We thank Victor Marquez for providing DZNep.HCl and Scotty Bolling for manufacturing the bloodfeeders. The Mopti and SUA2La strains of An. gambiae were obtained from the Malaria Research and Reference Reagent Resource Center (MR4). This work was supported by the Fralin Life Science Institute and the grant from National Institutes of Health 1R21AI094289 to Igor V. Sharakhov.

注:该协议的三个部分描述DZNep药物的水溶液暴露于幼虫蚊子，DZNep的基于血液的暴露于成年女性进行研究繁殖力其效果，并且使用简单的比色技术SAH水解酶抑制DZNep测量。这些测定法的示意图示于图1。 1.未成熟蚊子发展和存活率测定注:本节介绍使用水溶性药物，影响蚊子幼虫的发展和生存。 <ol><li>哈奇A.冈比亚鸡蛋在28°C的蒸馏水（DH 2 O）和后他们第二龄幼虫。 </li><li>取一个六孔DNA酶，RNA酶游离细胞培养板和倾10毫升的dh 2 O至每个孔中。 </li><li>选择蚊子的幼虫在2 次龄每孔加入15幼虫。然后加入第二龄幼虫直接制版，PUT斯达康它们在纸巾上为2-3秒，以除去多余的水。 </li><li>标签井用颜色编码的磁带随机实验。标签上的每个板（ 图2），每个测试浓度2孔中。 </li><li>通过在1ml的dh 2 O中溶解为0.3mg DZNep制备1mM的DZNep盐酸盐（MW = 298.73）原液添加DZNep.HCl的原液到标孔中，以达到0.5μM和5.0μM的终浓度。 注:原液可以储存在4 °下进行短期（1天至一个月）或-20 ℃下长期（超过一个月）。 注:加入的储备溶液为上述的浓度不会改变水的体积在孔中显著。然而，在情况下要高得多的浓度，最好是溶解化合物在幼虫介质，以避免任何不必要的稀释的化合物。 </li><li>加的片状鱼食等量至每个孔中。覆盖板s和培养他们在28 °下，在孵化器。 </li><li>记录的死亡率为24小时暴露时间后DZNep处理的和未处理的幼虫蚊子。取下并丢弃任何死幼虫。每天重复这个记录直到所有的幼虫模或化蛹并脱颖而出，成为成年人。记录幼虫的大小，每隔一天， 即，第0天，2，4，6，8，直到它们化蛹。 </li><li>分析使用适当的统计分析软件的幼体发育和死亡数据。 注:条形图，可以用于表示死亡率数据在Microsoft Excel中。多元方差分析（MANOVA）进行了软件如JMP或SPSS就会发现，如果不同剂量的药物导致显著不同的生存蚊子幼虫。 </li></ol> 2.繁殖力测定通过给予药品通过人工进纸器雌蚊注:本部分详细介绍了另外的药物，以血次使用人工馈线系统它馈送到成年雌性蚊子。 <ol><li>设置成蚊笼同等数量的男性和女性的蚊子控制和测试笼。提供10％的糖溶液中浸泡棉球两个笼子里，直到雌性准备喂养（3-5天是最佳的冈比亚按蚊 ）。 </li><li> 2小时前的血液供给，取出棉球，以确保女性是饿了。 </li><li>装配两个人工血液供料器，它由一个倒宽底部玻璃漏斗（直径= 50毫米，长度= 70毫米）和周围的塑料工业胶密封。附加塑料管（直径= 7毫米）至2馈线"网点（直径= 10mm）的通过连接器，用于流入和流出的水​​。标记两个馈线为"控制"和"5μMDZNep"。 </li><li>使用市售的加热元件，设置程序觅食。在"菜单"进入"设置"并选择"设定点";预装的程序将会出现。选择"SP1"，并设置该温度37 ℃。 注:加热元件确保了血液施用维持在一个恒定的温度。可用于不同的蚊子或昆虫物种附加程序设置。 </li><li>填充有水的水桶和浸入加热元件到水中。 </li><li>切一块方形封口膜（50毫米×50毫米），并伸展它，使薄的膜状的膜。涵盖人工饲养的封口膜的底部。切另一片封口膜（50毫米×10mm的）的，它伸展并密封该馈线的边缘。 </li><li>组装系统中，通过管连接馈线和加热元件。一旦完成，切换系统，选择"SP1"。按"Enter"来启动系统。 注:水会加热到37 °C和保持在该温度。 </li><li>监测日水用水桶温度计é温度。 注:连接器和管道有助于通过该系统保持恒定的水循环，使血液的温度保持在37 ℃。 </li><li>填平底托盘与将用于净化任何血液泄漏的10％漂白溶液。 </li><li>加入2毫升除颤羊的血液离心管。这个标签为"控制"。重复该过程为实验管标记为"5μMDZNep"。新增6微升DZNep原液（见步骤1.5）的实验管标有"5μMDZNep"轻轻颠倒几次混合血液和药品。在室温下孵育该管10分钟。 </li><li>使用移液管，加2ml对照和测试血液到相应标记的馈线的顶部。丢弃在漂白溶液中的吸移管。盖上笼用深色袋子，呼吸的空气笼定期鼓励˚Females喂养。 </li><li>让蚊子喂约30分钟。一旦加料完毕，关掉加热元件，把馈线出来，剥离封口膜。浸泡在漂白剂溶液馈线约5分钟，并在去离子水冲洗干净。 </li><li>取出馈线，把棉球用白糖水的笼子里48小时。将含有水和滤纸在笼子里过夜，以方便产卵一个鸡蛋的菜。标记每个蛋盘与相应的测试或对照的浓度。 </li><li>取出蛋菜第二天检查蛋的数量和结构的立体显微镜下。获得使用Q-colors5软件映像。计数的蛋的数量。分析在从测试和对照笼获得蛋的数量差异。 注意:一个非配对t检验可以用于确定是否蛋的数量是显著差异（p≤0.05）。 </li></ol>蚊子酶抑制3.生化测定通过药物注意:本节介绍使用DTNB作为指标来确定DZNep对成年雄性和雌性蚊子SAH水解酶的抑制效果的酶活性测定。 <ol><li>通过加入Na 214.19克HPO 4到1升的dh 2 O的制备的0.1M的Na 2 HPO 4（磷酸氢二钠）溶液接着，通过添加13.79克的NaH 2 PO 4至1升的dh 2 O的制备的0.1M的NaH 2 PO 4（磷酸二氢钠）溶液</li><li>滴定的0.1M的Na 2 HPO 4溶液与所述的0.1M的NaH 2 PO 4的溶液至pH 8.5。 注意:这将是为0.1M的Na 2 HPO 4（pH为8.5）的工作原液。 </li><li>通过加入0.3％的Triton X-100的制备的的0.1M的Na 2 HPO 4（pH为8.5）均质化的溶液。 注:同质化的解决方案可以保存在4℃。 </li><li>公关epare 1.6毫SAH（MW = 384.4）通过在10毫升的0.1M的Na 2 HPO 4（pH为8.5）中溶解6.15毫克SAH的溶液。 </li><li>通过在10毫升的0.1M的Na 2 HPO 4（pH为8.5）中溶解6.3毫克DTNB的制备1.6毫DTNB（MW = 396.4）溶液。置于冰上新鲜DTNB液备用。 </li><li>通过在1毫升的dh 2 O中溶解为0.3mg DZNep的制备1mM的DZNep.HCl（MW = 298.73）的溶液接下来，稀释1mM的DZNep溶液成一系列浓度从1000微米至0.002微米的dh 2 O. </li><li>为了测量SAH水解酶抑制DZNep，准备蚊子粗酶提取:均质10个非血液喂养成蚊1毫升冰冷的0.1M的NaH 2 PO 4（pH值8.5），含0.3％的Triton X- 100，使用玻璃组织匀浆器。匀浆液转移至1.5ml离心管中。 </li><li>离心匀浆5分钟以10,000 xg离心在4℃下。上清转移到一个干净的1.5 ml离心管。使用上清液作为酶源SAH生物化学测定。 </li><li>对于空白处理（ 即无DZNep，无酶）中，添加50微升1.6毫SAH，50微升1.6毫DTNB，并加入100μl的0.1M的Na 2 HPO 4（pH为8.5），以的96孔的各孔，平底微孔板。 </li><li>对于控制（ 即没有DZNep），加入50微升1.6毫米SAH，50微升1.6毫米DTNB，50微升的0.1M的Na 2 HPO 4（pH值8.5）和50微升酶（从原油中提取SAH水解获得）个别井。 </li><li>对于DZNep治疗，加50微升1.6毫米SAH，50微升1.6毫米DTNB，50微升酶和50微升选择DZNep集中到个别井。准备每次治疗和控制4个重复。 </li><li>使用96孔酶标仪读取SAH酶样品的​​光密度（OD）在405nm进行5分钟，在20秒的间隔。 </li><li>减去控制空白OD次治疗OD从每个获得良好。计算使用下面的等式其余SAH水解酶活性的百分比:％残留活性=（处理的OD /对照OD值）×100。 </li></ol>

<ol>
	<li>Gatton, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+importance+of+mosquito+behavioural+adaptations+to+malaria+control+in+Africa.">The importance of mosquito behavioural adaptations to malaria control in Africa.</a> Evolution; International Journal of Organic Evolution. 67, 1218-1230 (2013).</li><li>Sternberg, E. D., Thomas, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+adaptation+to+temperature+and+the+implications+for+vector-borne+diseases.">Local adaptation to temperature and the implications for vector-borne diseases.</a> Trends in Parasitology. , (2014).</li><li>Rocca, K. A., Gray, E. M., Costantini, C., Besansky, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=2La+chromosomal+inversion+enhances+thermal+tolerance+of+Anopheles+gambiae+larvae.">2La chromosomal inversion enhances thermal tolerance of Anopheles gambiae larvae.</a> Malaria Journal. 8, 147 (2009).</li><li>Coluzzi, M., Sabatini, A., Petrarca, V., Di Deco, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosomal+differentiation+and+adaptation+to+human+environments+in+the+Anopheles+gambiae+complex.">Chromosomal differentiation and adaptation to human environments in the Anopheles gambiae complex.</a> Transactions of the Royal Society of Tropical Medicine and Hygiene. 73, 483-497 (1979).</li><li>Donepudi, S., Mattison, R. J., Kihslinger, J. E., Godley, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Update+on+Cancer+Therapeutics.">Update on Cancer Therapeutics.</a> Science Direct. 2 (4), 157-206 (2007).</li><li>Greer, E. L., Shi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+methylation:+a+dynamic+mark+in+health,+disease+and+inheritance.">Histone methylation: a dynamic mark in health, disease and inheritance.</a> Nature Reviews. Genetics. 13, 343-357 (2012).</li><li>Kouzarides, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+modifications+and+their+function.">Chromatin modifications and their function.</a> Cell. 128, 693-705 (2007).</li><li>Dawson, M. A., Kouzarides, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+epigenetics:+from+mechanism+to+therapy.">Cancer epigenetics: from mechanism to therapy.</a> Cell. 150, 12-27 (2012).</li><li>Clough, E., Tedeschi, T., Hazelrigg, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+regulation+of+oogenesis+and+germ+stem+cell+maintenance+by+the+Drosophila+histone+methyltransferase+Eggless/dSetDB1.">Epigenetic regulation of oogenesis and germ stem cell maintenance by the Drosophila histone methyltransferase Eggless/dSetDB1.</a> Developmental Biology. 388, 181-191 (2014).</li><li>Conrad, T., Akhtar, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dosage+compensation+in+Drosophila+melanogaster:+epigenetic+fine-tuning+of+chromosome-wide+transcription.">Dosage compensation in Drosophila melanogaster: epigenetic fine-tuning of chromosome-wide transcription.</a> Nature Reviews. Genetics. 13, 123-134 (2011).</li><li>Miranda, T. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DZNep+is+a+global+histone+methylation+inhibitor+that+reactivates+developmental+genes+not+silenced+by+DNA+methylation.">DZNep is a global histone methylation inhibitor that reactivates developmental genes not silenced by DNA methylation.</a> Molecular Cancer Therapeutics. 8, 1579-1588 (2009).</li><li>Cui, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PRIMA-1,+a+Mutant+p53+Reactivator,+Restores+the+Sensitivity+of+TP53+Mutant-type+Thyroid+Cancer+Cells+to+the+Histone+Methylation+Inhibitor+3-Deazaneplanocin+A+(DZNep).">PRIMA-1, a Mutant p53 Reactivator, Restores the Sensitivity of TP53 Mutant-type Thyroid Cancer Cells to the Histone Methylation Inhibitor 3-Deazaneplanocin A (DZNep).</a> J. Clin. Endocrinol. Metab. 99 (11), E962 (2014).</li><li>Fujiwara, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3-Deazaneplanocin+A+(DZNep),+an+inhibitor+of+S-adenosyl-methionine-dependent+methyltransferase,+promotes+erythroid+differentiation.">3-Deazaneplanocin A (DZNep), an inhibitor of S-adenosyl-methionine-dependent methyltransferase, promotes erythroid differentiation.</a> The Journal of Biological Chemistry. , (2014).</li><li>Li, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+polycomb+group+protein+EZH2+is+a+novel+therapeutic+target+in+tongue+cancer.">The polycomb group protein EZH2 is a novel therapeutic target in tongue cancer.</a> Oncotarget. 4, 2532-2549 (2013).</li><li>Nakagawa, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+therapy+with+the+histone+methyltransferase+EZH2+inhibitor+3-deazaneplanocin+A+inhibits+the+growth+of+cholangiocarcinoma+cells.">Epigenetic therapy with the histone methyltransferase EZH2 inhibitor 3-deazaneplanocin A inhibits the growth of cholangiocarcinoma cells.</a> Oncology Reports. 31, 983-988 (2014).</li><li>Crea, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacologic+disruption+of+Polycomb+Repressive+Complex+2+inhibits+tumorigenicity+and+tumor+progression+in+prostate+cancer.">Pharmacologic disruption of Polycomb Repressive Complex 2 inhibits tumorigenicity and tumor progression in prostate cancer.</a> Molecular Cancer. 10, 40 (2011).</li><li>Vella, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EZH2+down-regulation+exacerbates+lipid+accumulation+and+inflammation+in+in+vitro+and+in+vivo+NAFLD.">EZH2 down-regulation exacerbates lipid accumulation and inflammation in in vitro and in vivo NAFLD.</a> International Journal of Molecular Sciences. 14, 24154-24168 (2013).</li><li>Tan, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacologic+disruption+of+Polycomb-repressive+complex+2-mediated+gene+repression+selectively+induces+apoptosis+in+cancer+cells.">Pharmacologic disruption of Polycomb-repressive complex 2-mediated gene repression selectively induces apoptosis in cancer cells.</a> Genes &amp; Development. 21, 1050-1063 (2007).</li><li>Chiang, P. K., Cantoni, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perturbation+of+biochemical+transmethylations+by+3-deazaadenosine+in+vivo.">Perturbation of biochemical transmethylations by 3-deazaadenosine in vivo.</a> Biochemical Pharmacology. 28, 1897-1902 (1979).</li><li>Lu, Y. X., Denlinger, D. L., Xu, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polycomb+repressive+complex+2+(PRC2)+protein+ESC+regulates+insect+developmental+timing+by+mediating+H3K27me3+and+activating+prothoracicotropic+hormone+gene+expression.">Polycomb repressive complex 2 (PRC2) protein ESC regulates insect developmental timing by mediating H3K27me3 and activating prothoracicotropic hormone gene expression.</a> The Journal of Biological Chemistry. 288, 23554-23564 (2013).</li></ol>

The authors have nothing to disclose.

有几个步骤，这个协议的成功应用至关重要。对于幼虫检测，应注意正确的标签和复制每个测试浓度。随机化的测试样品和添加药物的指定量的各试验孔中是本实验装置的一个重要部分。之前加入第二龄蚊子幼虫到96孔微量，每个幼虫可以放在纸巾2-3秒，以除去多余的水。为了防止干燥，幼虫必须立即转移到用钝钳子的微孔板。这使水在各微孔板的体积以及与药物的浓度保持为意。当用于...

疟疾是负责的最高数目昆虫有关的死亡，在世界。每年发生大约2.19亿箱子全球，造成约66万死亡病例，主要是在非洲1。尽管共同努力，疟疾项目面临着一些挑战。而杀虫处理的蚊帐和程序室内滞留喷洒形式的关键部件，阻力在当地居民杀虫剂阻碍这些努力2。在杀虫剂抗性蚊子种群的快速增长在很大程度上归因于疟蚊的快速适应变化的环境，并利用不同的壁龛3,4,5的能力。为了克服抗药性的现有机制，新型杀虫剂目标和下一代化合物的探索是必要的。一个简单的，一步一步的协议确定的疟疾米不同人生阶段的实验杀虫剂的功效osquitoes将显著加强这些努力。 对细胞系和动物模型中药物作用的药理学研究已经建立的使用后生药物作为用于调节细胞和生物体的遗传学和生理学的有用工具。 DNA甲基化和组蛋白修饰是影响在多细胞生物体的基因表达不改变基本的DNA序列6 2后生机制。翻译后修饰，例如甲基化在维持细胞完整性和基因表达至关重要的作用，并且可影响几个基本过程7,8,9。研究在一些昆虫物种突出表观遗传学的重要性在涉及卵子发生过程和干细胞维持10，以及剂量补偿11。然而，这些方面在病媒尚待开发。用化合物调节该系统可蚊子为我们提供了在景点融入新颖杀虫剂目标的途径。 3- Deazaneplanocin A（DZNep）是一种已知的组蛋白甲基化抑制剂，其在不同类型的癌症的影响进行了研究12,13,14,15,16。 DZNep是一种稳定的水溶性后生药物间接抑制组蛋白赖氨酸 N- -methyltransferase（EZH2），在哺乳动物细胞中的Polycomb压制性复杂2（PRC2）的一个组件。 PRC2在调节干细胞的多细胞生物的生长中起重要作用，并且组蛋白甲基化是将PRC2介导的基因沉默的一个关键方面。在免疫缺陷小鼠中，细胞预先用DZNep已经显示出少致瘤17。这种药物正在成为用于研究其他疾病，如非酒精性脂肪肝疾病，其中EZH2牵连18。 DZNep是一个既定的S -adenosylhomocysteine（SAH）水解酶抑制剂19，20。的SAH水解导致一个抑制SAH的累积和，反过来，通过限制可用的甲基供体基团导致甲基转移酶活性的抑制。 SAH是由许多生物，包括昆虫，在他们的代谢途径利用的氨基酸衍生物。最近的一项研究表明，DZNep低剂量可能会影响滞育和延缓发展的昆虫21。 这里，一个强大的协议来调查对蚊子的各种生命阶段的水溶性化合物的效果十分发达。这个协议的三个部分，包括:用于检查在未成熟的蚊子，成人吸血雌性和雄性和雌性蚊子的酶活性的水溶性化合物的效果的说明。首先，DZNep溶解在水中以研究未成熟蚊发育和存活。这被执行以两种浓度进行比较，从在药物暴露10倍的增加所引起的任何差异。探索成人的雌蚊，DZNep药物的效果被添加到除颤羊血和供给血液中人为地女性。接着，对生殖力的药物的结果进行检测。最后，使用5,5&#39;- dithiobis-（2-硝基苯甲酸）（DTNB）作为指标来确定DZNep对成年雄性和雌性蚊子SAH水解酶抑制效果进行的酶活性测定。虽然该协议被显影用疟蚊， 冈比亚按蚊 ，它可以容易地适应于学习的任何种类的蚊子或其他昆虫感兴趣化合物的作用。在这个协议中详述的技术可能不被有效地应用到具有在水中或含水介质有限或没有溶解性的药物。

<table><tbody><tr><td>96-well microplate</td><td>Fisher Scientific</td><td>12565561</td><td></td></tr><tr><td>Cell culture plate</td><td>CytoOne</td><td>CC7682-7506</td><td></td></tr><tr><td>Centrifuge</td><td>Sorvall Fresco</td><td>76003758</td><td>A different centrifuge can be used</td></tr><tr><td>Colored tape rolls</td><td>Fisher</td><td>S68134</td><td></td></tr><tr><td>Dissection microscope</td><td>Olympus</td><td>SZ</td><td></td></tr><tr><td>DTNB</td><td>Sigma Aldrich</td><td>D8130</td><td></td></tr><tr><td>DZNep.HCl</td><td>Sigma Aldrich</td><td>SMLO305</td><td></td></tr><tr><td>Egg dish cups</td><td></td><td></td><td></td></tr><tr><td>Filter papers</td><td>Fisher</td><td>09-795E</td><td></td></tr><tr><td>Glass feeders</td><td>Virginia Tech</td><td></td><td></td></tr><tr><td>Glass tissue homogenizer</td><td></td><td></td><td></td></tr><tr><td>Heating element</td><td>Fisher Scientific</td><td>NC0520091</td><td></td></tr><tr><td>Incubator</td><td>Percival scientific</td><td>I36VLC8</td><td>A different incubator can be used</td></tr><tr><td>Microcentrifuge tube, 2&amp;nbsp;ml</td><td>Axygen</td><td>22-283</td><td></td></tr><tr><td>Microcentrifuge tube, 1.5&amp;nbsp;ml</td><td>Axygen</td><td>MCT-150-C</td><td></td></tr><tr><td>Micropipette</td><td>Eppendorf</td><td>4910 000.069</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Fisher Scientific</td><td>M-3154</td><td></td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Fisher Scientific</td><td>M-8643</td><td></td></tr><tr><td>pH meter&amp;nbsp;</td><td>Mettler Toledo 7easy</td><td>S20</td><td></td></tr><tr><td>Plate reader</td><td>Spectramax</td><td>M2</td><td></td></tr><tr><td>SAH</td><td>Sigma Aldrich</td><td>A9384</td><td>store at -20 &amp;#176;C</td></tr><tr><td>Triton X-100</td><td>Sigma Aldrich</td><td>T8787</td><td></td></tr></tbody></table>

toxicological assays for testing effects of an epigenetic drug on development, fecundity and survivorship of malaria mosquitoes

杀虫性带来的疟疾控制规划的一大难题。蚊子适应范围广的变化迅速的环境中，使得疟疾控制一个无所不在的问题，在热带国家。杀虫剂抗性种群的出现值得新型药物靶点的途径和化合物矢量控制蚊子的探索。后生药物确立在癌症研究，但没有多少人知道他们对昆虫的影响。这项研究提供了一个简单的协议，用于检查3 Deazaneplanocin A（DZNep），实验后生药物用于癌症治疗的毒性作用，对疟蚊， 冈比亚按蚊 。在死亡率和降低尺寸的浓度依赖性增加，观察暴露于DZNep未成熟蚊子，而化合物还原成蚊相对于对照治疗的繁殖力。此外，还有S中的药物依赖性降低-腺苷高半胱氨酸（SAH）水解酶的蚊子暴露于DZNep相对于对照治疗活动。这些协议提供研究者用一个简单的，一步一步的程序，以评估多个毒理学终点的实验性药物，反过来，表现出探索的水溶性后生药物或化合物的毒性效应独特的多管齐下的方法利率对蚊子和其他昆虫。

图1是测定的示意图;它描述了各个步骤本文中列出的程序。作为该协议基于蚊不同的生命阶段，没有特定的顺序应遵循此处详述的实验。用户可以选择要进行的一个或多个测定中的同时，这取决于样品的可用性。 图2演示了该板设置为未成熟蚊子发展和存活测定法。测试浓度和重复的数量可以根据用户的要求和药物的可用性而改变。对于未成熟?...

Watch this Scientific Journal Video about Toxicological Assays for Testing Effects of an Epigenetic Drug on Development, Fecundity and Survivorship of Malaria Mosquitoes at JoVE.com

Toxicological Assays for Testing Effects of an Epigenetic Drug on Development, Fecundity and Survivorship of Malaria Mosquitoes

A protocol is developed to examine the effects of an epigenetic drug DZNep on the development, fecundity and survivorship of mosquitoes. Here we describe procedures for the aqueous exposure of DZNep to immature mosquitoes and a blood-based exposure of DZNep to adult mosquitoes in addition to measuring SAH hydrolase inhibition.

毒理学化验一个后生药物对疟蚊的发育，繁殖和存活率测试效果

Insecticidal resistance poses a major problem for malaria control programs. Mosquitoes adapt to a wide range of changes in the environment quickly, making ...

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Research

JoVE Journal

Biology

8.4K 次观看.  Virginia Tech. A protocol is developed to examine the effects of an epigenetic drug DZNep on the development, fecundity and survivorship of mosquitoes. Here we describe procedures for the aqueous exposure of DZNep to immature mosquitoes and a blood-based exposure of DZNep to adult mosquitoes in addition to measuring SAH hydrolase inhibition. 

视频: Toxicological Assays for Testing Effects of an Epigenetic Drug on Development, Fecundity and Survivorship of Malaria Mosquitoes

Transfection and Mutagenesis of Target Genes in Mosquito Cells by Locked Nucleic Acid-modified Oligonucleotides

Plasmodium parasites, the causative agent of malaria, are transmitted through the bites of infected Anopheles mosquitoes resulting in over 250 million new infections each year. Despite decades of research, there is still no vaccine against malaria, highlighting the need for novel control strategies. One innovative approach is the use of genetically modified mosquitoes to effectively control malaria parasite transmission. Deliberate alterations of cell signaling pathways in the mosquito, via targeted mutagenesis, have been found to regulate parasite development 1. From these studies, we can begin to identify potential gene targets for transformation. Targeted mutagenesis has traditionally relied upon the homologous recombination between a target gene and a large DNA molecule. However, the construction and use of such complex DNA molecules for generation of stably transformed cell lines is costly, time consuming and often inefficient. Therefore, a strategy using locked nucleic acid-modified oligonucleotides (LNA-ONs) provides a useful alternative for introducing artificial single nucleotide substitutions into episomal and chromosomal DNA gene targets (reviewed in 2). LNA-ON-mediated targeted mutagenesis has been used to introduce point mutations into genes of interest in cultured cells of both yeast and mice 3,4. We show here that LNA-ONs can be used to introduce a single nucleotide change in a transfected episomal target that results in a switch from blue fluorescent protein (BFP) expression to green fluorescent protein (GFP) expression in both Anopheles gambiae and Anopheles stephensi cells. This conversion demonstrates for the first time that effective mutagenesis of target genes in mosquito cells can be mediated by LNA-ONs and suggests that this technique may be applicable to mutagenesis of chromosomal targets in vitro and in vivo.

Oligonucleotides can be used to site specifically substitute a single nucleotide of transfected target genes in both Anopheles gambiae and Anopheles stephensi cells.

酸修饰的寡核苷酸锁定核酸转染和蚊虫细胞的靶基因突变

Plasmodium parasites, the causative agent of malaria, are transmitted through the bites of infected Anopheles mosquitoes resulting in over 250 million new ...

科研

免疫与感染

Protocol for Production of a Genetic Cross of the Rodent Malaria Parasites

Variation in response to antimalarial drugs and in pathogenicity of malaria parasites is of biologic and medical importance. Linkage mapping has led to successful identification of genes or loci underlying various traits in malaria parasites of rodents1-3 and humans4-6. The malaria parasite Plasmodium yoelii is one of many malaria species isolated from wild African rodents and has been adapted to grow in laboratories. This species reproduces many of the biologic characteristics of the human malaria parasites; genetic markers such as microsatellite and amplified fragment length polymorphism (AFLP) markers have also been developed for the parasite7-9. Thus, genetic studies in rodent malaria parasites can be performed to complement research on Plasmodium falciparum. Here, we demonstrate the techniques for producing a genetic cross in P. yoelii that were first pioneered by Drs. David Walliker, Richard Carter, and colleagues at the University of Edinburgh10.

Genetic crosses in P. yoelii and other rodent malaria parasites are conducted by infecting mice Mus musculus with an inoculum containing gametocytes of two genetically distinct clones that differ in phenotypes of interest and by allowing mosquitoes to feed on the infected mice 4 days after infection. The presence of male and female gametocytes in the mouse blood is microscopically confirmed before feeding. Within 48 hrs after feeding, in the midgut of the mosquito, the haploid gametocytes differentiate into male and female gametes, fertilize, and form a diploid zygote (Fig. 1). During development of a zygote into an ookinete, meiosis appears to occur11. If the zygote is derived through cross-fertilization between gametes of the two genetically distinct parasites, genetic exchanges (chromosomal reassortment and cross-overs between the non-sister chromatids of a pair of homologous chromosomes; Fig. 2) may occur, resulting in recombination of genetic material at homologous loci. Each zygote undergoes two successive nuclear divisions, leading to four haploid nuclei. An ookinete further develops into an oocyst. Once the oocyst matures, thousands of sporozoites (the progeny of the cross) are formed and released into mosquito hemoceal. Sporozoites are harvested from the salivary glands and injected into a new murine host, where pre-erythrocytic and erythrocytic stage development takes place. Erythrocytic forms are cloned and classified with regard to the characters distinguishing the parental lines prior to genetic linkage mapping. Control infections of individual parental clones are performed in the same way as the production of a genetic cross.

Genetic crosses of rodent malaria parasites are performed by feeding two genetically distinct parasites to mosquitoes. Recombinant progeny are cloned from mouse blood after allowing mosquitoes to bite infected mice. This video shows how to produce genetic crosses of Plasmodium yoelii and is applicable to other rodent malaria parasites.

生产的灭鼠疟疾寄生虫的遗传交叉议定书“

Variation in response to antimalarial drugs and in pathogenicity of malaria parasites is of biologic and medical importance. Linkage mapping has led to ...

Building a Better Mosquito: Identifying the Genes Enabling Malaria and Dengue Fever Resistance in A. gambiae and A. aegypti Mosquitoes

In this interview, George Dimopoulos focuses on the physiological mechanisms used by mosquitoes to combat Plasmodium falciparum and dengue virus infections.   Explanation is given for how key refractory genes, those genes conferring resistance to vector pathogens, are identified in the mosquito and how this knowledge can be used to generate transgenic mosquitoes that are unable to carry the malaria parasite or dengue virus.

In this interview, George Dimopoulos focuses on the physiological mechanisms used by mosquitoes to combat Plasmodium falciparum and dengue virus infections. Explanation is given for how key refractory genes, those genes conferring resistance to vector pathogens, are identified in the mosquito and how this knowledge can be used to generate transgenic mosquitoes that are unable to carry the malaria parasite or dengue virus.

建设一个更美好的蚊子：确定基因在冈比亚按蚊和埃及斑蚊蚊子启用疟疾和登革热的电阻

In this interview, George Dimopoulos focuses on the physiological mechanisms used by mosquitoes to combat Plasmodium falciparum and dengue virus ...

生物学

CRISPR/Cas9 Gene Editing to Make Conditional Mutants of Human Malaria Parasite P. falciparum

Malaria is a significant cause of morbidity and mortality worldwide. This disease, which primarily affects those living in tropical and subtropical regions, is caused by infection with Plasmodium parasites. The development of more effective drugs to combat malaria can be accelerated by improving our understanding of the biology of this complex parasite. Genetic manipulation of these parasites is key to understanding their biology; however, historically the genome of P. falciparum has been difficult to manipulate. Recently, CRISPR/Cas9 genome editing has been utilized in malaria parasites, allowing for easier protein tagging, generation of conditional protein knockdowns, and deletion of genes. CRISPR/Cas9 genome editing has proven to be a powerful tool for advancing the field of malaria research. Here, we describe a CRISPR/Cas9 method for generating glmS-based conditional knockdown mutants in P. falciparum. This method is highly adaptable to other types of genetic manipulations, including protein tagging and gene knockouts.

CRISPR/Cas9 Gene Editing to Make Conditional Mutants of Human Malaria Parasite P. falciparum

We describe a method for generating glmS-based conditional knockdown mutants in Plasmodium falciparum using CRISPR/Cas9 genome editing.

CRISPR/Cas9 基因编辑制作人类疟疾寄生虫的条件突变体

Malaria is a significant cause of morbidity and mortality worldwide. This disease, which primarily affects those living in tropical and subtropical ...

遗传学

Phenotypic Analysis of Rodent Malaria Parasite Asexual and Sexual Blood Stages and Mosquito Stages

Recent advances in genetics and systems biology technologies have promoted our understanding of the biology of malaria parasites on the molecular level. However, effective malaria parasite targets for vaccine and chemotherapy development are still limited. This is largely due to the unavailability of relevant and practical in vivo infection models for human Plasmodium species, most notably for P.&#160;falciparum and P. vivax. Therefore, rodent malaria species have been extensively used as practical alternative in vivo models for malaria vaccine, drug targeting, immune response, and functional characterization studies of conserved Plasmodiumspp. genes. Indeed, rodent malaria models have proven to be invaluable, especially for exploring mosquito transmission and liver stage biology, and were indispensable for immunological studies. However, there are discrepancies in the methods used to evaluate the phenotypes of transgenic and wild-type asexual and sexual blood-stage parasites.&#160;Examples of these discrepancies are the choice of an intravenous vs. intraperitoneal infection of rodents with blood-stage parasites and the evaluation of male gamete exflagellation. Herein, we detail standardized experimental methods to evaluate the phenotypes of asexual and sexual blood stages in transgenic parasites expressing reporter-gene or wild-type rodent malaria parasite species. We also detail the methods to evaluate the phenotypes of malaria parasite mosquito stages (gametes, ookinetes, oocysts, and sporozoites) inside Anopheles mosquito vectors. These methods are detailed and simplified here for the lethal and non-lethal strains of P. berghei and P. yoelii but can also be applied with some adjustments to P. chabaudi and P. vinckei rodent malaria species.

Due to the striking similarities of the life cycle and biology of rodent malaria parasites to human malaria parasites, rodent malaria models have become indispensable for malaria research. Herein, we standardized some of the most important techniques used in the phenotypic analysis of wild-type and transgenic rodent malaria species.

啮齿动物疟原虫无性、性血期和蚊子阶段的类状分析

Recent advances in genetics and systems biology technologies have promoted our understanding of the biology of malaria parasites on the molecular level. ...

Standard Membrane Feeding Assay for the Detection of Plasmodium falciparum Infection in Anopheles Mosquito Vectors

Malaria remains one of the most devastating diseases worldwide and, to date, the African region is still responsible for 94% of all cases worldwide. This parasitic disease requires a protozoan parasite, an Anopheles mosquito vector, and a vertebrate host. The Anopheles genus comprises more than 500 species, of which 60 are known as vectors of the parasite. The Plasmodium parasite genus consists of 250 species, and 48 of these are involved in disease transmission. Furthermore, the Plasmodium falciparum parasite has contributed toward an estimated 99.7% of malaria cases in sub-Saharan Africa in recent years. 
Gametocytes form part of the sexual stage of the parasite and are ingested by the female mosquito upon feeding on an infected human host. Further development of the parasite within the mosquito is enhanced by favorable environmental conditions in the midgut of the mosquito. Here, the fusion of the female and male gametes takes place, and the motile ookinetes originate. The ookinetes enter the midgut epithelium of the mosquito, and mature ookinetes form oocysts, which, in turn, produce motile sporozoites. These sporozoites migrate to the mosquito's salivary glands and are injected as a mosquito takes a blood meal. 
For drug discovery purposes, mosquitoes were artificially infected with gametocyte-infected blood in the standard membrane feeding assay (SMFA). To detect infection within the mosquito and/or to assess the efficacy of antimalarial compounds, the midguts of the female mosquitoes were removed post infection and were stained with mercurochrome. This method was used to enhance the visual detection of oocysts under the microscope for the accurate determination of oocyst prevalence and intensity.

Standard Membrane Feeding Assay for the Detection of Plasmodium falciparum Infection in Anopheles Mosquito Vectors

The standard membrane feeding assay (SMFA) is regarded as the gold standard for the assessment and identification of potential antimalarial compounds. This artificial feeding system is used to infect mosquitoes to further evaluate the effects of such compounds on the intensity and prevalence of the Plasmodium falciparum parasite.

用于检测按蚊媒介中恶性疟原虫感染的标准膜喂养测定

Malaria remains one of the most devastating diseases worldwide and, to date, the African region is still responsible for 94% of all cases worldwide. This ...

荧光 原位 杂交研究蚊子精子发生

Whole-Mount Fluorescence In Situ Hybridization to Study Spermatogenesis in the Anopheles Mosquito

Spermatogenesis is a complex biological process during which diploid cells undergo successive mitotic and meiotic division followed by large structural changes to form haploid spermatozoa. Besides the biological aspect, studying spermatogenesis is of paramount importance for understanding and developing genetic technologies such as gene drive and synthetic sex ratio distorters, which, by altering Mendelian inheritance and the sperm sex ratio, respectively, could be used to control pest insect populations. These technologies have proven to be very promising in lab settings and could potentially be used to control wild populations of Anopheles mosquitoes, which are vectors of malaria. Due to the simplicity of the testis anatomy and their medical importance, Anopheles gambiae, a major malaria vector in sub-Saharan Africa, represents a good cytological model for studying spermatogenesis. This protocol describes how whole-mount fluorescence in situ hybridization (WFISH) can be used to study the dramatic changes in cell nuclear structure through spermatogenesis using fluorescent probes that specifically stain the X and Y chromosomes. FISH usually requires the disruption of the reproductive organs to expose mitotic or meiotic chromosomes and allow the staining of specific genomic regions with fluorescent probes. WFISH enables the preservation of the native cytological structure of the testis, coupled with a good level of signal detection from fluorescent probes targeting repetitive DNA sequences. This allows researchers to follow changes in the chromosomal behavior of cells undergoing meiosis along the structure of the organ, where each phase of the process can clearly be distinguished. This technique could be particularly useful for studying chromosome meiotic pairing and investigating the cytological phenotypes associated with, for example, synthetic sex ratio distorters, hybrid male sterility, and the knock-out of genes involved in spermatogenesis.

作者聚焦:全安装荧光原位杂交研究按蚊精子发生  

Given their simple anatomy, Anopheles testes offer a good cytological model for studying spermatogenesis. This protocol describes whole-mount fluorescence in situ hybridization, a technique used to investigate this biological process, as well as the phenotype of transgenic strains harboring mutations in the genes involved in sperm production.

全安装荧光 原 位杂交研究 按 蚊精子发生

Spermatogenesis is a complex biological process during which diploid cells undergo successive mitotic and meiotic division followed by large structural ...

Protocols for Testing the Toxicity of Novel Insecticidal Chemistries to Mosquitoes

New classes of insecticides with novel modes of action are needed to control insecticide resistant populations of mosquitoes that transmit diseases such as Zika, dengue and malaria. Assays for rapid, high-throughput analyses of unformulated novel chemistries against mosquito larvae and adults are presented. We describe protocols for single point-dose and dose response assays to evaluate the toxicity of small molecule chemistries to the Aedes aegypti vector of Zika, dengue and yellow fever,&#160;the malaria vector, Anopheles gambiae and the northern house mosquito, Culex quinquefasciatus, on contact and via ingestion. As an example, we evaluated the toxicity of amitriptyline, a small molecule antagonist of G protein-coupled receptors, via larval, adult topical and adult blood-feeding assay. The protocols provide a starting point to investigate insecticide potential. Results are discussed in the context of additional experiments to explore product applications and mechanisms for delivery.

Protocols are described for assessing the toxicity of chemistries to immature and adult mosquitoes for development as new classes of larvicides, adulticides and endectocides. The protocols enable high-throughput testing of multiple chemistries at single-point dose and subsequent evaluation via dose response assay to determine toxicity on contact or via ingestion.

新型杀虫化学对蚊子毒性测试规程

New classes of insecticides with novel modes of action are needed to control insecticide resistant populations of mosquitoes that transmit diseases such ...

毒理学化验一个后生药物对疟蚊的发育，繁殖和存活率测试效果

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酸修饰的寡核苷酸锁定核酸转染和蚊虫细胞的靶基因突变

生产的灭鼠疟疾寄生虫的遗传交叉议定书“

建设一个更美好的蚊子：确定基因在冈比亚按蚊和埃及斑蚊蚊子启用疟疾和登革热的电阻

CRISPR/Cas9 基因编辑制作人类疟疾寄生虫的条件突变体

啮齿动物疟原虫无性、性血期和蚊子阶段的类状分析

用于检测按蚊媒介中恶性疟原虫感染的标准膜喂养测定

全安装荧光原位杂交研究按蚊精子发生

全安装荧光原位杂交研究按蚊精子发生

全安装荧光原位杂交研究按蚊精子发生

新型杀虫化学对蚊子毒性测试规程

毒理学化验一个后生药物对疟蚊的发育，繁殖和存活率测试效果

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