一种精确且可量化的小型节肢动物血淋巴收集方法
Summary
我们描述了一种从小型节肢动物中有效收集可量化血淋巴以进行后续分析的方法。
Abstract
众所周知，节肢动物通过其血淋巴传播各种具有医学和农业重要性的病毒，这对于病毒传播至关重要。血淋巴采集是研究病毒-载体相互作用的基本技术。在这里，我们描述了一种新颖而简单的方法，用于定量收集小型节肢动物的血淋巴，使用 Laodelphax striatellus （小棕色飞虱，SBPH）作为研究模型，因为这种节肢动物是水稻条纹病毒（RSV）的主要载体。在该协议中，该过程首先用细尖镊子轻轻捏掉冷冻节肢动物的一条腿，然后将血淋巴从伤口中压出。然后，根据毛细管力原理，使用由毛细管和移液器灯泡组成的简单微量移液器从伤口收集漏出性血淋巴。最后，可以将收集的血淋巴溶解到特定的缓冲液中以供进一步研究。这种从小型节肢动物收集血淋巴的新方法是进一步研究虫媒病毒和载体-病毒相互作用的有用且有效的工具。
Introduction
动植物病毒都可以通过节肢动物传播，这些病毒对人类健康构成严重威胁，给农业造成巨大的经济损失1，2，3。重要的是，节肢动物血淋巴作为节肢动物的循环系统和免疫系统的重要组成部分，在调节虫媒病毒传播方面起着重要作用。通过节肢动物肠道获得的病毒只有在成功逃脱不良血淋巴环境后才会转运到其他组织4，5，6，7。节肢动物血淋巴中病毒的生命周期涉及病毒在血浆中存活、进入血细胞并转运到其他组织，血淋巴中发生各种病毒-载体相互作用机制8、9、10、11、12。例如，SBPH 对 RSV 的垂直传播取决于 SBPH 卵黄素蛋白和 RSV（米条纹病毒）衣壳蛋白13，14 之间的分子相互作用。一些病毒可能通过结合特定的载体因子15，16，17，18来逃避血淋巴的免疫反应。因此，研究节肢动物血淋巴中的载体-病毒相互作用对于更好地了解虫媒病毒传播非常重要。
一些小昆虫的血淋巴，如飞虱、叶蝉和一些蚊子，由于其体型的原因，很难收集。为了解决这个问题，已经开发了几种收集血淋巴的方法，包括将注射器针头直接插入昆虫体内以提取微量的血淋巴，用细尖镊子从伤口部位收集渗出物，以及直接离心。这些方法能够测量血淋巴内的相对基因表达水平和病毒滴度19，20，21。然而，对于这些小昆虫，目前还没有一种有效的血淋巴体积定量方法，这是血细胞计数、蛋白质定量和酶活性分析所必需的。
SBPH（小棕色飞虱）是一种体长约2-4毫米的小型昆虫媒介。SBPH能够传播多种植物病毒，包括RSV，玉米粗矮病毒和水稻黑条纹矮病毒22，23，24。在过去十年中，对SBPH和RSV之间的相互作用进行了深入研究。为了便于使用SBPH，我们开发了一种新颖而简单的血淋巴收集方法。这种方法基于毛细管力原理，使用带有刻度标记的毛细管以精确和可量化的方式获取昆虫的血淋巴。这使我们能够有效地从小昆虫中收集特定体积的血淋巴，并更详细地研究小载体的血淋巴环境。
Protocol
1. 昆虫饲养 提高本实验中使用的水稻幼苗（Oryza sativa cv. Japanbonbare）的SBPH。在培养箱（65mm x 200mm）中种植20棵水稻幼苗，并在16小时光照/ 8小时黑暗光周期下在25°C下生长。 2. 解剖 SBPH 以收集血淋巴 将SBPH放入离心管中，并将它们置于冰浴中10-30分钟。注意：不要将SBPH放入冰浴中少于10分钟，否则昆虫可能会复活。 …
Representative Results
微量移液器模型和血淋巴收集我们开发了一种简单的微量移液器，其作用基于毛细管的毛细管力。微量移液器由毛细管和移液器灯泡组成（图1A）。毛细管有不同的体积尺寸，范围从 1 μL 到 20 μL，毛细管体积可根据要求选择。不建议使用体积较小的毛细管，因为较小体积的毛细管的额外细孔会使吸收血淋巴等液体变得困难。移液器灯泡顶部有一个孔，在血淋…
Discussion
血淋巴是节肢动物循环系统的介质，虫媒病毒只有在恶劣的血淋巴环境中生存下来，才能侵入其他节肢动物组织。收集高质量的血淋巴样本是研究血淋巴中发生的载体-病毒相互作用的第一步。据报道，昆虫血淋巴可以从昆虫身体的几个部位获得，包括前腿上的伤口、头部区域的小切口或腹部的撕裂伤口26、27、28、<sup class="xref"…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项工作得到了国家重点研发计划（编号：2022YFD1401700）和国家自然科学基金（编号：32090013和32072385）的支持。
Materials
| 10% SDS-PAGE protein gel | Bio-rad | 4561035 | Protein separation and detection |
| 4% paraformaldehyde | Solarbio | P1110 | For fixation of the cells or tissues |
| Bradford dye reagent | Bio-rad | 5000205 | Protein concentration detection |
| Capillary | Hirschmann | 9000101 | For collecting hemolymph |
| Cell counting chamber | ACMEC | AYA0810 | Hemocytes counting |
| Glass slide | Gitoglas | 10127105A | For holding insects |
| Glass slide coated with silane | Sigma | S4651-72EA | For holding microscope samples |
| Gold antifade reagent with DAPI | Invitrogen | P36935 | Nucleus staining |
| Microscope cover glass | Gitoglas | 10212424C | For microscopic observation |
| Pipette bulb | Hirschmann | 9000101 | For collecting hemolymph |
| Prism 8.0 software | GraphPad Software | / | Statistical analyses |
| Stereomicroscope | Motic | SMZ-168 | For insect dissection |
| Tweezers | Tianld | P5622 | For insect dissection |
| Zeiss inverted microscope | Zeiss | Observer Z1 | Hemocytes observation |
Referências
- Hogenhout, S. A., Ammar el, D., Whitfield, A. E., Redinbaugh, M. G. Insect vector interactions with persistently transmitted viruses. Annual Review of Phytopathology. 46, 327-359 (2008).
- Ray, S., Casteel, C. L. Effector-mediated plant-virus-vector interactions. Plant Cell. 34 (5), 1514-1531 (2022).
- Islam, W., et al. Plant-insect vector-virus interactions under environmental change. Science of the Total Environment. 701, 135044 (2020).
- Cory, J. S. Insect virus transmission: Different routes to persistence. Current Opinion in Insect Science. 8, 130-135 (2015).
- Wang, X. W., Blanc, S. Insect transmission of plant single-stranded DNA viruses. Annual Review of Entomology. 66, 389-405 (2021).
- Yi, H. Y., Chowdhury, M., Huang, Y. D., Yu, X. Q. Insect antimicrobial peptides and their applications. Applied Microbiology and Biotechnology. 98 (13), 5807-5822 (2014).
- Liu, W. W., et al. Proteomic analysis of interaction between a plant virus and its vector insect reveals new functions of hemipteran cuticular protein. Molecular & Cellular Proteomics. 14 (8), 2229-2242 (2015).
- Wang, L., Van Meulebroek, L., Vanhaecke, L., Smagghe, G., Meeus, I. The bee hemolymph metabolome: A window into the impact of viruses on bumble bees. Viruses. 13 (4), 600 (2021).
- Jia, D., et al. Vector mediated transmission of persistently transmitted plant viruses. Current Opinion in Virology. 28, 127-132 (2018).
- Anderson, J. F., Main, A. J., Ferrandino, F. J. Horizontal and vertical transmission of West Nile Virus by Aedes vexans (Diptera: Culicidae). Journal of Medical Entomology. 57 (5), 1614-1618 (2020).
- Gadhave, K. R., et al. Low frequency of horizontal and vertical transmission of cucurbit leaf crumple virus in whitefly Bemisia tabaci Gennadius. Phytopathology. 110 (6), 1235-1241 (2020).
- Logan, R. A. E., et al. Vertical and horizontal transmission of cell fusing agent virus in Aedes aegypti. Applied and Environmental Microbiology. 88 (18), e0106222 (2022).
- Huo, Y., et al. Transovarial transmission of a plant virus is mediated by vitellogenin of its insect vector. PLoS Pathogens. 10 (3), e1003949 (2014).
- Wei, J., et al. Vector development and vitellogenin determine the transovarial transmission of begomoviruses. Proceedings of the National Academy of Sciences of the United States of America. 114 (26), 6746-6751 (2017).
- Medzhitov, R. Toll-like receptors and innate immunity. Nature Reviews Immunology. 1 (2), 135-145 (2001).
- Kingsolver, M. B., Huang, Z., Hardy, R. W. Insect antiviral innate immunity: Pathways, effectors, and connections. Journal of Molecular Biology. 425 (24), 4921-4936 (2013).
- Pei, R. J., Chen, X. W., Lu, M. J. Control of hepatitis B virus replication by interferons and Toll-like receptor signaling pathways. World Journal of Gastroenterology. 20 (33), 11618-11629 (2014).
- Kao, Y. T., Lai, M. M. C., Yu, C. Y. How dengue virus circumvents innate immunity. Frontiers in Immunology. 9, 2860 (2018).
- Gilliam, M., Shimanuki, H. Coagulation of hemolymph of the larval honey bee (Apis mellifera L). Experientia. 26 (8), 908-909 (1970).
- Huo, Y., et al. Insect tissue-specific vitellogenin facilitates transmission of plant virus. PLoS Pathogens. 14 (2), e1006909 (2018).
- Chen, X., et al. A plant virus ensures viral stability in the hemolymph of vector insects through suppressing prophenoloxidase activation. mBio. 11 (4), e01453 (2020).
- Vidano, C. Phases of maize rough dwarf virus multiplication in the vector Laodelphax striatellus (Fallén). Virology. 41 (2), 218-232 (1970).
- Yu, Y. L., et al. Laodelphax striatellus Atg8 facilitates Rice stripe virus infection in an autophagy-independent manner. Journal of Insect Science. 28 (2), 315-329 (2021).
- Zhang, J. H., et al. Cytochrome P450 monooxygenases CYP6AY3 and CYP6CW1 regulate Rice black-streaked dwarf virus replication in Laodelphax striatellus (Fallen). Viruses. 13 (8), 1576 (2021).
- Ribeiro, C., Brehelin, M. Insect haemocytes: What type of cell is that. Journal of Insect Physiology. 52 (5), 417-429 (2006).
- Butolo, N. P., et al. A high quality method for hemolymph collection from honeybee larvae. PLoS One. 15 (6), e0234637 (2020).
- Nesa, J., et al. Antimicrobial potential of a ponericin-like peptide isolated from Bombyx mori L. hemolymph in response to Pseudomonas aeruginosa infection. Scientific Reports. 12 (1), 15493 (2022).
- Mahmoud, S., et al. Curcumin-injected Musca domestica larval hemolymph: Cecropin upregulation and potential anticancer effect. Molecules. 27 (5), 1570 (2022).
- Patton, T. G., et al. salivary gland, and hemolymph collection from Ixodes scapularis ticks. Journal of Visualized Experiments. (60), e3894 (2012).
- Piyankarage, S. C., Augustin, H., Featherstone, D. E., Shippy, S. A. Hemolymph amino acid variations following behavioral and genetic changes in individual Drosophila larvae. Amino Acids. 38 (3), 779-788 (2010).
- Fiorotti, J., et al. Disclosing hemolymph collection and inoculation of metarhizium blastospores into Rhipicephalus microplus ticks towards invertebrate pathology studies. Journal of Visualized Experiments. (148), e59899 (2019).
Citar este artigo
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