用于 老德法纹 状体唾液采集的双层膜夹层法
Summary
本方案描述了一种使用人工介质从穿刺-吸吮昆虫中收集足够唾液的方法。这是一种收集昆虫唾液和研究唾液对昆虫进食行为和媒介传播病毒传播的有用方法。
Abstract
稻条病毒（RSV）在东亚造成农业的重大经济损失，其在宿主水稻之间的有效传播完全依赖于昆虫媒介。 Laodelphax striatellus（ 小型棕色飞虱，SBPH）是主要昆虫媒介，在从韧皮部吸食汁液时水平传播RSV。唾液在昆虫的进食行为中起着重要作用。这里描述了一种方便的方法，可用于研究具有穿刺 – 吸食进食行为的昆虫唾液。在这种方法中，允许昆虫以夹在两个拉伸的石蜡膜层之间的人造饮食为食。每天收集含有唾液的饮食，过滤并浓缩以进行进一步分析。最后，通过蛋白质染色和免疫印迹检查收集的唾液的质量。该方法通过检测SBPH唾液中RSV和粘蛋白样蛋白的存在来举例说明。这些人工摄食和唾液收集方法将为进一步研究昆虫唾液中与摄食行为和病毒传播相关的因素奠定基础。
Introduction
水稻条纹病毒（RSV）是Tenuivirus属中的一种阴性链RNA病毒，在东亚的水稻生产中引起严重疾病1，2，3。RSV从受感染的水稻植物传播到健康的水稻植物取决于昆虫载体，主要是Laodelphax striatellus，它以持久繁殖的方式传播RSV。SBPH在以RSV感染的植物为食后获得病毒。一旦进入昆虫体内，RSV在进食后一天感染中肠上皮细胞，然后穿过中肠屏障穿透血淋巴。随后，RSV通过血淋巴扩散到不同的组织中，然后传播。在获得后约10-14天的潜伏期后，唾液腺内的病毒可以通过分泌的唾液传播给健康的寄主植物，而SBPH则从韧皮部4，5，6，7，8，9，10中吸收汁液.有效的摄食过程和唾液中的各种因素对于RSV从昆虫传播到寄主植物至关重要。
唾液腺分泌的昆虫唾液被认为介导昆虫，病毒和寄主植物。半翅目昆虫通常产生两种类型的唾液：胶状唾液和水性唾液11，12，13。胶凝唾液主要分泌到脑浆中，以维持寄主细胞间的蠕动，也与克服植物抗性和免疫反应有关14、15、16、17。在进食的探测阶段，昆虫间歇性地分泌凝胶状唾液，立即被氧化形成表面法兰。然后，单层或分支鞘包裹样式以保留管状通道18，19，20。表皮上的表面法兰被认为通过充当锚点来促进样式的穿透，而样式周围的护套可以提供机械稳定性和润滑16，21，22，23。Nlshp被确定为唾液鞘形成和成功喂养棕色飞虱（Nilaparvata lugens，BPH）的必需蛋白质。抑制由蚜虫Acyrthosiphon pisum分泌的结构鞘蛋白（SHP）的表达通过破坏宿主筛管24的进食来减少其繁殖。此外，在一些昆虫物种中，凝胶唾液因子应该通过形成所谓的食草动物相关分子模式（HAMPs）来触发植物的免疫反应。在N. lugens中，NlMLP，一种与鞘形成相关的粘蛋白样蛋白质，诱导植物防御进食，包括细胞死亡，防御相关基因的表达和胼脂沉积25，26。 此外，蚜虫中的一些凝胶唾液因子已被证明可以通过类似于病原体相关分子模式的基因间相互作用触发植物防御反应12，15，27。
为了研究昆虫喂养和/或病原体传播所必需的唾液因子，有必要分析分泌的唾液。这里描述了人工喂养和收集方法以获得足够量的唾液，以便进一步分析。使用仅含有单一营养元素的培养基，通过银染和蛋白质印迹收集和分析许多唾液蛋白。该方法将有助于进一步研究唾液中对SBPH传播RSV至关重要的因素。
Protocol
1. SBPH维护 在实验室的每个玻璃室中，将具有毒性和无RSV的SBPH个体置于玻璃培养箱（65 x 200 mm）中，每个玻璃室中含有5-6个大米（Oryza sativa cv. Nipponbare）幼苗。在25°C下在16小时光照/ 8小时黑暗光周期下种植水稻植物。注：这些有毒和无RSV的SBPH个体最初是在中国江苏省捕获的。 通过点酶联免疫吸附测定（dot-ELISA）检测SBPH中的RSV，该测定与针对RSV核糖核蛋白（RNPs）的兔RSV特…
Representative Results
人工饲喂装置及唾液采集示意图图1A 描绘了用作收集唾液的进食室的玻璃圆筒（15厘米×2.5厘米）。首先，将SBPH幼虫饥饿数小时以提高收集效率，然后通过冷却5 min固定。将昆虫转移到玻璃圆筒中后，腔室的两个开口端都覆盖着拉伸的石蜡膜。在一端，将200μL5%蔗糖夹在两层石蜡膜之间，延伸至其原始面积的约两倍（图1B）。腔室被铝箔?…
Discussion
1962年首次报道了人工日粮成功饲养昆虫，当时米特勒和爸爸描述了石蜡膜技术来保持人工日粮29，30。这种方法已经在昆虫生物学和行为的许多方面进行了探索，例如营养补充，dsRNA喂养和病毒获取。根据唾液分析的要求，本研究以5%蔗糖作为一般人工饮食，收集SBPH的唾液。为了成功收集唾液，这里有几个关键步骤值得注意。首先，在将实验昆虫引入腔?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
本研究由国家重点研发计划（编号：2019YFC1200503）、国家自然科学基金（编号：32072385）和中国科学院青年创新促进会（2021084）资助。
Materials
| 10-KD centrifugal filter | Merck Millipore | R5PA83496 | For concentration |
| 10x Protein Transfer Buffer(wet) | macGENE | MP008 | Transfer buffer for western blotting |
| 10x TBST buffer | Coolaber | SL1328-500mL×10 | Wash buffer for western blotting |
| Azure c600 biosystems | Azure Biosystems | Azure c600 | Imaging system for western blotting and silver staining |
| Color Prestained protein ladder | GenStar | M221-01 | Protein marker for western blotting |
| ECL western blotting detection reagents | GE Healthcare | RPN2209 | Western blotting detection |
| Enchanced HRP-DAB Chromogenic Kit | TIANGEN | #PA110 | Chromogenic reaction |
| Horseradish peroxidase-conjugated goat anti-rabbit antibodies | Sigma | 401393-2ML | Polyclonal secondary antibody for western blotting |
| Immobilon(R)-P Polyvinylidene difluoride membrane | Merck Millipore | IPVH00010 | Transfer membrane for western blotting |
| iTaq Universal SYBR Green Supermix | Bio-Rad | 1725125 | For quantitative real-time PCR (qRT-PCR) |
| KIT,iSCRIPT cDNA SYNTHES | Bio-Rad | 1708891 | For Reverse-transcriptional PCR (RT-PCR) |
| Millex-GP Filter, 0.22 µm | Merck Millipore | SLGP033RB | For filtration |
| Mini-PROTEAB TGX Gels | Bio-Rad | 4561043 | For SDS-PAGE |
| NanoDrop One | Thermo Scientific | ND-ONEC-W | Detection of protein concentration |
| Nylon membrane | PALL | T42754 | Membrane for dot-ELISA |
| Parafilm M Membrane | Sigma | P7793-1EA | Making artifical diet sandwichs |
| Rabbit anti-LssgMP polyclonal antibody against LssgMP peptides | Genstript | Rabbit primary anti-LssgMP polyclonal antibody for western blotting | |
| Rabbit anti-RSV polyclonal antibody | Genstript | Rabbit primary anti-RSV polyclonal antibody for western blotting and dot-ELISA | |
| RNAprep pure Micro Kit | TIANGEN | DP420 | For RNA Extraction |
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Cite This Article
Zhao, J., Yang, J., Zhang, L., Fang, R., Huo, Y. Two-layered Membrane Sandwich Method for Laodelphax striatellus Saliva Collection. J. Vis. Exp. (174), e62831, doi:10.3791/62831 (2021).