Name: functional characterization of carboxylesterases in insecticide resistant house flies, musca domestica
Uploaded: 2018-08-23T14:30:00.000+00:00
Duration: 8 min 17 s
Description: Watch this Scientific Journal Video about Functional Characterization of Carboxylesterases in Insecticide Resistant House Flies, Musca Domestica at JoVE.com

1. 用杆状病毒介导的昆虫细胞表达系统表达和分离靶蛋白
<ol><li>定向克隆的钝端 PCR 产品的目标蛋白质从房子苍蝇。<ol>
<li>设计了绿色荧光蛋白 (GFP) 和 MdαE7 基因的 PCR 引物, 并根据其序列和选择向量的特殊要求 (表 1)。</li>
		<li>使用耐热, 校对 dna 聚合酶和引物从步1.1.1 执行150µL PCR 反应 (30 µL 反应缓冲器, 3 µL 10 毫米 dNTPs, 1.5 µL dna 聚合酶, 6 µL 的房子飞行模板 DNA, 7.5 µL 向前底漆, 7.5 µL 的反向底漆, 与水的最终体积150µL)。热 PCR 反应到98°c 三十年代, 其次是98°c 的35个周期为十年代, 53 °c 为三十年代和72°c 为六十年代, 然后最后的引伸在72°c 为2分钟)。<ol>
<li>运行1% 琼脂糖凝胶与150µL PCR 产品。</li>
		</ol>
</li>
		<li>用锋利、干净的手术刀将目标 DNA 片段从琼脂糖凝胶中清除: 1317 bp 用于 MdαE7 和 858 bp 用于 GFP。根据制造商的协议 (材料表), 使用商业上可用的凝胶萃取试剂盒净化 DNA。将纯化的 DNA 溶解在15µL 的蒸馏水中。</li>
		<li>运行1% 琼脂糖凝胶与1µL 的纯化 DNA, 以检查完整性。使用另外1µL 的纯化 DNA 测量分光光度计的浓度。</li>
	</ol>
</li>
	<li>构建靶蛋白的入门质粒<ol>
<li>建立克隆反应。混合新鲜纯化的 DNA 产品从步1.1.3 和商业上可利用的入口载体包含的 L 站点 (材料表) 以摩尔比率 1:1 (0.5-2 µL 新鲜 PCR 产品在 70-200 ng 或µL 浓度: 0.5 µL 传染媒介)。然后添加1µL 的商业可用盐溶液 (1.2 米氯化钠2和0.06 米氯化镁2), 并增加水的最后一卷6µL. 在室温下轻轻混合, 并孵育1小时。</li>
		<li>在步骤1.2.1 中转移4µL 克隆反应产物, 转化为50µL 化学能力的大肠杆菌细胞。在冰上孵育30分钟热休克细胞三十年代在42°c 水浴没有震动。</li>
		<li>把管子放回冰中再加2分钟. 添加250µL 室温 S.O.C. 培养基 (2% 胰蛋白胨; 0.5% 酵母萃取物; 10 毫米氯化钠; 2.5 毫米氯化钾; 10 毫米氯化镁2; 10 毫米 MgSO4; 20 mm 葡萄糖)。37摄氏度孵育1小时, 轻轻摇动。</li>
		<li>在选择性 LB 板 (1 克的胰蛋白胨; 1 克的氯化钠; 0.5 克的酵母萃取物中, 50-200 µL 细菌培养的 1.2.3; 1.5 克琼脂溶解在100毫升蒸馏水中。蒸压釜和添加 0.1% 50 毫克/毫升卡那霉素)。孵化 LB 板16小时在37°c 为殖民地生长。</li>
		<li>选取5-10 个菌落, 将它们分别重新悬浮到5µL 的蒸馏水中。</li>
		<li>通过添加5µL 反应缓冲剂进行 PCR, 0.5 µL 10 毫米 dNTPs, 0.25 µL 的脱氧核糖核酸聚合酶, 1 µL 悬浮的殖民地, 1.25 µL µM 向前底漆, 10 M13 1.25 µL 反向底漆的目标基因, 和水到最后容量10µM. 热 PCR反应到98°c 三十年代, 其次是35个周期 98° C 为十年代, 53 °c 为三十年代和72°c 为六十年代, 然后最后的引伸在72°c 为2分钟 (桌 1)。</li>
		<li>再培养3µL 悬浮的殖民地从结核媒介的步 1.2.5 (100 毫升磷酸盐缓冲包含 0.17 m 的2PO4和 0.72 m K2HPO4与450毫升基本的肉汤培养基包含 6 g 胰蛋白胨, 12 g 酵母和2毫升甘油, 灭菌, 含 0.1% 50 毫克/毫升卡那霉素) 为 16 h. 提取超纯质粒 DNA 后, 制造商的协议。</li>
		<li>使用200µL 超纯质粒从步进1.2.7 为商业的序列与 M13 正向和反向引物。根据制造商提供的序列图验证目标基因在矢量中的正确插入。</li>
		<li>将 MdαE7 和 GFP 的序列验证的超纯质粒 DNA 样品贮存在-20 摄氏度。 注: 这些是 MdαE7 和 GFP 的进入质粒 DNA。</li>
	</ol>
</li>
	<li>通过执行 Lambda 重组 (LR) 反应构建重组杆状病毒。<ol>
<li>分别将1µL 300 ng/µL 进入质粒 dna 的 GFP, MdαE7 从步进1.2.7 或入口质粒 dna 的氯霉素乙酰转移酶基因 (CAT) 提供的细胞转染试剂盒和5µL 的商业可用(含 L 的网站)250µL PCR 管中的 C 期线性 DNA (包含的 R 点)。添加 TE 缓冲区, 使总容量为8µL。 注意: GFP 和猫的反应被用作对照组。</li>
		<li>添加2µL 的商业可用的 Lambda 重组 (LR) 酶混合物 (物质表) 到每一个混合物的步骤 1.3.1, 以催化 LR 反应。轻轻地混合和孵化在25°c 过夜 (≈16 h)。 注意: LR 反应促进了一项含有含 R 的 C 期线性 dna 的 l-含L 型进入质粒 dna 的重组, 从而产生一种含 B 的表达病毒. 这一步为每个靶蛋白生成重组杆状病毒。</li>
		<li>从步骤 1.3.2 20X 稀释1µL 的 LR 反应产物。使用多角体前底漆和 V5 反向底漆进行 PCR (表 1)。使用5µL PCR 产品运行1% 琼脂糖凝胶检查的质量。图 1显示了 MdαE7 基因的 LR 反应产物的例子。</li>
	</ol>
</li>
</ol><img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58106/58106fig1.jpg"> 图 1: PCR 分析 MdαE7 LR 反应的示例结果.稀释2µL 的 LR 反应200倍, 并使用2µL 稀释连同多角体前底漆和 V5 反向底漆执行25µL PCR。使用5µL PCR 产品运行1% 琼脂糖凝胶, 以检查 LR 反应的质量。一个
<ol start="4"><li>染昆虫 Sf9 细胞<ol>
<li>培养昆虫 Sf9 细胞与5毫升完全细胞生长培养基 (无血清培养基和10% 胎牛血清 (血清)) 在 T25 治疗烧瓶27˚C。</li>
		<li>用3毫升新鲜完整细胞生长培养基去除上清和冲洗底附着细胞。将0.5 毫升的重悬浮细胞转移到一个新的 T25 治疗瓶中。加入4.5 毫升的完全生长培养基。孵育27°c 3-4 天, 直到下一个转移。</li>
		<li>种子2毫升的对数相生长昆虫 Sf9 细胞培养 (≈3.0-5.0×106细胞) 均匀地在细胞培养上。允许单元格在引擎盖的室温下附加至少3小时。</li>
		<li>检查细胞附件通过观察与倒置相显微镜在250X。移除细胞培养培养基, 并用2毫升的宽限期昆虫培养基代替。</li>
		<li>准备转染混合物 (8 µL 的商业可用细胞感染试剂 (材料表) 与100µL 的恩典的昆虫培养基) 和转染混合物 B 溶液的每个目标基因 (9 µL 的 LR 反应产品从步骤1。3与100µL unsupplemented 宽的昆虫培养基) 在1.5 毫升离心管, 分别。</li>
		<li>轻轻地混合转染混合物 A 和 B 一起通过攻管。在室温下孵育35分钟的引擎盖。</li>
		<li>均匀地添加从步骤1.4.6 滴到种子细胞从步骤1.4.4 的混合物。封井与磁带和孵化在27°c 过夜。</li>
		<li>用2毫升完全生长培养基取代2毫升的宽限期昆虫培养基。添加100µM 更昔洛韦到每个井对非重组杆状病毒阴性选择。用胶带封井, 在27°c 处孵育72小时。</li>
		<li>从各井收集 72 h 后感染细胞培养培养基, 转移至1.5 毫升离心管。离心机在 1500 x g 为5分钟在4°c 去除细胞或大残骸。</li>
		<li>将上清液转化为新的1.5 毫升离心管。储存在4摄氏度与保护从光。 注意: 这些是针对每个目标基因的 P1 病毒库存解决方案。</li>
		<li>将低效 P1 病毒性股 (1×105-1×106 pfu/毫升) 放大至高滴度 P2 病毒性股 (5×107-1×108 pfu/毫升)。</li>
		<li>种子2毫升的对数相生长昆虫 Sf9 细胞培养 (≈3.0-5.0×106细胞) 均匀地在细胞培养上。允许单元格在引擎盖的室温下附加至少3小时。</li>
		<li>接种5µL 的 P1 病毒, 分别在步进1.4.12 的细胞播种井中获得1.4.10。然后, 增加100µM 更昔洛韦对每个井。密封井和孵育在27°c 为72小时。</li>
		<li>收集 P2 病毒储存液72小时后感染。存放在4°c 与保护从光。 注意: 这些是针对每个目标基因的 P2 病毒库存解决方案。</li>
		<li>选重复步骤 1.4. 12-1. 4.14 与5µL P2 病毒库存收集 P3 病毒库存解决方案。 注意: 这些是针对每个目标基因的 P3 病毒库存解决方案。</li>
		<li>存储所有构造的杆状病毒在4°c 与保护免受光照。 注: GFP 和猫基因作为对照组。图 2显示了细胞在不同放大阶段被 GFP-重组杆状病毒感染的迹象。</li>
	</ol>
</li>
</ol><img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58106/58106fig2.jpg"> 图 2: 不同杆状病毒扩增阶段 Sf9 细胞感染迹象的例子.该图显示了 GFP-重组杆状病毒细胞在自然光和荧光光。在 P1 感染期, 感染率低。在 P2 感染期, 感染率明显提高。在 P3 感染阶段, 几乎所有的细胞都表现出从细胞培养板脱落的症状, 细胞直径的增加, 以及细胞生长的停止。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58106/58106fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<ol start="5"><li>昆虫 Sf9 细胞中靶蛋白的大尺度表达<ol>
<li>培养10毫升的记录相生长昆虫 Sf9 细胞与完全生长培养基在 T25 不处理烧瓶。</li>
		<li>添加200µL P2 病毒库存解决方案 (在步骤1.4.14 中获得), 以在步骤1.5.1 中感染细胞培养72小时。</li>
		<li>收集 72 h 后感染细胞培养基。离心机在 1500 x g 5 分钟在4摄氏度。</li>
		<li>用1毫升0.1 米的 PBS 缓冲器 (pH 7.5) 丢弃上清和洗两次颗粒。</li>
		<li>用1毫升的昆虫细胞蛋白萃取 & 裂解缓冲 (材料表) 完全溶解细胞颗粒。在细胞裂解中加入10% 甘油。立即存储在-80 摄氏度。</li>
		<li>重复步骤 1.5. 1-1. 5.5 与 P2 病毒库存解决方案的猫蛋白作为对照组。 注: 对不同的蛋白质制剂重复步骤1.5。</li>
	</ol>
</li>
</ol>2. 以细胞为基础的 MTT 法细胞毒性试验
<ol><li>培养5毫升的对数相生长 (1.5-2.5×106细胞/毫升) 昆虫 Sf9 细胞与完全生长培养基在 T25 未处理的烧瓶。</li>
	<li>添加25µL 的 P1 病毒库存解决方案 (获得在步骤 1.4.14), 以感染48小时的步骤2.1 的细胞培养, 在27˚C 的温和晃动。</li>
	<li>在乙腈中制备100毫米菊酯标准库存溶液。使用乙腈稀释到50毫米, 25 毫米, 12.5 毫米和6.25 毫米, 通过添加500µL, 250 µL, 125 µL 和62.5 µL 氯菊酯库存解决方案, 在总容量1000µL, 分别。</li>
	<li>从步骤2.2 中将受感染细胞培养物的200µL 均匀地辅以300µL 的完全生长培养基, 在 2×105细胞/毫升的密度下加入24井板。</li>
	<li>添加4µL 氯菊酯标准溶液 (6.25 毫米, 12.5 毫米, 25 毫米和50毫米) 到每个井, 使最终浓度50µM, 100 µM, 200 µM 和400µM, 分别。仅将4µL 乙腈添加到井中进行细胞生存能力计算 (图 3)。</li>
	<li>带胶带的密封板, 在27摄氏度处孵化48小时, 保护免受光照。</li>
	<li>准备5毫克/毫升 MTT (噻唑蓝四氮唑溴) 试剂溶解在缓冲器中 (2.0 克氯化钠, 0.05 克氯化钾, 0.36 克 NaH2po4∙2H2O, 0.28 克 NaH2po4, 0.05 g2PO4 溶解在200毫升蒸馏水, pH 值 7.5)。</li>
	<li>在48小时孵化后从步骤2.6 移除细胞培养培养基, 而不接触底附着细胞层。在步骤2.7 中添加200µL 的 MTT 试剂到每个井中。孵育在37°c 为4小时直到深紫色 formazan 沉淀形成在每个井 (图 3)。</li>
	<li>孵育在37°c 为4小时直到深紫色 formazan 沉淀形成在每个井。添加500µL 的亚砜完全溶解沉淀 (图 3)。</li>
</ol><img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58106/58106fig3.jpg"> 图 3: MTT 结果的示例.在 48 h 后感染 P1 病毒库存解决方案的猫重组病毒的解决方案, 均匀种子500µL 细胞表达猫基因的24井细胞培养板。分别添加4µL 氯菊酯, 在不同剂量 (6.25 毫米, 12.5 毫米, 25 毫米和50毫米) 在每行, 使最终浓度为50µM, 100 µM, 200 µM 和400µM。控制行只用乙腈处理, 并在盘中标记为 CK。(A) 在27摄氏度孵化48小时后, 丢弃上层的细胞培养基, 并用200µL 的黄色 MTT 试剂代替。然后孵育在37°c 为 48 h. 深紫彩色还原沉淀形式在每个井表明, 存活细胞能够代谢黄色的 MTT 试剂, 以深紫色的彩色沉淀。(B) 在每个井中加入二甲基亚砜溶剂的500µL, 以溶解形成的沉淀。用 540 nm 分光光度法测定了各井的吸光度值。随着氯菊酯浓度的增加, 颜色将逐渐从暗红色变为浅红色, 表明细胞可行性逐渐减少。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58106/58106fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<ol start="10"><li>将200µL 溶解溶液转化为96井板。使用微板块读取器 (材料表) 测量 540 nm 的吸光度值。</li>
	<li>通过比较氯菊酯处理细胞与仅乙腈处理细胞的吸光度值, 计算细胞活力。</li>
	<li>对于对照组, 请重复步骤1.4 中获得的氯霉素乙酰转移酶 (CAT) 重组杆状病毒 P2 的所有步骤。</li>
	<li>重复4次不同的病毒制剂。</li>
</ol>3.体外代谢测定
<ol><li>在乙腈中制备100毫米菊酯标准库存溶液。使用乙腈稀释到50毫米, 25 毫米, 12.5 毫米和6.25 毫米。用 HPLC 法测定各浓度下的相应峰值面积。根据不同氯菊酯浓度的峰值面积, 建立和计算氯菊酯的标准曲线。</li>
	<li>测量步骤1.5 中的蛋白质浓度与布拉德福德方法21。</li>
	<li>制备700µL 代谢反应与40µM 氯菊酯标准和1毫克的蛋白质在步骤1.5 溶解在0.2 米三盐酸缓冲 (pH 7.4)。在30摄氏度孵育2小时, 轻轻摇动。保护光线。</li>
	<li>加入700µL 冷乙腈, 淬火反应。在30˚C 中孵育30分钟, 轻轻摇动。保护光线。</li>
	<li>在室温下, 离心反应混合物在 1.6万 x g 处为2分钟。收集上清和过滤通过0.45 µm 膜。将过滤液转移到 ultraclean 棕色玻璃瓶中进行 HPLC 分析。</li>
	<li>在最佳色谱条件下运行高效液相色谱 (流动相 A: 90% 乙腈和10% 水;移动相 B: 5% 乙腈调整到 pH 2.3 与85% 磷酸)。梯度洗脱的流速为1毫升/分钟, 测量波长为 232 nm。</li>
	<li>通过与不添加蛋白质样品的反应进行比较, 计算氯菊酯的消耗率。</li>
	<li>采用步进1.5.6 中获得的猫蛋白作为控制。</li>
	<li>在步骤1.5.7 中重复使用不同蛋白质制剂的所有步骤。</li>
</ol>

<ol>
	<li>Scott, J. 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近几十年来, 异基因表达系统被广泛用于表达和分离大量的蛋白质, 从而允许生化和功能的测定和酶的体外鉴定。到目前为止, 几种不同的模型系统, 包括大肠杆菌,毕赤酵母, Sacccharomyces 酵母和斜纹夜蛾 frugiperda已被改编为重组蛋白表达, 并选择体外系统是重要的大规模产生感兴趣的蛋白质22,23,24,25。在本研究中, 选择了杆状病毒介导的昆虫细胞表达系统来生成 carboxylesterases 蝇, 因为它为昆虫细胞提供了相似的细胞内环境, 并保持了一些重要的真核处理能力。9. 尽管杆状病毒介导的表达系统具有优势, 但也应解决其局限性。杆状病毒介导的昆虫细胞表达系统只有在昆虫细胞被构造重组杆状病毒感染的情况下才能产生靶蛋白。目前建立稳定转化的昆虫细胞系的发展, 不仅使组成性在杆状病毒感染的情况下产生感兴趣的蛋白质, 而且能提高 glycosylating 和折叠的细胞能力。通过工程修改产生的蛋白质9。
为了更好地研究 carboxylesterases 在细胞内代谢杀虫剂中的作用, 从而保护细胞免受化学损伤, mtt 法对不同氯菊酯治疗的细胞毒性进行了测定。在此过程中, 许多与细胞代谢相关的实验参数, 如细胞培养基组成、细胞生长速率、能量供应代谢物的浓度和消耗等, 都会影响 MTT 的降低, 从而导致不准确的结果。为了最大限度地减少 mtt 试验的过度/低估, 保持细胞培养基、细胞生长状态和不同实验治疗前后的培养条件一致26。为了确定杆状病毒感染而非氯菊酯对细胞活力的影响, 我们发现 GFP-重组杆状病毒对昆虫 Sf9 细胞的感染过程, 在 P2 感染阶段, 细胞具有最高感染率与其他感染阶段相比, 细胞死亡率最低 (图 2)。这可以更好地解释在 P2 感染阶段选择细胞进行 MTT 试验的原因。
通过对 MdαE7 和猫重组杆状病毒在 MTT 研究中感染细胞可行性的比较, 我们发现细胞在表达 MdαE7 蛋白时有增强的生存能力, 可以更好地支持 carboxylesterases 对氯菊酯的代谢作用。在昆虫细胞和保护细胞免受化学损伤 (图 4)。体外代谢研究被认为是反映 carboxylesterases 对杀虫剂代谢能力的直接方法, 也应探讨 MTT 法在更好表征蛋白质中的局限性。代谢功能。
本研究以杆状病毒介导的表达系统 carboxylesterases 在昆虫 Sf9 细胞中的异种表达, 用 MTT 法测定不同氯菊酯的细胞可行性, 并表征carboxylesterases 通过体外代谢试验, 共同为研究昆虫解毒酶的作用提供系统、科学、准确的策略, 从而有助于更好地了解杀虫剂抵抗机制, 从而制定害虫管理的创新战略。

杀虫剂抗药性目前是一个主要问题的房子飞行控制全球1,2。确定杀虫剂抗药性机制的努力有助于更好地了解这一问题, 从而提供了新的战略, 有效地防止或尽量减少抗药性发展3的蔓延。Carboxylesterases 作为一种主要的解毒酶, 在各种昆虫4、5、6中对杀虫剂的螯合和代谢中的作用引起了人们的极大关注。我们以前的研究已经确定了多 carboxylesterases 的房子苍蝇和他们的表达水平不仅组成性在抗 ALHF 菌株, 但也可以诱导到较高水平的反应氯菊酯治疗7.然而, 这些羧酸基因在代谢杀虫剂中的功能特征仍有待探讨。
自从8二十世纪八十年代初的第一次报告以来, 由于其高蛋白的生产效率和真核蛋白处理能力9, 一种杆状病毒介导的国外基因表达系统得到了广泛的应用。该二元体系由两个基本要素组成: 构建的重组杆状病毒将外来基因传递给宿主细胞, 以及由重组杆状病毒感染的细胞大规模表达感兴趣的蛋白质。在过去的几十年中, 杆状病毒介导的细胞表达系统已被广泛用于生产数以千计的重组蛋白, 从胞浆酶到膜结合蛋白的昆虫和哺乳动物细胞10。我们以前的研究成功地表达了昆虫 Sf9 细胞中的多种 CYP450 酶, 这个系统11。在本研究中, 我们构建了羧酸重组杆状病毒感染昆虫 Sf9 细胞, 探讨了细胞对不同氯菊酯治疗的耐受性, 以及大尺度表达的羧酸蛋白在体外功能探索。而不是从昆虫组织匀浆的多种羧酸同工酶混合物的研究12,13, 这种杆状病毒介导的昆虫细胞表达系统允许特定的表达和分离靶蛋白, 更好地表征其生物化学和结构特性。
四氮唑盐基检测 (MTT) 是一种高通量比色法, 用于测量细胞活力。这一分析的基础上, 只有活细胞能够代谢黄色的 MTT 试剂到深紫色的彩色 formazan 沉淀, 可以 colorimetrically 分析后溶解在有机溶剂14, 15。近年来, 一些比较准确但耗时的方法, 如台盼蓝排斥和胸苷滴定法16,17。然而, 基于细胞的 mtt 检测目前仍然被认为是最快速和最容易操作的方法, 快速检测细胞的生存能力。在这里, 我们使用 MTT 法来探讨细胞对杀虫剂治疗的耐受性。羧酸重组杆状病毒感染后细胞的增强耐受性强烈支持 carboxylesterases 对杀虫剂的代谢作用, 这反过来又表明它们参与了杀虫剂抗药性的研究。
此外, 本研究还进行了体外代谢试验。与一般的羧酸, 如α-napthyl 醋酸酯 (α钠) 和β萘乙酸 (β钠) 共同基质, 以反映 carboxylesterases 的水解活动,体外代谢研究被认为是一个准确的方法直接测量 carboxylesterases 对杀虫剂的活动18。该方法已成功地应用于各种昆虫, 以表征多种细胞色素 P450s 与杀虫剂抗药性11,19,20。然而, 这种方法尚未在羧酸研究中得到应用。利用杆状病毒介导的表达系统所产生的羧酸蛋白, 可以对氯菊酯 carboxylesterases 进行体外代谢研究, 进一步提供有力的证据证明参与carboxylesterases 在给拟除虫菊酯抗性的房子苍蝇。

<table><tbody><tr><td>Q5 High-Fidelity DNA Polymerase</td><td>New England Biolabs inc.</td><td>M0491L</td><td></td></tr><tr><td>QIAquick Gel Extraction Kit</td><td>QIAGEN</td><td>28704</td><td></td></tr><tr><td>pENTR/D-TOPO Cloning Kit, with One Shot TOP10 Chemically Competent E. coli</td><td>Invitrogen by life technology</td><td>K240020</td><td>S.O.C medium and universal M13 sequence primers were included in this kit.</td></tr><tr><td>PureLink HiPure Plasmid Miniprep Kit</td><td>Invitrogen by life technology</td><td>K210002</td><td></td></tr><tr><td>Gateway LR Clonase II Enzyme mix for BaculoDirect&lt;sup&gt;TM&lt;/sup&gt; Kits</td><td>Invitrogen by life technology</td><td>11791-023</td><td></td></tr><tr><td>BaculoDirect C-Term Linear DNA Transfection Kit</td><td>Invitrogen by life technology</td><td>12562-019</td><td>Cellfectin transfection reagent and ganciclovir were included in this kit</td></tr><tr><td>pENTR-CAT plasmid</td><td>Invitrogen by life technology</td><td></td><td>Included in BaculoDirect C-Term Linear DNA Transfection Kit, concentration: 0.5 ug/uL</td></tr><tr><td>Heat inactivated Fetal Bovine Serum, Certified</td><td>Gibco by Life Technologies</td><td>10082-139</td><td></td></tr><tr><td>Sf9 cells in Sf-900 III SFM</td><td>Gibco by Life Technologies</td><td>12659017</td><td></td></tr><tr><td>Insect Cell-PE LB Insect Cell Protein Extraction &amp;amp; Lysis Buffer</td><td>G Biosciences by A Geno Technology Inc</td><td>786-411</td><td></td></tr><tr><td>Sf-900 III SFM (1&amp;times;) Serum Free Medium Complete</td><td>Gibco by Life Technologies</td><td>12658-019</td><td></td></tr><tr><td>Grace&#039;s Insect Medium, unsupplemented</td><td>Gibco by Life Technologies</td><td>11595030</td><td></td></tr><tr><td>Permethrin (isomers) analytical standard</td><td>SUPELCO by Solutions Within&lt;sup&gt;TM&lt;/sup&gt;</td><td>442748</td><td></td></tr><tr><td>Methanol (analytical graded)</td><td>Sigma-Aldrich</td><td>67-56-1</td><td></td></tr><tr><td>Acetonitrile (analytical graded)</td><td>Sigma-Aldrich</td><td>75-05-8</td><td></td></tr><tr><td>GHP Acrodisc 25 mm Syringe Filters with 0.45 &amp;mu;m GHP Membrane (HPLC Certified)</td><td>Pall Life Sciences</td><td>21890388</td><td></td></tr><tr><td>Alliance Waters 2695 HPLC System</td><td>Waters</td><td></td><td></td></tr><tr><td>T100 Thermal Cycle</td><td>Bio-Rad Laboratories Inc.</td><td>1861096</td><td></td></tr><tr><td>Nanodrop 2000/2000c Spectrophotometers</td><td>ThermoFisher Scientific</td><td>ND2000CLAPTOP</td><td></td></tr><tr><td>Cytation 5 Cell Imaging Multi-Mode Reader</td><td>BioTek</td><td></td><td></td></tr></tbody></table>

functional characterization of carboxylesterases in insecticide resistant house flies, musca domestica

羧酸介导的代谢在各种昆虫的抗药性中起着重要的作用。在耐药宫蝇株中发现了几种羧酸基因, 而它们在赋予杀虫剂抗药性方面的作用仍有待探讨。在这里, 我们设计了一个 carboxylesterases 功能特性的协议。三例实验表明: (1) 羧酸蛋白的表达和分离, 通过杆状病毒介导的昆虫斜纹 frugiperda (Sf9) 细胞表达系统;(2) 以细胞为基础的 MTT 法 (3-[4, 5-dimethykthiazol 2-基] 2, 5-diphenyltetrazolium 溴化物) 细胞毒性试验, 测定不同氯菊酯处理对昆虫细胞的耐受性;(3)体外代谢研究, 探讨 carboxylesterases 对氯菊酯的代谢能力。羧酸基因 MdαE7 是克隆自抗性家飞应变 ALHF, 并用于构建重组杆状病毒 Sf9 细胞感染。用 MTT 法测定了不同氯菊酯治疗可行性细胞的含量。实验组 (MdαE7-recombinant 杆状病毒感染细胞) 的增强耐受性与对照组 (CAT 重组杆状病毒感染细胞和 GFP-重组杆状病毒感染细胞) 相比, 对氯菊酯治疗建议的能力, MdαE7 在代谢杀虫剂, 从而保护细胞免受化学损害。此外, 羧酸蛋白在昆虫 Sf9 细胞中表达, 并被分离出来进行体外代谢研究。结果表明, MdαE7 对氯菊酯的体外代谢效率显著, 直接表明 carboxylesterases 参与了杀虫剂的代谢, 从而在室内蝇类中具有抗药性。

不同氯菊酯治疗的细胞生存能力 (MTT 法)
MdαE7-recombinant 杆状病毒感染的 Sf9 细胞 (实验组) 和猫重组杆状病毒 (由杆状病毒感染的试剂盒提供) 感染细胞 (对照组) 检测氯菊酯的细胞毒性。MdαE7 表达细胞对氯菊酯的增强细胞耐受性强烈支持这种羧酸对杀虫剂的代谢作用, 从而保护细胞免受化学损伤。在本研究中, 对不同氯菊酯浓度 (50、100、200和400µM) 的细胞活力进行了比较, 并与乙腈单独处理的细胞进行了对比计算。结果表明, MdαE7 表达细胞的生存能力显著提高 (从86.00% 到 101.92%)。(图 4B) 比控制单元 (范围从67.59% 到 81.43%)(图 4A) 当暴露在不同浓度的氯菊酯时, 表明 MdαE7 在昆虫细胞中对氯菊酯代谢的重要作用。
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58106/58106fig4.jpg"> 图 4: 不同氯菊酯处理下昆虫 Sf9 细胞的可行性.(a) 由猫重组杆状病毒感染的控制细胞的生存能力 (B) MdαE7 重组杆状病毒感染实验细胞的可行性。对每种氯菊酯浓度进行了四次复制。数据显示为平均值 "电子扫描电镜<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58106/58106fig4large.jpg" target="_blank">". 请单击此处查看此图的较大版本.</a>
MdαE7 对氯菊酯体外代谢的研究
氯菊酯的新陈代谢是通过孵化40µM 氯菊酯标准溶液以及从感染的 Sf9 细胞提取的 MdαE7 蛋白来测定的。反应与猫蛋白质担当了控制。计算了氯菊酯的消耗率, 并与不添加酶的反应进行了比较。120分钟潜伏期后, 反相高效液相色谱仪对反应进行监测。由于氯菊酯标准实际上是顺式和反式异构体的混合物, 在 HPLC 色谱图谱中观察到两个峰值, 分别为10.67 分钟和10.87 分钟的反氯菊酯和顺氯菊酯的洗脱次数 (图 5)。MdαE7 蛋白 (39.18±3.78%) 对氯菊酯的耗竭率显著高于 CAT 蛋白 (7.29±0.81%)(图 6), 这不仅与 MTT 结果一致, 而且直接证明了 MdαE7 在体外对氯菊酯代谢的能力。
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58106/58106fig5.jpg"> 图 5: 氯菊酯标准的 HPLC 图谱.本研究采用的氯菊酯标准为反氯菊酯和顺氯菊酯异构体的混合物。红色箭头分别表示了反氯菊酯异构体和顺氯菊酯异构体的峰值。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58106/58106fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/58106/58106fig6.jpg"> 图 6: MdαE7 和猫重组蛋白对氯菊酯的体外代谢.计算了 MdαE7 和猫蛋白对氯菊酯的消耗百分比。对每种氯菊酯浓度进行了三次复制。数据显示为平均值 "电子扫描电镜<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58106/58106fig6large.jpg" target="_blank">". 请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Functional Characterization of Carboxylesterases in Insecticide Resistant House Flies, Musca Domestica at JoVE.com

Functional Characterization of Carboxylesterases in Insecticide Resistant House Flies, Musca Domestica

在这里, 我们提出了一个协议, 生产的房子飞羧酸蛋白的体外与杆状病毒介导的昆虫细胞表达系统, 后来功能表征其在代谢氯菊酯的作用, 从而, 赋予以传导细胞为基础的 mtt 法和体外代谢研究的抗性。

Carboxylesterases 在抗杀虫剂家蝇、家蝇中的功能表征

Carboxylesterase-mediated metabolism is thought to play a major role in insecticide resistance in various insects. Several carboxylesterase genes were ...

functional-characterization-carboxylesterases-insecticide-resistant

Nanyang Technological University

Research

JoVE Journal

Biochemistry

Functional Characterization of Carboxylesterases in Insecticide Resistant House Flies, Musca Domestica

7.4K 次观看.  Auburn University. 在这里, 我们提出了一个协议, 生产的房子飞羧酸蛋白的体外与杆状病毒介导的昆虫细胞表达系统, 后来功能表征其在代谢氯菊酯的作用, 从而, 赋予以传导细胞为基础的 mtt 法和体外代谢研究的抗性。

视频: Functional Characterization of Carboxylesterases in Insecticide Resistant House Flies, Musca Domestica

A Calcium Bioluminescence Assay for Functional Analysis of Mosquito (Aedes aegypti) and Tick (Rhipicephalus microplus) G Protein-coupled Receptors

Arthropod hormone receptors are potential targets for novel pesticides as they regulate many essential physiological and behavioral processes. The majority of them belong to the superfamily of G protein-coupled receptors (GPCRs). We have focused on characterizing arthropod kinin receptors from the tick and mosquito. Arthropod kinins are multifunctional neuropeptides with myotropic, diuretic, and neurotransmitter function. Here, a method for systematic analyses of structure-activity relationships of insect kinins on two heterologous kinin receptor-expressing systems is described. We provide important information relevant to the development of biostable kinin analogs with the potential to disrupt the diuretic, myotropic, and/or digestive processes in ticks and mosquitoes.
The kinin receptors from the southern cattle tick, Boophilus microplus (Canestrini), and the mosquito Aedes aegypti (Linnaeus), were stably expressed in the mammalian cell line CHO-K1. Functional analyses of these receptors were completed using a calcium bioluminescence plate assay that measures intracellular bioluminescence to determine cytoplasmic calcium levels upon peptide application to these recombinant cells. This method takes advantage of the aequorin protein, a photoprotein isolated from luminescent jellyfish. We transiently transfected the aequorin plasmid (mtAEQ/pcDNA1) in cell lines that stably expressed the kinin receptors. These cells were then treated with the cofactor coelenterazine, which complexes with intracellular aequorin. This bond breaks in the presence of calcium, emitting luminescence levels indicative of the calcium concentration. As the kinin receptor signals through the release of intracellular calcium, the intensity of the signal is related to the potency of the peptide.
This protocol is a synthesis of several previously described protocols with modifications; it presents step-by-step instructions for the stable expression of GPCRs in a mammalian cell line through functional plate assays (Staubly et al., 2002 and Stables et al., 1997). Using this methodology, we were able to establish stable cell lines expressing the mosquito and the tick kinin receptors, compare the potency of three mosquito kinins, identify critical amino acid positions for the ligand-receptor interaction, and perform semi-throughput screening of a peptide library. Because insect kinins are susceptible to fast enzymatic degradation by endogenous peptidases, they are severely limited in use as tools for pest control or endocrinological studies. Therefore, we also tested kinin analogs containing amino isobutyric acid (Aib) to enhance their potency and biostability. This peptidase-resistant analog represents an important lead in the development of biostable insect kinin analogs and may aid in the development of neuropeptide-based arthropod control strategies.

A Calcium Bioluminescence Assay for Functional Analysis of Mosquito Aedes aegypti and Tick Rhipicephalus microplus G Protein-coupled Receptors

This protocol provides instructions for clonal-cell line selection and a calcium bioluminescence assay to analyze the structure-activity relationships of synthesized arthropod neuropeptides on their cognate GPCRs. This assay can be used for receptor deorphanization and structure-activity relationship studies for synthetic analog design and peptide/drug-lead discovery.

一个钙生物发光检测蚊虫的功能分析（埃及伊蚊）和刻度（镰形扇头microplus）G蛋白偶联受体

Arthropod hormone receptors are potential targets for novel pesticides as they regulate many essential physiological and behavioral processes.  The ...

科研

免疫与感染

A Method for Evaluating Insecticide Efficacy against Bed Bug, Cimex lectularius, Eggs and First Instars

Standard toxicity evaluations of insecticides against insect pests are primarily conducted on adult insects. Evaluations are based on a dose-response or concentration-response curve, where mortality increases as the dose or concentration of an insecticide is increased. Standard lethal concentration (LC50) and lethal dose (LD50) tests that result in 50% mortality of a test population can be challenging for evaluating toxicity of insecticides against non-adult insect life stages, such as eggs and early instar or nymphal stages. However, this information is essential for understanding insecticide efficacy in all bed bug life stages, which affects control and treatment efforts. This protocol uses a standard dipping bioassay modified for bed bug eggs and a contact insecticidal assay for treating nymphal first instars. These assays produce a concentration-response curve to further quantify LC50 values for insecticide evaluations.

A Method for Evaluating Insecticide Efficacy against Bed Bug,  Cimex lectularius, Eggs and First Instars

Insecticide evaluations are often targeted against adult insects, rather than immature stages. Here, we present a protocol for evaluating insecticides against bed bug eggs with a comparison to the first nymphal bed bug stage. These protocols could be adjusted for other insects to evaluate insecticide efficacy in non-adult life stages.

一个反对臭虫评估杀虫剂效果的方法，<em&gt;臭虫臭虫</em&gt;，鸡蛋和一龄期

Standard toxicity evaluations of insecticides against insect pests are primarily conducted on adult insects. Evaluations are based on a dose-response or ...

环境科学

Crystal Structure of the N-terminal Domain of Ryanodine Receptor from Plutella xylostella

Development of potent and efficient insecticides targeting insect ryanodine receptors (RyRs) has been of great interest in the area of agricultural pest control. To date, several diamide insecticides targeting pest RyRs have been commercialized, which generate annual revenue of 2 billion U.S. dollars. But comprehension of the mode of action of RyR-targeting insecticides is limited by the lack of structural information regarding insect RyR. This in turn restricts understanding of the development of insecticide resistance in pests. The diamondback moth (DBM) is a devastating pest destroying cruciferous crops worldwide, which has also been reported to show resistance to diamide insecticides. Therefore, it is of great practical importance to develop novel insecticides targeting the DBM RyR, especially targeting a region different from the traditional diamide binding site. Here, we present a protocol to structurally characterize the N-terminal domain of RyR from DBM. The x-ray crystal structure was solved by molecular replacement at a resolution of 2.84 &#197;, which shows a beta-trefoil folding motif and a flanking alpha helix. This protocol can be adapted for the expression, purification and structural characterization of other domains or proteins in general.

Crystal Structure of the N-terminal Domain of Ryanodine Receptor from Plutella xylostella

In this article, we describe the protocols of protein expression, purification, crystallization and structure determination of the N-terminal domain of&#160;ryanodine receptor from diamondback moth (Plutella xylostella).

龙苷受体 n 端域的晶体结构

Development of potent and efficient insecticides targeting insect ryanodine receptors (RyRs) has been of great interest in the area of agricultural pest ...

生物化学

Embryo Microinjection and Knockout Mutant Identification of CRISPR/Cas9 Genome-Edited Helicoverpa Armigera (H&#252;bner)

The cotton bollworm, Helicoverpa armigera, is one of the most destructive pests in the world. A combination of molecular genetics, physiology, functional genomics, and behavioral studies has made H. armigera a model species in Lepidoptera Noctuidae. To study the in vivo functions of and interactions between different genes, clustered regularly interspaced short palindromic repeats (CRISPR)/ associated protein 9 (Cas9) genome editing technology is a convenient and effective method used for performing functional genomic studies. In this study, we provide a step-by-step systematic method to complete gene knockout in H. armigera using the CRISPR/Cas9 system. The design and synthesis of guide RNA (gRNA) are described in detail. Then, the subsequent steps consisting of gene-specific primer design for guide RNA (gRNA) creation, embryo collection, microinjection, insect rearing, and mutant detection are summarized. Finally, troubleshooting advice and notes are provided to improve the efficiency of gene editing. Our method will serve as a reference for the application of CRISPR/Cas9 genome editing in H. armigera as well as other Lepidopteran moths.

Embryo Microinjection and Knockout Mutant Identification of CRISPR/Cas9 Genome-Edited Helicoverpa Armigera H&#252;bner

Presented here is a protocol of Helicoverpa armigera (H&#252;bner) embryo microinjection and knockout mutant identification created by CRISPR/Cas9 genome editing. Mutant insects enable further research of gene function and interaction among different genes in vivo.

CRISPR/Cas9基因组编辑的半球 藻（ Hübner）的胚胎显微注射和敲除突变体鉴定

The cotton bollworm, Helicoverpa armigera, is one of the most destructive pests in the world. A combination of molecular genetics, physiology, functional ...

行为学

Bioassays for Monitoring Insecticide Resistance

Pest resistance to pesticides is an increasing problem because pesticides are an integral part of high-yielding production agriculture. When few products are labeled for an individual pest within a particular crop system, chemical control options are limited. Therefore, the same product(s) are used repeatedly and continual selection pressure is placed on the target pest. There are both financial and environmental costs associated with the development of resistant populations. The cost of pesticide resistance has been estimated at approximately $ 1.5 billion annually in the United States. This paper will describe protocols, currently used to monitor arthropod (specifically insects) populations for the development of resistance. The adult vial test is used to measure the toxicity to contact insecticides and a modification of this test is used for plant-systemic insecticides. In these bioassays, insects are exposed to technical grade insecticide and responses (mortality) recorded at a specific post-exposure interval. The mortality data are subjected to Log Dose probit analysis to generate estimates of a lethal concentration that provides mortality to 50% (LC50) of the target populations and a series of confidence limits (CL's) as estimates of data variability. When these data are collected for a range of insecticide-susceptible populations, the LC50 can be used as baseline data for future monitoring purposes. After populations have been exposed to products, the results can be compared to a previously determined LC50 using the same methodology.

This manuscript demonstrates and discusses techniques used to survey pesticide susceptibility and detect resistance to contact and systemic pesticides in arthropod pests.

生物测定对杀虫剂的抗药性监测

Pest resistance to pesticides is an increasing problem because pesticides are an integral part of high-yielding production agriculture. When few products ...

生物学

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 ...

Topical Application Bioassay to Quantify Insecticide Toxicity for Mosquitoes and Fruit Flies

The continued use of insecticides for public health and agriculture has led to widespread insecticide resistance and hampering of control methods. Insecticide resistance surveillance of mosquito populations is typically done through Centers for Disease Control and Prevention (CDC) bottle bioassays or World Health Organization (WHO) tube tests. However, these methods can result in a high degree of variability in mortality data due to variable insecticide contact with&#160;the insect, the relatively small numbers of organisms tested, extensive variation in mass between populations, and constantly changing environmental conditions, leading to variable outcomes. This paper presents the topical application bioassay, adapted as a high-throughput phenotypic bioassay for both mosquitoes and fruit flies, to test large numbers of insects along&#160;a range of insecticide concentrations.
This assay 1) ensures consistent treatment and insecticide contact with every organism, 2) produces highly specific dose-response curves that account for differences in average mass between strains and sexes (which is particularly important for field-collected organisms), and 3) allows for the calculation of statistically rigorous median lethal doses (LD50), which are necessary for resistance ratio comparisons-an alternative surveillance approach from diagnostic dose mortality, which is also used for larvicide resistance surveillance. This assay will be a complementary tool for accurately phenotyping mosquito populations and, as illustrated using fruit flies, is easily adaptable for use with other insects. We argue that this assay will help fill the gap between genotypic and phenotypic insecticide resistance in multiple insect species.

We describe the methodology and importance of the topical application bioassay to measure insecticide susceptibility in mosquitoes and fruit flies. The presented assay is high-throughput, utilizes insect mass-thus allowing for calculating a mass-relativized lethal dose instead of concentration-and likely has lower variability than other similar methods.

局部应用生物测定以量化蚊子和果蝇的杀虫剂毒性

The continued use of insecticides for public health and agriculture has led to widespread insecticide resistance and hampering of control methods. ...

Control of Aedes albopictus Mosquito Larvae with Carpesium abrotanoides L.

As a vital vector of dengue fever, yellow fever, and other mosquito-borne diseases, Aedes albopictus (Diptera: Culicidae) can be broadly distributed worldwide and cause a severe threat to public health. To date, considering the fast-emerging insecticide resistance in the mosquito, the development of new botanical insecticides to control and reduce Ae. albopictus is urgent and crucial. Therefore, to investigate the decoction effect of the plant C. abrotanoides L. on mosquito larvae killing, we detected the mortality of larvae after treatment with different concentrations (60 mg/mL, 120 mg/mL, and 180 mg/mL) of decoction within a series of time points (12 h, 24 h, 36 h, and 48 h). We found that 24 h with 180 mg/mL C. abrotanoides L. decoction treatment killed 92.35% of mosquitoes relative to the control treatment. Meanwhile, 36 h with 120 mg/mL could also kill more than 90% of mosquitoes. Furthermore, Carassius auratus populations were exposed to 120 mg/mL C. abrotanoides L. decoction for 36 h and were not dead. The mortality evaluation indicated that this concentration is not a harmful level of ecological environmental pollution. This study provides a possible plant candidate that could be used for designing plant-derived insecticides. Additionally, these methods can be altered and applied to other mosquito species.

Control of Aedes albopictus Mosquito Larvae with Carpesium abrotanoides L.

Using the Carpesium abrotanoides L. plant for mosquito larvae control can effectively reduce the Aedes albopictus mosquito population and provide a foundation for designing plant-derived insecticides.

As a vital vector of dengue fever, yellow fever, and other mosquito-borne diseases, Aedes albopictus (Diptera: Culicidae) can be broadly distributed ...

Protocols for CRISPR/Cas9 Mutagenesis of the Oriental Fruit Fly Bactrocera dorsalis

The Oriental fruit fly, Bactrocera dorsalis, is a highly invasive and adaptive pest species that causes damage to citrus and over 150 other fruit crops worldwide. Since adult fruit flies have great flight capacity and females lay their eggs under the skins of fruit, insecticides requiring direct contact with the pest usually perform poorly in the field. With the development of molecular biological tools and high-throughput sequencing technology, many scientists are attempting to develop environmentally friendly pest management strategies. These include RNAi or gene editing-based pesticides that downregulate or silence genes (molecular targets), such as olfactory genes involved in searching behavior, in various insect pests. To adapt these strategies for Oriental fruit fly control, effective methods for functional gene research are needed. Genes with critical functions in the survival and reproduction of B. dorsalis serve as good molecular targets for gene knockdown and/or silencing. The CRISPR/Cas9 system is a reliable technique used for gene editing, especially in insects. This paper presents a systematic method for CRISPR/Cas9 mutagenesis of B. dorsalis, including the design and synthesis of guide RNAs, collecting embryos, embryo injection, insect rearing, and mutant screening. These protocols will serve as a useful guide for generating mutant flies for researchers interested in functional gene studies in B. dorsalis.

Protocols for CRISPR/Cas9 Mutagenesis of the Oriental Fruit Fly Bactrocera dorsalis

This paper presents the step-by-step protocols for CRISPR/Cas9 mutagenesis of the Oriental fruit fly Bactrocera dorsalis. Detailed steps provided by this standardized protocol will serve as a useful guide for generating mutant flies for functional gene studies in B. dorsalis.

东方果蝇背侧芽孢杆菌CRISPR/Cas9诱变实验方案

The Oriental fruit fly, Bactrocera dorsalis, is a highly invasive and adaptive pest species that causes damage to citrus and over 150 other fruit crops ...

通过饲喂测定评估植物化学对 Helicoverpa armigera 的影响

Developing a Feeding Assay System for Evaluating the Insecticidal Effect of Phytochemicals on Helicoverpa armigera

Helicoverpa armigera, a lepidopteran insect, is a polyphagous pest with a worldwide distribution. This herbivorous insect is a threat to plants and agricultural productivity. In response, plants produce several phytochemicals that negatively impact the insect's growth and survival. This protocol demonstrates an obligate feeding assay method to evaluate the effect of a phytochemical (quercetin) on insect growth, development, and survival. Under controlled conditions, the neonates were maintained until the second instar on a pre-defined artificial diet. These second-instar larvae were allowed to feed on a control and quercetin-containing artificial diet for 10 days. The insects' body weight, developmental stage, frass weight, and mortality were recorded on alternate days. The change in body weight, the difference in feeding pattern, and developmental phenotypes were evaluated throughout the assay time. The described obligatory feeding assay simulates a natural mode of ingestion and can be scaled up to a large number of insects. It permits one to analyze phytochemicals' effect on the growth dynamics, developmental transition, and overall fitness of H. armigera. Furthermore, this setup can also be utilized to evaluate alterations in nutritional parameters and digestive physiology processes. This article provides a detailed methodology for feeding assay systems, which may have applications in toxicological studies, insecticidal molecule screening, and understanding chemical effects in plant-insect interactions.

作者聚焦:害虫管理饲喂分析的发展和挑战 

This protocol describes the obligate feeding assay to evaluate the potentially toxic effect of a phytochemical on the lepidopteran insect larvae. This is a highly scalable insect bioassay, easy to optimize the sublethal and lethal dose, deterrent activity, and physiological effect. This could be used for screening eco-friendly insecticides.

开发一种用于评估植物化学物质对 Helicoverpa armigera 的杀虫作用的摄食试验系统

Helicoverpa armigera, a lepidopteran insect, is a polyphagous pest with a worldwide distribution. This herbivorous insect is a threat to plants and ...

Carboxylesterases 在抗杀虫剂家蝇、家蝇中的功能表征

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开发一种用于评估植物化学物质对 Helicoverpa armigera 的杀虫作用的摄食试验系统

Carboxylesterases 在抗杀虫剂家蝇、家蝇中的功能表征

摘要

探索更多视频

此视频中的章节

一个钙生物发光检测蚊虫的功能分析（埃及伊蚊）和刻度（镰形扇头microplus）G蛋白偶联受体

一个反对臭虫评估杀虫剂效果的方法，

龙苷受体 n 端域的晶体结构

CRISPR/Cas9基因组编辑的半球 藻（ Hübner）的胚胎显微注射和敲除突变体鉴定

生物测定对杀虫剂的抗药性监测

新型杀虫化学对蚊子毒性测试规程

局部应用生物测定以量化蚊子和果蝇的杀虫剂毒性

Control of Aedes albopictus Mosquito Larvae with Carpesium abrotanoides L.

东方果蝇背侧芽孢杆菌CRISPR/Cas9诱变实验方案

开发一种用于评估植物化学物质对 Helicoverpa armigera 的杀虫作用的摄食试验系统

CRISPR/Cas9基因组编辑的半球藻（ Hübner）的胚胎显微注射和敲除突变体鉴定