumulate and drive our understanding of particular mechanisms or processes explaining natural phenomena. If we fail to reject our null hypothesis, then it becomes necessary to revisit the initial stages of the scientific method and try to reformulate our questions and understand why an anticipated result was not attained.</p>
<h4>Application of the Scientific Method</h4>
<p>The only difference between the use of this method in every-day life and in the laboratory is that scientists carefully document their work, from observation to hypothesis to experiment, and finally conclusions and peer review. In addition, unlike problem solving outside the lab, the scientific method in the lab includes controlled conditions and variables.</p>
<p>Let&#8217;s investigate the scientific method using an example from the lab. It is known that plant growth is affected by microbes, such as bacteria and fungi, living in their soil. It is possible to figure out what microbes have which effects by potting plants in completely sterile soil, then adding in microbes one at a time, or in different combinations and measuring the growth of the plant. Now let&#8217;s fit this into the terms used to describe the scientific method:</p>
<p><strong>Observation and Question</strong>: There are microbes in the soil&#8230;do these affect plant growth?</p>
<p>HYPOTHESES:</p>
<p>Experimental: One particular microbe of interest will cause the plants to grow more slowly.</p>
<p>Null: The presence or absence of microbes will have no effect on plant growth</p>
<p><strong>Experiment</strong>: set up groups of plants in 1) sterile soil, 2) soil with the microbe added in, and 3) natural soil. Measure the growth of the plants over time, using a ruler.</p>
<p><strong>Conclusion</strong>: if the plants in group 2 grow more slowly than the other two, the hypothesis is supported. This needs to be backed up with statistical analysis from many plants to be considered significant. An experiment like this is not legitimate with just one plant per group.</p>
<p>Group 1 is a<strong> control</strong> which shows the plants can grow in the sterile soil. Group 3 is a control that shows the plants can grow under normal conditions. Group 2 is the experimental group. It would be possible to add different amounts of the microbe, or different microbes, to introduce more variables. The main point is that the researcher has something to which to compare the experimental group- the control group. If the experiment included only group 2 and the researcher determined that the plants &#8220;looked sick,&#8221; that would be a matter of opinion. The only way to make that observation scientific is to have healthy plants to measure. The type or amount of microbe used is the <strong>independent variable</strong>, because the researcher has control over it. The size of the plant at the end of the experiment is the <strong>dependent or response variable</strong> because it is the result.</p>
<p>Ultimately, work like this is published in scientific journals so that other researchers can read about the methods used and conclusions drawn. Publications like this are subject to peer-review, which means that an article won&#8217;t be published in a journal until other researchers have checked it out and agree it is well-done. As a community of scientists, general concepts are developed based on observed patterns in the experiments that individual scientists conduct. This results in the development of a scientific <strong>theory</strong>. This term means that there is a consensus among researchers that a particular concept or process exists. It is important to note that the word theory does not mean the same thing as hypothesis. Once scientists label a concept with this term, it is considered to be true, considering all currently available data. Of course, if a large body of experimentation demonstrates information to the contrary, theories can be modified.</p>

Scientific Method: Steps and Applications - Concept

Método Científico

The scientific method is used to solve problems and explain phenomena. The development of the scientific method coincided with changes in philosophy ...

Education

JoVE Lab Manual

Biologia

Fundamentais

Group Behavior

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">The Hawk-Dove Game
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">NOTE: In this activity, you will play the hawk and dove game with a single partner and then with multiple partners to observe hypothetical costs and benefits of the antagonist or cooperative behavior respectively. 
</div></li><li data-sub-note-item="1-2"><div class="lab-step">Before the activity starts, review the cost-benefit table so that you understand how to score the game. <a href="https://www-jove-com.remotexs.ntu.edu.sg/CDNSource/lm/labs/33/Table_1_Group_behavior.xlsx" style="display:block;margin:20px;" target="_blank">Click Here to download Table 1</a>
</div></li><li data-sub-note-item="1-3"><div class="lab-step">HYPOTHESES: The experimental hypothesis for this exercise could be that the dove strategy will become more frequent between repeated partners, and the hawk strategy will become more frequent when interactions are with a variety of partners. The null hypothesis could be that there will be no change in strategy over time with repeat or different partner combinations. NOTE: Benefit can be determined by looking at the left column and choosing the row that corresponds to the card you played. The box that corresponds to the choice of your partner can be found in the top row. For the purposes of this game, B equals four and C equals three. So depending on the cards played, player one here, which is you, would gain a score of two, four, zero or one half. 
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Collect a set of two game cards. 
</div></li><li data-sub-note-item="1-5"><div class="lab-step">Take out the cards labeled hawk and dove and place them face down on the table or conceal them in your hands. 
</div></li><li data-sub-note-item="1-6"><div class="lab-step">When the instructor gives the signal show either of the two cards to the student sitting across from you. Try to strategize how to receive the highest benefit from every interaction on your own but do not communicate with your partner. 
</div></li><li data-sub-note-item="1-7"><div class="lab-step">In the Single Partner Recording Table (Table 2) provided by your instructor, note the cards played as well as the benefit you received for that round.
</div></li><li data-sub-note-item="1-8"><div class="lab-step">Repeat showing cards to the same partner and determining benefit on your instructor&#39;s signal for a total of 15 rounds.
</div></li><li data-sub-note-item="1-9"><div class="lab-step">At the end of those rounds, the student with the highest total benefit value was determined to be the &#8220;winner&#8221;, or the most fit.
</div></li><li data-sub-note-item="1-10"><div class="lab-step">As a class, determine the percentages of hawks and doves played as well as the average benefit received for each round, using this formula: Class average benefit=(sum total benefit)/(number of players)
</div></li><li data-sub-note-item="1-11"><div class="lab-step">For the second game, and then for each round of play, one row of students should rotate to their right, leaving all players with a new partner.
</div></li><li data-sub-note-item="1-12"><div class="lab-step">Continue playing for 15 rounds or until students end up back in their starting positions, recording the data in the Multiple Partner Recording Table (Table 3) given to you by your instructor.
</div></li><li data-sub-note-item="1-13"><div class="lab-step">For each round, determine the percentages of hawks and doves played that round, as well as the average benefit received.
</div></li></ul></li><li data-note-item="2" data-parent-collapse="">Cheating and Cooperation Exercise
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="2-1"><div class="lab-step">NOTE: In this activity, you will exchange benefits, represented by colored or similar items, with your classmates. In this simulation, a cheater is defined as a person who gives fewer benefits than they receive. During this activity, you will observe the frequency of cheating behavior over time. 
</div></li><li data-sub-note-item="2-2"><div class="lab-step">HYPOTHESES: The experimental hypothesis is that cheaters will receive fewer benefits over time and cooperators will continue to provide equal benefits to one another. The null hypothesis is that the frequency of cheaters and cooperators does not change over time. 
</div></li><li data-sub-note-item="2-3"><div class="lab-step">Each person should collect beads of one color to represent a tangible benefit, as well as one paper bag and a three-ring binder.
</div></li><li data-sub-note-item="2-4"><div class="lab-step">Get into a group of three so that all members have objects of the same color.
</div></li><li data-sub-note-item="2-5"><div class="lab-step">Now find another group with a different color to pair with. In each round, partners will exchange benefits without knowing what they are receiving from their partner and without discussion within their group.  NOTE: Benefits of a different color from your starting color are worth twice as much.
</div></li><li data-sub-note-item="2-6"><div class="lab-step">Turn face to face with a member from the other group while still being able to conceal the objects in your possession.  TIP:<strong> </strong>Setting up a screen using a notebook or sitting behind a desk can be helpful here.
</div></li><li data-sub-note-item="2-7"><div class="lab-step">When given a signal from the instructor, place zero to two benefits into the paper bag and pass it to your partner.
</div></li><li data-sub-note-item="2-8"><div class="lab-step">Simultaneously, receive the bag from your partner and then remove the benefits from the bag.
</div></li><li data-sub-note-item="2-9"><div class="lab-step">Make a note of how many benefits you gave away and how many you received in the Cheating and Cooperation Table (Table 4) provided by your instructor.
</div></li><li data-sub-note-item="2-10"><div class="lab-step">Turn to a new member from the other group and repeat the exchange.
</div></li><li data-sub-note-item="2-11"><div class="lab-step">Rotate through all of the members of the other group for as many rounds as the instructor determines - which will not be revealed until the game ends so as not to alter player behavior.
</div></li><li data-sub-note-item="2-12"><div class="lab-step">Once the game has ended, note for each round in the cheating and cooperation table whether you gave more than your partner, received more from them or if you gave the same amount.
</div></li><li data-sub-note-item="2-13"><div class="lab-step">Now total up all of the benefits in your possession, with benefits you&#39;ve received from the other group being worth twice as much.
</div></li><li data-sub-note-item="2-14"><div class="lab-step">For each round, the whole class should total up the number of cheaters - with cheaters defined as those who gave less than they received.
</div></li></ul></li><li data-note-item="3" data-parent-collapse="">Results
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="3-1"><div class="lab-step">For both the single partner hawk-dove game and the multiple partners hawk-dove game, use the whole class data to plot the percentages as two separate lines - one for the percentage of hawks, and one for the percentage of doves, for each of the 15 rounds.
</div></li><li data-sub-note-item="3-2"><div class="lab-step">Take note of whether the cards played changed as more rounds were played.
</div></li><li data-sub-note-item="3-3"><div class="lab-step">Manipulate the values for B and C to see if you can find the point at which values of B and C would render the hawk strategy no longer effective (this would be the point when there is no benefit to choosing the hawk strategy over always choosing the dove strategy).
</div></li><li data-sub-note-item="3-4"><div class="lab-step">As a class, use the data from the cheating and cooperation activity to make a graph showing the percentage of rounds each individual player cheated versus the amount of benefits they ended with.
</div></li><li data-sub-note-item="3-5"><div class="lab-step">Consider whether it is better to be a cheater or a cooperator in this scenario and discuss as a class what makes this scenario different from the hawk-dove game.
</div></li><li data-sub-note-item="3-6"><div class="lab-step">Now, use the class data to graph the percentage of cheaters against the number of rounds, and look for any trends over time.
</div></li><li data-sub-note-item="3-7"><div class="lab-step">Consider which type of strategy would most likely evolve in a setting where partners interact repeatedly.
</div></li><li data-sub-note-item="3-8"><div class="lab-step">Then, note which tactic might be beneficial for a species that relies on cooperation. 
</div></li><li data-sub-note-item="3-9"><div class="lab-step">Discuss as a class what would likely happen if you increased or decreased the relative value of the benefits you receive.
</div></li></ul></li></ol>

Group Behavior: Hawk-Dove Game and Trading Exercise - Procedure

Comportamento do grupo

The Hawk-Dove Game
ExpandNOTE: In this activity, you will play the hawk and dove game with a single partner and then with multiple partners to observe ...

Ecologia

Lab Manual Sub Articles

Optimal Foraging

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Simulating Foraging with Artificial Habitats and Prey
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">NOTE: The foragers will hunt for prey represented by pinto beans in four buckets of rice with varying prey densities. Without knowing what these densities are, foragers must obtain as many prey items as possible in as little time as possible. 
</div></li><li data-sub-note-item="1-2"><div class="lab-step">HYPOTHESES: In this experiment, the experimental hypothesis could be that foragers will catch the most prey in the higher prey density bucket and also spend the most time foraging there.  The null hypothesis would then be that foragers will catch an equal number of prey in each bucket and that foragers will spend an equal amount of time foraging in each patch. 
</div></li><li data-sub-note-item="1-3"><div class="lab-step">To begin the foraging activity, record the names and role of each participant at the top of the student data collection sheet.  NOTE: Before starting the experiment, the forager is allowed to become familiar with the foraging process &#8211; the following steps are a practice round. 
</div></li><li data-sub-note-item="1-4"><div class="lab-step">The forager should kneel or sit in front of the average density bucket. 
</div></li><li data-sub-note-item="1-5"><div class="lab-step">Once the timer says start, the forager may begin looking for the pinto beans representing the prey. The forager can spend as much time as they like foraging in this bucket. 
</div></li><li data-sub-note-item="1-6"><div class="lab-step">Once a prey item is located, the forager must signal that one has been found and place the prey in the cup next to the bucket.
</div></li><li data-sub-note-item="1-7"><div class="lab-step">Once they believe they have acquired most of the prey in the patch, they should tell the timer to stop the time and the practice round is over. 
</div></li><li data-sub-note-item="1-8"><div class="lab-step">To begin the simulation proper, the forager should sit or kneel in front of bucket A. 
</div></li><li data-sub-note-item="1-9"><div class="lab-step">The timer must be ready to start the stopwatch. 
</div></li><li data-sub-note-item="1-10"><div class="lab-step">The recorder should be ready to record the time each prey item is located, in addition to the time that the forager arrived at and left each bucket. 
</div></li><li data-sub-note-item="1-11"><div class="lab-step">When all parties are ready, the timer should start the stopwatch and say &#8220;start&#8221;,  and the forager should begin foraging. NOTE: The stopwatch will run through the rest of the round for buckets B and C.
</div></li><li data-sub-note-item="1-12"><div class="lab-step">As the forager signals finding pinto beans and places them in the cup next to the bucket, the timer should tell the recorder the time.
</div></li><li data-sub-note-item="1-13"><div class="lab-step">The recorder should then write the time in the student data collection sheet.  <a href="https://www-jove-com.remotexs.ntu.edu.sg/CDNSource/lm/labs/34/Table_1_Optimal_foraging.xlsx" style="display:block;margin:20px;" target="_blank">Click Here to download Table 1</a>
</div></li><li data-sub-note-item="1-14"><div class="lab-step">Once the forager has decided to move on to the next bucket, they should tell the timer &#8220;done&#8221;, and begin moving to the next patch. 
</div></li><li data-sub-note-item="1-15"><div class="lab-step">The recorder should record the time the forager left and the time they arrived at bucket B in the student data collection sheet.
</div></li><li data-sub-note-item="1-16"><div class="lab-step">Repeat the same procedure for buckets B and C. Once the forager has completed the activity, it is time for the next forager to begin using the same procedure. 
</div></li></ul></li><li data-note-item="2" data-parent-collapse="">Results
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="2-1"><div class="lab-step">Before performing any data analysis, review the data collection sheet and compile your individual data for captured prey, foraging time, and travel time for each bucket in your student calculations table. <a href="https://www-jove-com.remotexs.ntu.edu.sg/CDNSource/lm/labs/34/Table_2_Optimal_foraging.xlsx" style="display:block;margin:20px;" target="_blank">Click Here to download Table 2</a>
</div></li><li data-sub-note-item="2-2"><div class="lab-step">After this, calculate your individual capture rate for each bucket by dividing the number of prey captured by the sum of the foraging time and travel time and record these values in your table. Individual capture rate =  Number of prey captured / (foraging time + travel time)
</div></li><li data-sub-note-item="2-3"><div class="lab-step">To calculate your Giving-Up-Time or a GUT for each bucket, subtract the time that the last prey item was acquired from the time you left the bucket.  GUT = time forager left bucket  -  time last prey item acquired
</div></li><li data-sub-note-item="2-4"><div class="lab-step">Next, calculate the mean GUT for your foraging group and record the values in your table.
</div></li><li data-sub-note-item="2-5"><div class="lab-step">Finally, calculate your scaled GUT for each bucket by dividing the bucket GUT by your group&#39;s mean GUT. Scaled GUT =  Bucket GUT / Group mean GUT NOTE: The scaled GUT will allow class data to be pooled later on without differences in individual foragers skewing the data. 
</div></li><li data-sub-note-item="2-6"><div class="lab-step">The instructor should now compile the data into the classroom spreadsheet.  <a href="https://www-jove-com.remotexs.ntu.edu.sg/CDNSource/lm/labs/34/Table_3_Optimal_foraging.xlsx" style="display:block;margin:20px;" target="_blank">Click Here to download Table 3</a>
</div></li><li data-sub-note-item="2-7"><div class="lab-step">Plot a bar graph of the average number of prey collected in each bucket. On the x-axis, A should represent the low prey density bucket, B should represent the medium prey density bucket, and C the high prey density bucket. Plot the y-axis as the number of prey items caught. 
</div></li><li data-sub-note-item="2-8"><div class="lab-step">Next, plot the graph of the class average scaled GUT, where the x-axis represents the buckets with varying levels of prey density and the y-axis shows the values for the average scaled GUT. 
</div></li><li data-sub-note-item="2-9"><div class="lab-step">Plot a third graph of the average time spent foraging in each bucket. 
</div></li><li data-sub-note-item="2-10"><div class="lab-step">Now review the Marginal Value Theorem (MVT) from the concepts video and examine the three graphs that you generated from your class data. Take note of whether the graphs conform to the predictions of MVT. 
</div></li></ul></li></ol>

Optimal Foraging: Foraging with Artificial Habitats and Prey - Procedure

Forrageamento ideal

Simulating Foraging with Artificial Habitats and Prey
ExpandNOTE: The foragers will hunt for prey represented by pinto beans in four buckets of rice with ...

Species Distribution and Biogeography

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Simulating Colonization and Extinction on Islands
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">Form groups of three. One member of the group will simulate colonization by throwing balls into the groups of cups representing the islands. Another will simulate extinction by rolling dice to randomly remove balls from the cups. The third group member will record the data.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">Take time to familiarize yourself with the four island stations. NOTE: There are two large islands with 12 cups and two small islands with 6 cups. Two of the stations will have tape lines that are 2 m away from the cups. These are the far islands. The other two stations will have tape lines that are 1 m away from the cups. These are the near islands. Thus, the stations are far large and near large and far small and near small. HYPOTHESES: In this experiment, one alternate hypothesis might be that the larger islands with more cups will support a greater number of species than the smaller islands. The null hypothesis might be that all islands will support this same number of species regardless of size. Another alternate hypothesis might be that islands closer to the mainland will have a higher colonization rate than islands farther from the mainland. The null hypothesis might be that all islands will have the same colonization rate regardless of their distance from the mainland. Additionally, in all trials we may hypothesize that the colonization rate will be expected to decrease as more species inhabit an island whereas the extinction rate will increase as more species are found on the island. The null hypothesis might be that the colonization rate and extinction rates are expected to stay the same across all numbers of species on the island.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">First, choose one of the four stations as a starting place.
</div></li><li data-sub-note-item="1-4"><div class="lab-step">From the line of tape at this location, the colonization simulator must throw the ball to the island and try to get the ball into a cup. NOTE: One round consists of five throws, each throw representing a colonization attempt. If the ball lands in a cup that does not already have a ball in it, this is a successful colonization attempt and the ball should be left in the cup.
</div></li><li data-sub-note-item="1-5"><div class="lab-step">The data collector should place a tally on the colonizers box for the round in the recording sheet found at the station. <a href="https://www-jove-com.remotexs.ntu.edu.sg/CDNSource/lm/labs/29/Tables_1-4_Island_biogeography.xlsx" style="display:block;margin:20px;" target="_blank">Click Here to download Tables 1-4</a>
</div></li><li data-sub-note-item="1-6"><div class="lab-step">If the ball does not land in a cup, retrieve it. This is an unsuccessful colonization attempt.
</div></li><li data-sub-note-item="1-7"><div class="lab-step">If a ball lands in a cup that already has a ball in it, retrieve it. This is also an unsuccessful colonization attempt.
</div></li><li data-sub-note-item="1-8"><div class="lab-step">Add the number of species at the beginning of the round with the tally of colonizers.
</div></li><li data-sub-note-item="1-9"><div class="lab-step">The data collector should record this number in the column entitled &#8220;Number of species after colonization&#8221; in the recording sheet found at the station.
</div></li><li data-sub-note-item="1-10"><div class="lab-step">The extinction simulator should now roll a die. If the trial is occurring at a large island site, choose one of the two colored dice at random with eyes closed.
</div></li><li data-sub-note-item="1-11"><div class="lab-step">Remove the ball if there is one present from the cup that is of the same color as the die and labeled with the number rolled by that die.
</div></li><li data-sub-note-item="1-12"><div class="lab-step">The data collector should count the removal as an extinction in the station recording table. NOTE: In a trial at the small island station, follow the same procedure except that it is only necessary to match the number rolled by the die to the cup. If there is no ball in the cup, no extinction is recorded. For both the large and small islands, repeat the extinction roll for a total of two rolls if there are 3 - 8 species present on the island and three rolls if there are 9 - 12 species on the island. If there are fewer than three species on the island, do not roll the die again.
</div></li><li data-sub-note-item="1-13"><div class="lab-step">This is the end of one round. After this, count the total number of species currently in the cups at this station and then tally this number into the &#8220;Number of species at beginning of round&#8221; column on the station recording sheet for the next round.
</div></li><li data-sub-note-item="1-14"><div class="lab-step">Groups should switch stations after five rounds at one table to control for the throwing ability of the colonization simulators. Repeat the trials and rotation through the stations until 20 rounds have been recorded in total for all groups combined at all four stations.
</div></li><li data-sub-note-item="1-15"><div class="lab-step">Count the tally marks for the number of species at the beginning of the round, colonizers, total number of species after colonization, and extinctions for each round at each island station.
</div></li><li data-sub-note-item="1-16"><div class="lab-step">Look at your data recording sheet. How many times were there zero species at the beginning of a round? How many times was there only one species at the beginning of a round? Repeat this calculation for 0 through 12 species present at the beginning of a round.
</div></li><li data-sub-note-item="1-17"><div class="lab-step">Then, tally these values in the corresponding rows of the &#8220;Rounds with this many species&#8221; column in the appropriate colonization rate calculation table. <a href="https://www-jove-com.remotexs.ntu.edu.sg/CDNSource/lm/labs/29/Tables_5-8_Island_biogeography.xlsx" style="display:block;margin:20px;" target="_blank">Click Here to download Tables 5-8</a>
</div></li><li data-sub-note-item="1-18"><div class="lab-step">In the colonization attempts column, multiply these values by five to represent the total number of Ping-Pong balls thrown.
</div></li><li data-sub-note-item="1-19"><div class="lab-step">Look at your data recording sheet once more and calculate the total number of colonizers for each value in the number of species at beginning of round column.
</div></li><li data-sub-note-item="1-20"><div class="lab-step">Record these values in the &#8220;Successful colonization attempts&#8221; column of the appropriate colonization rate calculation table.
</div></li><li data-sub-note-item="1-21"><div class="lab-step">Divide the successful colonization attempts by the colonization attempts for each initial species value and record this in the &#8220;Colonization rate&#8221; column of the appropriate colonization rate calculation table.
</div></li><li data-sub-note-item="1-22"><div class="lab-step">Repeat the process for determining the colonization rate for all of the island trials. 
</div></li><li data-sub-note-item="1-23"><div class="lab-step">To calculate the extinction rate for each of the trials, return to the data recording sheet for the near large island trial. Count the number of times that the number of species after colonization matched each number from 1 to 12, as previously described.
</div></li><li data-sub-note-item="1-24"><div class="lab-step">Record these values in the &#8220;Rounds with this many species&#8221; column in the appropriate extinction rate calculation table. <a href="https://www-jove-com.remotexs.ntu.edu.sg/CDNSource/lm/labs/29/Tables_9-12_Island_biogeography.xlsx" style="display:block;margin:20px;" target="_blank">Click Here to download Tables 9-12</a>
</div></li><li data-sub-note-item="1-25"><div class="lab-step">To calculate extinction attempts, multiply the value in the &#8220;Rounds with this many species&#8221; cells by 1 if the species after colonization value is 0 &#8211; 2, by 2 if the value is 3 &#8211; 8, and by 3 if the value is 9 or above. NOTE: These values correspond with the number of times the die was rolled during the extinction simulation.
</div></li><li data-sub-note-item="1-26"><div class="lab-step">Then, using the data contained in the data recording sheet, count the number of extinctions that occurred for each value in the &#8220;Number of species after colonization&#8221; column.
</div></li><li data-sub-note-item="1-27"><div class="lab-step">Record these values in the &#8220;Extinctions&#8221; column of the appropriate extinction rate calculation table.
</div></li><li data-sub-note-item="1-28"><div class="lab-step">Record these values in the &#8220;Extinctions&#8221; column of the appropriate extinction rate calculation table.
</div></li><li data-sub-note-item="1-29"><div class="lab-step">Repeat the process for calculating the extinction rate for all of the island trials. 
</div></li><li data-sub-note-item="1-30"><div class="lab-step">After this, plot the colonization and extinction rates versus the number of species for each of the island stations.
</div></li><li data-sub-note-item="1-31"><div class="lab-step">Draw the best-fit lines for the colonization and extinction rates in each of the four graphs.
</div></li><li data-sub-note-item="1-32"><div class="lab-step">Compare the colonization rate between the near and far islands. Determine the distance that had a greater dynamic equilibrium of species and develop a hypothesis to explain your observation. 
</div></li><li data-sub-note-item="1-33"><div class="lab-step">Next, compare the extinction rates between the large and small islands. Determine the size that had a greater dynamic equilibrium number of species.
</div></li><li data-sub-note-item="1-34"><div class="lab-step">Observe the slopes of the colonization rates in the plots. Determine if they are positive or negative. 
</div></li><li data-sub-note-item="1-35"><div class="lab-step">Observe the slopes of the extinction rates in the plots. Determine if they are positive or negative. 
</div></li></ul></li></ol>

Distribuição de Espécies e Biogeografia

Simulating Colonization and Extinction on Islands
ExpandForm groups of three. One member of the group will simulate colonization by throwing balls into ...

Extinction

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Diversity and Extinction during the Paleogene Epoch
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">Begin this procedure by reading the extinction reading supplement that has been printed out for you. <br /><strong>NOTE:</strong> Alternatively, you may download the reading supplement at the link below.&nbsp; <a href="https://www-jove-com.remotexs.ntu.edu.sg/CDNSource/lm/labs/24/Lab_20_Reading Supplement.docx" style="display:block;margin:20px;" target="_blank">Click here to download the extinction reading supplement</a>
</div></li><li data-sub-note-item="1-2"><div class="lab-step">Using the information contained in the extinction reading supplements pertaining to the taxa of interest and the Chicxulub impact event, make predictions for each taxon listed in Table 1. Consider whether diversity in each taxon increased, decreased, or stayed the same immediately following the Cretaceous-Paleogene boundary and later on in the Paleogene epoch. <a href="https://www-jove-com.remotexs.ntu.edu.sg/CDNSource/lm/labs/24/Table_1_Extinction.xlsx" style="display:block;margin:20px;" target="_blank">Click Here to download Table 1</a>
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Then, fill out the reasons for each of your predictions in the Contributing Factors column. HYPOTHESES: The experimental hypothesis is that many of the different taxa will show changes in diversity after the Cretaceous-Paleogene event, and again at the end of the Paleogene, with some increasing in diversity, while others decrease. The null hypothesis is that there will be no changes in diversity between taxa following the Cretaceous-Paleogene event, or at the end of the Paleogene.
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Gather diversity data by going to the Paleobiology Database: <a href="http://www.paleobiodb.org" target="_blank">www.paleobiodb.org</a>.
</div></li><li data-sub-note-item="1-5"><div class="lab-step">In the center of the screen, click the yellow box labeled Download Data. Under the italicized heading, &#8220;What do you want to download?&#8221;, select Diversity over time on the left side and Comma-separated values on the right side.
</div></li><li data-sub-note-item="1-6"><div class="lab-step">Scroll down to the heading &#8220;Select by taxonomy&#8221;.
</div></li><li data-sub-note-item="1-7"><div class="lab-step">In the first box labeled 'Taxon or taxa to include', type in the name of the taxon that data is being gathered on.  NOTE: Ensure the spelling is correct.
</div></li><li data-sub-note-item="1-8"><div class="lab-step">Now, go to the dropdown menu right below that box labeled &#8220;Taxonomic resolution&#8221;, and select genera.
</div></li><li data-sub-note-item="1-9"><div class="lab-step">Under the next heading, Select by time, type Maastrichtian into the first box and Chattian in the box directly below. Make sure the dropdown menu labeled Time rule is set to major and the Temporal resolution is set to stage.
</div></li><li data-sub-note-item="1-10"><div class="lab-step">Select &#8220;Assume extant taxa range through to present&#8221;. Scroll back up to the middle of the page and click download. A save window will open.
</div></li><li data-sub-note-item="1-11"><div class="lab-step">Save the .csv file to a safe and accessible place and rename it to make it easy to identify.
</div></li><li data-sub-note-item="1-12"><div class="lab-step">Open the file and scroll down to the main dataset under Records.  NOTE: The data are arranged in reverse time order by geological stage. The second column, interval_name, gives the accepted name of the stage in question. The next two columns give the appropriate dates of the stage in millions of years before the present. The final column, number of occurrences, gives a total count of the genera extant during that geological stage as determined from occurrence of fossils.
</div></li><li data-sub-note-item="1-13"><div class="lab-step">To more easily work with this data, copy and paste each column into a new sheet. Be sure to label this sheet with the taxon in question, the data and time the data were obtained, and the data source.
</div></li><li data-sub-note-item="1-14"><div class="lab-step">Repeat this process for each of the taxa in Table 1 (steps 4 &#8211; 12). NOTE: Data from multiple taxa can be kept on the same sheet, so long as they are kept distinct and labeled appropriately.
</div></li><li data-sub-note-item="1-15"><div class="lab-step">To create line plots for each of the taxa showing the change in diversity over time, select the max_ma column and the number of occurrences column simultaneously.
</div></li><li data-sub-note-item="1-16"><div class="lab-step">In Excel, select Insert from the top toolbar and find the section labeled Charts. Select the bottom center icon, Insert Scatter Chart.
</div></li><li data-sub-note-item="1-17"><div class="lab-step">Then, select the Scatter with Straight Lines and Markers and a graph should appear. NOTE: Since the axis of this graph is in units of millions of years ago, the axis is in the reverse direction of time when put in numerical order.
</div></li><li data-sub-note-item="1-18"><div class="lab-step">To fix this, click on the green plus sign in the top-right corner of the graph and hover over Axes. This should reveal a small black arrow. Click the arrow to open up a small menu and select More Options.
</div></li><li data-sub-note-item="1-19"><div class="lab-step">This will open the Axis Options menu. Under Axis Options, click the box next to Values in reverse order. NOTE: It is also possible to change the axis scales from this menu, which can be used to eliminate the excess time that is not being analyzed in this lab. Keep any changes consistent between different graphs.
</div></li><li data-sub-note-item="1-20"><div class="lab-step">Double-click on the title at the top of the graph and change it to the taxon being represented.
</div></li><li data-sub-note-item="1-21"><div class="lab-step">Make a separate graph for each of the taxa in Table 1. Keep them organized and in one location. A separate sheet for graphs is recommended.
</div></li></ul></li><li data-note-item="2" data-parent-collapse="">Results
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="2-1"><div class="lab-step">For each graph, identify the location of the Cretaceous-Paleogene boundary and draw a vertical line through the graph at this point.
</div></li><li data-sub-note-item="2-2"><div class="lab-step">Using the information from the graphs, fill out Table 2. In some cases, it may be helpful to go back to the original dataset and compare actual values, particularly when they are small. <a href="https://www-jove-com.remotexs.ntu.edu.sg/CDNSource/lm/labs/24/Table_2_Extinction.xlsx" style="display:block;margin:20px;" target="_blank">Click Here to download Table 2</a>
</div></li><li data-sub-note-item="2-3"><div class="lab-step">Compare these values to the predictions made in Table 1 and discuss if the original contributing factors are still likely candidates.
</div></li><li data-sub-note-item="2-4"><div class="lab-step">Using the dataset, calculate the percentage of total taxa that went extinct over the Cretaceous-Paleogene boundary using this equation.
</div></li><li data-sub-note-item="2-5"><div class="lab-step">There is an event in the middle of the Paleogene known as the Paleocene-Eocene Thermal Maximum about 55.5 million years ago. This represents a high point in temperature during the Paleogene epoch at approximately 8 &#176;C higher than today&#39;s average temperature. Does this point seem to have an effect on diversity for any of the taxa? If yes, then which ones?
</div></li></ul></li></ol>

Extinction: Diversity and Extinction during the Paleogene Epoch - Procedure

Extinção

Diversity and Extinction during the Paleogene Epoch
ExpandBegin this procedure by reading the extinction reading supplement that has been printed out for ...

Animal Behavior

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Making Choice Chambers
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">Before beginning the experiments, use one of the Sharpies from the lab table to trace a line around the circumference of the bottom of both bottles. One line should be 1 cm from the base of the bottle and the other should be 1.5 cm from the base of the bottle.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">Use a razor blade to make a small cut in each bottle at the mark. 
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Then, insert the scissors and carefully cut along the line until the bottoms of the bottles are removed completely.
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Fit one bottle bottom into the other.
</div></li><li data-sub-note-item="1-5"><div class="lab-step">Tape the bottles together to make a secure chamber.
</div></li><li data-sub-note-item="1-6"><div class="lab-step">Use the razor blade and scissors to poke a hole into the center of the chamber. 
</div></li><li data-sub-note-item="1-7"><div class="lab-step">Take a piece of tape and fold the edge onto itself to make a tab and place the tape over the hole.  Note: The tab helps with easy removal and reattachment of the tape to the chamber.
</div></li><li data-sub-note-item="1-8"><div class="lab-step">Use the Sharpie to label one side of the chamber A and the other side B.
</div></li><li data-sub-note-item="1-9"><div class="lab-step">Upon receiving the colony of <em>Drosophila</em>, perform a count to make sure there are 40 living individuals in the vial.
</div></li></ul></li><li data-note-item="2" data-parent-collapse="">Geotaxis
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="2-1"><div class="lab-step">To perform a geotaxis assay, first attach a clean, empty bottle cap to each end of the choice chamber and turn the fly vial upside down onto its lid.
</div></li><li data-sub-note-item="2-2"><div class="lab-step">Open the tape flap in the center of the choice chamber.
</div></li><li data-sub-note-item="2-3"><div class="lab-step">Insert the funnel into the hole and tap all of the flies into the chamber. HYPOTHESES: The alternate hypothesis is that more flies will be moving against gravity, or up, regardless of the orientation of the choice chamber. Alternatively, if the flies are positively geotactic, then more flies will move down, or with gravity. The null hypothesis is that the flies will have no preference with regard to gravity and will move randomly within the chamber. 
</div></li><li data-sub-note-item="2-4"><div class="lab-step">Once the <em>Drosophila</em> are in the chamber, close the tape flap, rotate the chamber so that it stands vertically with side A on the bottom, and start the timer.
</div></li><li data-sub-note-item="2-5"><div class="lab-step">Immediately, count the number of <em>Drosophila</em> that are moving up and the number that are moving down. Record these data for time point 0 in the table. 
</div></li><li data-sub-note-item="2-6"><div class="lab-step">After 30 seconds, rotate the chamber so that side B is on the bottom and count the number of <em>Drosophila</em> that are moving up or down. Record these data for time point 30 in the table.
</div></li><li data-sub-note-item="2-7"><div class="lab-step">After 60 seconds, rotate the chamber so that side A is on the bottom again and record the number of moving flies for time point 60.  <a href="https://www-jove-com.remotexs.ntu.edu.sg/CDNSource/lm/labs/15/Table_1_Animal_behavior.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table 1</a>
</div></li><li data-sub-note-item="2-8"><div class="lab-step">Hold the chamber over the fly vial and carefully open the cap to return all of the <em>Drosophila</em> back to their empty vial. 
</div></li></ul></li><li data-note-item="3" data-parent-collapse="">Phototaxis
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="3-1"><div class="lab-step">To perform a phototaxis analysis, first cover side A of the choice chamber with tin foil, taking care that the foil reaches to the exact center between the two sides. 
</div></li><li data-sub-note-item="3-2"><div class="lab-step">Ensure the caps are secured on both ends of the chamber.  HYPOTHESES: In this exercise, the alternative hypothesis is that more flies will be found in the light side of the choice chamber, if the flues are attracted to light, or on the dark side if they are repelled by light. The null hypothesis is that <em>Drosophila</em> will have no preference with regard to either stimulus. Therefore, equal numbers of flies will be found on either side of the choice chamber.
</div></li><li data-sub-note-item="3-3"><div class="lab-step">Open the tape flap in the center of the choice chamber, insert the funnel into the hole, and tap all 40 flies into the chamber as previously described.
</div></li><li data-sub-note-item="3-4"><div class="lab-step">Set the timer to sound an alarm at 10 minutes.
</div></li><li data-sub-note-item="3-5"><div class="lab-step">When the alarm sounds, stop the timer, and count the number of flies in the open side B of the chamber.
</div></li><li data-sub-note-item="3-6"><div class="lab-step">Subtract that number from the total number of flies added to the choice chamber to calculate the number of flies in the covered side A.
</div></li><li data-sub-note-item="3-7"><div class="lab-step">Record these data in the table for time point 10.  <a href="https://www-jove-com.remotexs.ntu.edu.sg/CDNSource/lm/labs/15/Table_2_Animal_behavior.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table 2</a>
</div></li><li data-sub-note-item="3-8"><div class="lab-step">Carefully, return all of the flies back to their empty vial.
</div></li></ul></li><li data-note-item="4" data-parent-collapse="">Chemotaxis
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="4-1"><div class="lab-step">HYPOTHESES: In this exercise, the alternative hypothesis is that more flies will be found on the side of the chamber with the apple cider vinegar if the flies are attracted to the odor. Or, if the flies are repelled by the vinegar, then more of them will be found on the side of the chamber presenting the odorless water. The null hypothesis is that <em>Drosophila</em> will have no preference with regard to either stimulus and will move randomly in the chamber.  Use an individual disposable plastic pipette to dispense two drops of the apple cider vinegar onto one cotton ball for side A of the chamber. 
</div></li><li data-sub-note-item="4-2"><div class="lab-step">Use an individual disposable plastic pipette to dispense two drops of the distilled water onto a cotton ball for side B of the chamber. 
</div></li><li data-sub-note-item="4-3"><div class="lab-step">Fold two pieces of tape over themselves to create individual pieces of double-sided tape and use the tape to attach each cotton ball to the inside of the single bottle cap.
</div></li><li data-sub-note-item="4-4"><div class="lab-step">Screw the bottle caps with the cotton balls onto their corresponding sides of the chamber.
</div></li><li data-sub-note-item="4-5"><div class="lab-step">Transfer all of the flies into the chamber and set the timer to sound an alarm after 10 minutes. 
</div></li><li data-sub-note-item="4-6"><div class="lab-step">When the alarm sounds, stop the timer and record the number of flies in each side of the chamber. <a href="https://www-jove-com.remotexs.ntu.edu.sg/CDNSource/lm/labs/15/Table_3_Animal_behavior.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table 3</a>
</div></li><li data-sub-note-item="4-7"><div class="lab-step">At the end of the final experiment, funnel the flies into a morgue tube containing 70% ethanol. 
</div></li><li data-sub-note-item="4-8"><div class="lab-step">Dismantle the choice chambers for recycling.  NOTE: Since all of the chemicals used in these experiments are lab safe, the liquids can be disposed of down the sink, the chambers rinsed, and the solids can be thrown away in the trash. 
</div></li></ul></li><li data-note-item="5" data-parent-collapse="">Results
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="5-1"><div class="lab-step">Gather all of the data collected for the geotaxis, phototaxis, and chemotaxis experiments.
</div></li><li data-sub-note-item="5-2"><div class="lab-step">Plot these values as bar graphs for different conditions and different time points.
</div></li><li data-sub-note-item="5-3"><div class="lab-step">Use these data to draw inferences about the behavior of <em>drosophila melanogaster</em>. 
</div></li></ul></li></ol>

Animal Behavior: Studying Taxis Behavior of Drosophila - Procedure

Comportamento animal

Making Choice Chambers
ExpandBefore beginning the experiments, use one of the Sharpies from the lab table to trace a line around the circumference of the ...

Sexual Selection and Mate Choice

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Sexual Selection Class Simulation
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">Note: In this activity, you and your classmates will portray a sexually reproducing population consisting of an equal number of male and female animals. You will simulate several different mating scenarios to determine how relative quality, attractiveness, sex, and parental commitment affect mate choice and reproductive success. The goal of this activity is to maximize your progeny. 
</div></li><li data-sub-note-item="1-2"><div class="lab-step">Hypotheses: There are several overall hypotheses for female and male behavior for this activity. The main experimental hypothesis for female behavior is that females will mate with males of high quality more frequently than those of low quality, yielding a higher reproductive success. The null hypothesis is that females will not demonstrate preference towards high- or low-quality males in any of the four rounds of mating. For males, one experimental hypothesis is that males with a higher visible score will have greater reproductive success. Another could be that males will be more selective for females in scenarios in which they join females to care for offspring as opposed to scenarios in which they exhibit no parental commitment. The null hypothesis for male behavior might be that males will not demonstrate preference towards high- or low-quality females in any of the four rounds of mating. 
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Activity Overview: To begin, each student will be assigned a sex as well as a relative quality and attractiveness score. Remember - the goal is to maximize your reproductive success in each round. You do this by mating with partners of the highest possible quality which is rated on a scale of one to three with three being the highest possible score. Attractiveness scores, which follow the previously described scoring paradigm, correlate with quality but do not exactly predict the score. Though useful, they are not a perfect way to choose the best mate.  If you are assigned a male ID holder, you will have 16 gametes. However, if you are assigned a female ID holder, you will only have four. A successful mating occurs when one sperm and one egg are paper-clipped together and deposited at the fertilization area. 
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Note - Rules of the activity: 
<ul>
	<li>Assigned male and female identities for these experiments are not representative of students&#39; personal gender identities. </li>
	<li>Running is not permitted. </li>
	<li>Talking is not permitted.</li>
	<li>Hidden values cannot be shared but individuals can attempt to hide their visible scores. </li>
	<li>Respect the space around each individual and remain an arm&#39;s length away. </li>
	<li>Offspring will only be counted towards an individual score if both gametes are paper-clipped together and dropped off in the designated fertilization area before the end of the two-minute round. </li>
</ul>
</div></li></ul></li><li data-note-item="2" data-parent-collapse="">Round 1 &#8211; Hidden Quality Score
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="2-1"><div class="lab-step">To begin the first simulation, take the clip-on ID holder you were randomly assigned and make sure it contains four gametes if you are assigned female or sixteen gametes if male. Males should also have a strip of white paper and two paperclips. 
</div></li><li data-sub-note-item="2-2"><div class="lab-step">Next, males should paperclip the white strip of paper over their quality score to hide it, but keep the attractiveness score visible. 
</div></li><li data-sub-note-item="2-3"><div class="lab-step">Record your ID number for round one in table two.  <a href="https://www-jove-com.remotexs.ntu.edu.sg/CDNSource/lm/labs/35/Table_2_sexual_selection.xlsx" style="display:block;margin:20px;" target="_blank">Click Here to download Table 2</a>
</div></li><li data-sub-note-item="2-4"><div class="lab-step">Next, clip on the ID holder and move to the designated mating area. 
</div></li><li data-sub-note-item="2-5"><div class="lab-step">When the instructor says begin, you may commence the mating simulation. You will have exactly two minutes to produce as many offspring as possible. Males may freely give their sperm to females, and females may choose to use these sperm immediately after which they would walk to the fertilization area and paperclip it to one of their eggs, or they may choose to wait until they receive other sperm to do so. 
</div></li><li data-sub-note-item="2-6"><div class="lab-step">Once the gametes have been deposited in the fertilization area, females may resume mating. 
</div></li><li data-sub-note-item="2-7"><div class="lab-step">When the two minutes have elapsed, all matings must stop and any gametes not in the fertilization area at this time will not be counted. 
</div></li><li data-sub-note-item="2-8"><div class="lab-step">The instructor should now tally all of the matings and record the numbers of the individuals who produced each offspring.
</div></li><li data-sub-note-item="2-9"><div class="lab-step">Now, find all the gametes that came with your ID holder and repackage them for the next round. 
</div></li></ul></li><li data-note-item="3" data-parent-collapse="">Round 2 &#8211; Hidden Attractiveness Score
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="3-1"><div class="lab-step">For the second round, obtain a new randomly assigned ID holder and again ensure it contains the proper number of gametes. 
</div></li><li data-sub-note-item="3-2"><div class="lab-step">Next, males should use the paperclip and strip of white paper to hide their attractiveness score (instead of the quality score, which was covered in round one).
</div></li><li data-sub-note-item="3-3"><div class="lab-step">Record the ID number of your clip in table two under round two.  Note: The mating simulation process for round two will be the same as round one.
</div></li><li data-sub-note-item="3-4"><div class="lab-step">Move to the designated mating area and with for the instructor to signal the start of the round. As before, males may freely give away their sperm while females will choose to use it now or wait for different sperm. When a female decides to fertilize an egg, she will walk to the fertilization area and paperclip the gametes together. 
</div></li><li data-sub-note-item="3-5"><div class="lab-step">Proceed with the simulation for two minutes, and at the signal from the instructor all mating activity should stop. 
</div></li><li data-sub-note-item="3-6"><div class="lab-step">The instructor should now tally all of the matings from this second round.
</div></li><li data-sub-note-item="3-7"><div class="lab-step">Now, find and return the gametes to your ID holder, and then return the holder to the instructor.
</div></li></ul></li><li data-note-item="4" data-parent-collapse="">Round 3 &#8211; Visible Quality and Attractiveness Scores
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="4-1"><div class="lab-step">For the third simulation, you should again obtain a new clip-on ID holder and check that it contains the appropriate number of gametes. Record the ID number of your clip in table two.
</div></li><li data-sub-note-item="4-2"><div class="lab-step">For this round, leave both attractiveness and quality scores visible on male IDs. Note: The mating simulation process for round three will be the same as the previous two rounds.
</div></li><li data-sub-note-item="4-3"><div class="lab-step">Move to the mating area and wait for the signal to begin the simulation.
</div></li><li data-sub-note-item="4-4"><div class="lab-step">Once the instructor starts the timer and gives the signal, commence mating. As before, males are allowed to freely give away their sperm to females and females may use the sperm immediately or wait for different sperm. Again, fertilized eggs only count if placed in the fertilization area. 
</div></li><li data-sub-note-item="4-5"><div class="lab-step">After the two-minute time limit, the instructor should tally all matings. 
</div></li><li data-sub-note-item="4-6"><div class="lab-step">Now, find and return the gametes to your ID holder, and then return the holder to the instructor.
</div></li></ul></li><li data-note-item="5" data-parent-collapse="">Round 4 &#8211; Increased Male Parental Care
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="5-1"><div class="lab-step">In the final round, males will again leave their attractiveness and quality scores visible. 
</div></li><li data-sub-note-item="5-2"><div class="lab-step">Obtain an ID holder and ensure it contains the appropriate number of gametes. 
</div></li><li data-sub-note-item="5-3"><div class="lab-step">Clip on the ID holder and move to the mating area. Note: As before, males may freely give away their sperm to females. However, in this round, once a female chooses to mate with a male, both the male and female will walk to the fertilization area to paperclip their gametes together. After placing the paper-clipped gametes on the table, both male and female actors may return to the mating area. 
</div></li><li data-sub-note-item="5-4"><div class="lab-step">The instructor should signal the start of the round and begin the timer &#8211; at this point participants can begin the simulation.
</div></li><li data-sub-note-item="5-5"><div class="lab-step">Once the two-minute time limit is up, all matings must stop. 
</div></li><li data-sub-note-item="5-6"><div class="lab-step">The instructor should tally all mating events.
</div></li><li data-sub-note-item="5-7"><div class="lab-step">Find and return the gametes to your ID holder. Give your ID holder to the instructor and notify them if any gametes or supplies are missing. 
</div></li></ul></li><li data-note-item="6" data-parent-collapse="">Results
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="6-1"><div class="lab-step">To analyze the data generated during the mating activities, first split up into groups of four. 
</div></li><li data-sub-note-item="6-2"><div class="lab-step">Collect the class data from the instructor for ONE of the four rounds of mating. Note: In this activity, there were several types of females and males distinguished by different letters. These letter types correspond to specific combinations of quality and attractiveness scores. In the compiled data, the average number of matings and reproductive success has already been sorted by letter type for you by the instructor. 
</div></li><li data-sub-note-item="6-3"><div class="lab-step">Use these data to generate four graphs for the round you were assigned. First plot two graphs for the average number of matings against female and male type. 
</div></li><li data-sub-note-item="6-4"><div class="lab-step">Then plot the reproductive success for males and females against male or female type, giving a total of four graphs.
</div></li><li data-sub-note-item="6-5"><div class="lab-step">Once these graphs are complete, each group should present their data to the class, making sure to state the original hypothesis for your assigned round and whether or not the hypothesis was upheld.
</div></li><li data-sub-note-item="6-6"><div class="lab-step">After each group presents, discuss the overall results of the four rounds together. 
</div></li><li data-sub-note-item="6-7"><div class="lab-step">Note the affect that hiding the quality score had on the overall quality of mates chosen.
</div></li><li data-sub-note-item="6-8"><div class="lab-step">Note how hiding or revealing the attractiveness score affected the results.
</div></li><li data-sub-note-item="6-9"><div class="lab-step">Discuss the effect of male parental investment on the number and overall quality of mating pairs.
</div></li></ul></li></ol>

Seleção sexual e escolha do parceiro

Sexual Selection Class Simulation
ExpandNote: In this activity, you and your classmates will portray a sexually reproducing population consisting of an ...

Genetics of Organisms

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">REF and WEF Fly Genetics
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">To prepare for the experiment, be sure to obtain cultures of <em>Drosophila melanogaster</em> at least two weeks ahead of the planned experiment day. NOTE: Because adult females store sperm after mating, you will need this time to generate virgin females by breeding the flies.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">You should get two stock cultures: wildtype, that is homozygous dominant red eyed flies, and white chromosome one mutant, which are homozygous recessive white eyed flies.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Purchase or create <em>Drosophila </em>habitats consisting of a clean vial, 3 cm of prepared food media, and a stopper to close the open end of the vial.
</div></li><li data-sub-note-item="1-4"><div class="lab-step">When not being moved or counted, keep the flies in an incubator or room kept at 23 - 25 &#176;C, where the lifecycle will take around 12 days. NOTE: From the egg, <em>Drosophila </em>progress through four main stages of development: embryo, larva, pupa, and adult. The complete lifecycle takes about 10 days at 25 &#176;C, but it can be affected by temperature. For example, at 18 &#176;C, the lifecycle is about 19 days and at 29 &#176;C, the lifecycle is only seven days.
</div></li><li data-sub-note-item="1-5"><div class="lab-step">As an overview, to set up for the student experiment, you will generate vials for each student group containing white eyed virgin female flies only, white eyed males only, red eyed virgin females only, and red eyed males only. These will be referred to as the experimental vials.
</div></li><li data-sub-note-item="1-6"><div class="lab-step">When your vials from the supplier arrive, referred to as the stock vials, these will contain a mix of male and female flies, as well as larvae, pupae, and eggs.
</div></li><li data-sub-note-item="1-7"><div class="lab-step">Beginning with your stock vials, you will take around 3 - 5 males and the same number of females, keeping white eyed and red eyed flies separate at all times, and place these together into a new nursery vial containing food. NOTE: You can sex the flies using a magnifying glass. Look at the posterior of the fly. Males are slightly smaller and have darker solid pigment on the end of their abdomen. Females are larger and have stripes rather than a solid spot.
</div></li><li data-sub-note-item="1-8"><div class="lab-step">You will leave the flies in these nursery vials for around 3 - 5 days to mate and lay eggs.
</div></li><li data-sub-note-item="1-9"><div class="lab-step">After this, you will move them back to their stock vials.
</div></li><li data-sub-note-item="1-10"><div class="lab-step">Next, you will observe as the eggs and larvae in the nursery vials mature, checking the vials every 6 - 8 hours. As it takes females around 8 - 10 hours to become sexually mature, this should ensure that any females found in the nursery vials are virgin.
</div></li><li data-sub-note-item="1-11"><div class="lab-step">As the new adult flies appear, you should transfer them from the nursery to the appropriate experimental tube. For example, if it is a male, transfer it to the experimental male tube for the appropriate eye color. If it is a female, it should be placed in an experimental vial labeled with eye color and virgin female.
</div></li><li data-sub-note-item="1-12"><div class="lab-step">Keep adding flies to the appropriate vials until each experimental vial has at least five flies. NOTE: You will need to make enough experimental vials so that each student group has one of each eye color type and sex, with a few class spares. You should create enough nursery vials to account for this. A good rule of thumb is that it generally takes about two weeks for 25 healthy adults to produce about 50 new adults.
</div></li><li data-sub-note-item="1-13"><div class="lab-step">To begin the process, first label empty habitats to make the nursery vials to raise larvae and pupae for each strain of flies: red eyed and white eyed.
</div></li><li data-sub-note-item="1-14"><div class="lab-step">To move the adult flies into the nursery vial to lay eggs, use an anesthesia wand according to the manufacturer&#39;s instructions, placing it into the stock vial of mature flies. Wait until the animals become unconscious. Now they can be gently tapped onto a piece of white paper and sorted into piles of the same sex using a brush.
</div></li><li data-sub-note-item="1-15"><div class="lab-step">Transfer a few male and female flies to the appropriate vial labeled with their eye color phenotype and sex.
</div></li><li data-sub-note-item="1-16"><div class="lab-step">Place the nursery vials to incubate at 23 to 25 &#176;C and the flies will lay eggs.
</div></li><li data-sub-note-item="1-17"><div class="lab-step">Remove the adults after 3 - 4 days using anesthesia and put them back into the stock vial.
</div></li><li data-sub-note-item="1-18"><div class="lab-step">Remove any adult flies that have eclosed in the 6 - 8 hour period using an anesthesia wand.
</div></li><li data-sub-note-item="1-19"><div class="lab-step">Then, tip out the adults onto paper and sort them into groups based on their sex before placing each fly into an appropriate experimental vial marked male or virgin female and red or white for the phenotype. NOTE: Each experimental vial should contain five flies. Once this number is reached, then a new vial should be started. If you miss the 6 - 8 hour window, females may not be virgins, so these should be discarded into a morgue tube containing 70% ethanol or returned to the stock vial. Males do not need to be virgins, but do need to be kept in male only containers.
</div></li><li data-sub-note-item="1-20"><div class="lab-step">Each lab group will need one tube of five virgin red eyed females, one of five virgin white eyed females, one tube of red eyed males, and one of five white eyed males. In addition, they will need a morgue vial containing ethanol, two anesthesia wands, fly anesthetic, a paintbrush, a magnifying glass, some plain paper, and two empty fly vials for crosses.
</div></li></ul></li></ol>

Genetics of Organisms: REF and WEF Fly Genetics - Prep

Genética de Organismos

REF and WEF Fly Genetics
ExpandTo prepare for the experiment, be sure to obtain cultures of Drosophila melanogaster at least two weeks ahead of the ...

Genética

Artificial Selection

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Observing Artificial Selection in Plants 
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">To begin the experiment, collect five two-week old grown plants from the generation one population that appear healthy. Note: Plants should appear green, not wilted, and not have damaged leaves. Experimental Hypothesis: The experimental hypothesis for this lab might be that the number of lead trichomes varies naturally, but the average number will increase over generations if only plants with the highest number of trichomes are interbred or decrease if those with the least are interbred. Null Hypothesis: The null hypothesis might be that the average number of trichomes will not change over subsequent generations after artificial selection.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">Label each plant with a letter from A to E and with both partners&#8217; names.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Record any natural differences you see between your plant samples. Note: All of the plants are the same age, so any observed variation is due to expressed phenotype, not development.
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Use a hand lens to count the number of trichomes found on the petiole of the lowest leaf, not including the round, first-grown cotyledons for each plant. Note: It can be helpful to shine a light onto the plant and to hold it against a dark background, such as a lab bench, during the trichome analysis. 
</div></li><li data-sub-note-item="1-5"><div class="lab-step">Both partners will conduct counts on each plant independently and compare answers. If the counts are different, enlist a third counter or take the average of the two results.
</div></li><li data-sub-note-item="1-6"><div class="lab-step">After this, determine the median number of trichomes for the entire class.  <a target="__blank" style="display:block;padding:20px;" href="https://www-jove-com.remotexs.ntu.edu.sg/CDNSource/lm/labs/2/Lab_2_Table_A.xlsx">Click Here to download Table A</a>
</div></li><li data-sub-note-item="1-7"><div class="lab-step">Determine the top and bottom 10% of the plants from the class trichome count. These groups will make up the two artificially selected lines: high hairiness and low hairiness, respectively.
</div></li><li data-sub-note-item="1-8"><div class="lab-step">If more than 10% of the plants have no trichomes, select 10% of those plants randomly for the low hairiness plant line.
</div></li><li data-sub-note-item="1-9"><div class="lab-step">Separate the two groups of selected plants and label them clearly.
</div></li><li data-sub-note-item="1-10"><div class="lab-step">Move the plants to a well-lit area and keep them well watered until they develop flowers. Note: Flowers typically develop around 14 days.
</div></li><li data-sub-note-item="1-11"><div class="lab-step">When the flowers are fully developed, use a pollination wand or paintbrush to rub the stigma and anther of one plant&#8217;s flowers for a few seconds.
</div></li><li data-sub-note-item="1-12"><div class="lab-step">Using the same wand or brush, rub the inside of each flower of every other plant in the same selection line.
</div></li><li data-sub-note-item="1-13"><div class="lab-step">Using a different wand or brush, repeat steps 11 through 12 on the other line. Note: Take care not to cross the lines. Spread the pollen only among the appropriately selected plants.
</div></li><li data-sub-note-item="1-14"><div class="lab-step">Return the generation one plants to the growing areas. When the plants reach approximately 1 month old, they should produce mature seed pods.
</div></li><li data-sub-note-item="1-15"><div class="lab-step">Allow the plants to dry out once the seed pods turn yellow.
</div></li><li data-sub-note-item="1-16"><div class="lab-step">Break the seed pods open and collect the seeds.
</div></li><li data-sub-note-item="1-17"><div class="lab-step">Obtain new pots for the generation two seeds.
</div></li><li data-sub-note-item="1-18"><div class="lab-step">Fill the pots with moist potting soil about halfway and add a fertilizer pellet to each pot.
</div></li><li data-sub-note-item="1-19"><div class="lab-step">Fill the pots with more soil to fill just below the lip.
</div></li><li data-sub-note-item="1-20"><div class="lab-step">Make a shallow depression in the soil of each pot with a probe.
</div></li><li data-sub-note-item="1-21"><div class="lab-step">Touch a clean toothpick to a gluestick and use the toothpick to transplant the seeds into the soil one by one.
</div></li><li data-sub-note-item="1-22"><div class="lab-step">Cover the seeds with a thin layer of soil.
</div></li><li data-sub-note-item="1-23"><div class="lab-step">Label the pots with their hairiness line and place them in the designated growing area.
</div></li><li data-sub-note-item="1-24"><div class="lab-step">Use a plastic dropper to water the plants as needed to ensure they do not dry out.
</div></li><li data-sub-note-item="1-25"><div class="lab-step">When the plants are two weeks old, collect five plants from each line and label them A to E.
</div></li><li data-sub-note-item="1-26"><div class="lab-step">Then, count the trichomes on these plants as previously described (steps 4- 5).
<a target="__blank" style="display:block;padding:20px;" href="https://www-jove-com.remotexs.ntu.edu.sg/CDNSource/lm/labs/2/Lab_2_Table_B.xlsx">Click Here to download Table B</a>
</div></li></ul></li><li data-note-item="2" data-parent-collapse="">Data Analysis
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="2-1"><div class="lab-step">Create a frequency graph based on the generation one class data with the trichome count on the x axis and the number of plants on the y axis. Label the mean. 
</div></li><li data-sub-note-item="2-2"><div class="lab-step">Then, create frequency graphs for both of the selected hairiness lines.
</div></li><li data-sub-note-item="2-3"><div class="lab-step">Compare the frequency graphs for generation one and both hairiness lines.
</div></li><li data-sub-note-item="2-4"><div class="lab-step">Take note of phenotypic shifts observed in the selected hairiness lines.
</div></li></ul></li></ol>

Artificial Selection: Visualization in Plants and Data Analysis - Procedure

Seleção artificial

Observing Artificial Selection in Plants 
ExpandTo begin the experiment, collect five two-week old grown plants from the generation one population that ...

Evolução

Physiology of the Circulatory System

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Measuring Circulatory System Function in Humans
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">Before starting the experiment go to the front of the room and collect an alcohol swab, a sphygmomanometer, and a stethoscope.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">Clean the earpieces with the alcohol swab and then insert them into your ears.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">To check if the stethoscope is in the on position, gently tap the flat metal disc called the diaphragm.
</div></li><li data-sub-note-item="1-4"><div class="lab-step">If you hear a sound it&#39;s on. If not, turn the metal disc and tap again and keep turning and tapping the disc until you hear a sound. NOTE: For this experiment you will work in pairs. One person will measure their partner&#39;s blood pressure, while the person being measured will count their heartbeats over the course of one minute.
</div></li><li data-sub-note-item="1-5"><div class="lab-step">Fill out table one with the name of the person being monitored. HYPOTHESES: The experimental hypothesis is that systolic blood pressure will be higher when the person is sitting when compared to lying down because more pressure is required for blood to be moved around the body. The null hypothesis is that there will be no difference in systolic blood pressure whether a person is sitting or lying down.<a href="https://www-jove-com.remotexs.ntu.edu.sg/CDNSource/lm/labs/9/Table_1_Physiology_of_Circulatory_System.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table 1</a>
</div></li><li data-sub-note-item="1-6"><div class="lab-step">The person being monitored should begin by lying down. Next, they will need to locate their pulse by placing two fingers next to their windpipe. If a throbbing sensation is felt, this is the pulse.
</div></li><li data-sub-note-item="1-7"><div class="lab-step">Now, ready a time keeping device to count out 1 minute as the person taking their pulse will be having their blood pressure taken at the same time.
</div></li><li data-sub-note-item="1-8"><div class="lab-step">At this point, the person measuring blood pressure should place the cuff of the sphygmomanometer around the other arm of the subject and the stethoscope earpieces into their own ears.
</div></li><li data-sub-note-item="1-9"><div class="lab-step">Start the timer.
</div></li><li data-sub-note-item="1-10"><div class="lab-step">The person being monitored should begin counting the number of heartbeats felt over one full minute. While the subject is taking their pulse, the person taking blood pressure should place the diaphragm of the stethoscope under the cuff on the inside of their partner&#39;s elbow.
</div></li><li data-sub-note-item="1-11"><div class="lab-step">Then, pump the cuff to 200 mm of mercury.
</div></li><li data-sub-note-item="1-12"><div class="lab-step">Gently and slowly twist the metal knob on the sphygmomanometer to release air from the cuff.
</div></li><li data-sub-note-item="1-13"><div class="lab-step">As pressure is released the first Korotkoff sound should be heard. The corresponding value on the sphygmomanometer is the systolic blood pressure. Consistent sounds should be heard until they die out with the last sound signifying the diastolic blood pressure.
</div></li><li data-sub-note-item="1-14"><div class="lab-step">Record the systolic pressure, the diastolic pressure, and the heart rate in Table 1.
</div></li><li data-sub-note-item="1-15"><div class="lab-step">Now, repeat the blood pressure and pulse measurements with each partner keeping the same role but this time the subject should be sitting upright.
</div></li><li data-sub-note-item="1-16"><div class="lab-step">Record these data in the appropriate places in Table 1.
</div></li><li data-sub-note-item="1-17"><div class="lab-step">Finally, the partners should switch spots. So now, the person measuring blood pressure should be taking their pulse and acting as the subject.  NOTE: Be sure to wipe the earpieces of the stethoscope between users and don&#39;t forget to record your measurements in Table 1.
</div></li><li data-sub-note-item="1-18"><div class="lab-step">After finishing the experiment, discard all alcohol swabs into the trash. Then, bring the sphygmomanometer and stethoscope back to the front of the room.
</div></li></ul></li><li data-note-item="2" data-parent-collapse="">Measuring Respiratory Rate in Fish
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="2-1"><div class="lab-step">To prepare for this second experiment, collect two clear containers.
</div></li><li data-sub-note-item="2-2"><div class="lab-step">Begin by filling half of one container with aquarium water.
</div></li><li data-sub-note-item="2-3"><div class="lab-step">Then, use a fish net to gently catch a goldfish and place it into the cup. Pinch the net around the fish or else it may jump out of the net.
</div></li><li data-sub-note-item="2-4"><div class="lab-step">Familiarize yourself with the operculum of the fish. The operculum covers the gills and it is located behind and below the eye. When the fish respires the operculum moves in and out and this is one gill beat. HYPOTHESES: In this experiment you will measure the gill beats of the fish in different water temperatures. The hypothesis is that there will be a significant correlation between the temperature and respiration rate in goldfish. The null hypothesis is that there will be no correlation between temperature and respiration rate.
</div></li><li data-sub-note-item="2-5"><div class="lab-step">Take the other empty container with a screw cap, the cup with your fish, and the net to the water tanks.
</div></li><li data-sub-note-item="2-6"><div class="lab-step">Then, fill the empty container with the water from the lowest temperature water bath. This will act as your first respiration rate chamber.
</div></li><li data-sub-note-item="2-7"><div class="lab-step">Next, hold the fish net over a wastewater container and dump out the water from the cup holding the fish so that the fish falls into the net. Pinch the net around the fish without touching the fish itself.
</div></li><li data-sub-note-item="2-8"><div class="lab-step">Bring the net over the top of the container with the bath water and let go of the net to release the fish into the vial.
</div></li><li data-sub-note-item="2-9"><div class="lab-step">Once the fish is in the container, submerge it back into the water bath and then screw the cap on to prevent outside oxygen from entering the vial.
</div></li><li data-sub-note-item="2-10"><div class="lab-step">Next, remove the container from the controlled temperature water bath and place it on your workbench surface to avoid warming the water with body heat.
</div></li><li data-sub-note-item="2-11"><div class="lab-step">One partner should immediately begin counting the gill beats as soon as the second partner starts the stopwatch.
</div></li><li data-sub-note-item="2-12"><div class="lab-step">Continue counting the beats for 20 seconds. Record the number of beats in column A of Table 2. <a href="https://www-jove-com.remotexs.ntu.edu.sg/CDNSource/lm/labs/9/Table_2_Physiology_of_Circulatory_System.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table 2</a>
</div></li><li data-sub-note-item="2-13"><div class="lab-step">Repeat the measurement one more time and record the number in column B.
</div></li><li data-sub-note-item="2-14"><div class="lab-step">Next, hold the fish net over a wastewater container and dump out the water from the fish vial so that the fish falls into the net.  IMPORTANT: Move from lowest to highest temperature, so the fish is not stressed between trials.
</div></li><li data-sub-note-item="2-15"><div class="lab-step">Repeat the experiment for the remaining four temperature conditions (steps 8 &#8211; 13).
</div></li><li data-sub-note-item="2-16"><div class="lab-step">Record all the values for different temperatures in Table 2.
</div></li><li data-sub-note-item="2-17"><div class="lab-step">Once you are done, slowly mix water from the last respiration chamber with the fish tank water and then return the fish to the home tank. NOTE: This should be done over the course of approximately 10 minutes.
</div></li></ul></li><li data-note-item="3" data-parent-collapse="">Results
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="3-1"><div class="lab-step">It&#39;s time to analyze the data that you just collected. Look at the blood pressure and heart rate measurements for the different trials. If they were different, make a hypothesis to explain your observations. 
</div></li><li data-sub-note-item="3-2"><div class="lab-step">For the gill beat experiment, average the numbers in columns A and B and calculate the fish respiration rate as gill beats per minute.
</div></li><li data-sub-note-item="3-3"><div class="lab-step">Next, plot the graph of temperature versus gill beats per minute.
</div></li><li data-sub-note-item="3-4"><div class="lab-step">Record any correlations you see between the variables. If a correlation is observed, determine if it is positive or negative and provide an explanation for this result.  NOTE: Consider the fact that fishes are ectotherms and the impact of temperature on dissolved oxygen.
</div></li></ul></li></ol>