蜜蜂组织中的组织学基础和细胞死亡检测
Summary
免疫组织化学方法可用于蜜蜂研究，以检测和评估成年蜜蜂中肠和下咽腺的细胞凋亡和坏死水平。
Abstract
蜂巢内（护士和其他蜂巢蜜蜂）和蜂巢外（觅食者）的蜜蜂（Apis mellifera L.）暴露于气候和天气变化，各种杀虫剂，病原体和营养不良，主要通过口腔进入，主要影响成年蜜蜂的消化道。为了了解和防止这种外部和内部压力源对蜜蜂的影响，一种有用的研究方法是免疫组织化学方法。描述了准备成年蜜蜂的中肠（脑室）和下咽腺（HPG）以进行组织学分析的基本方案。描述了一种详细的方法来评估细胞损伤水平，并将坏死与作为组织再生自然过程的程序性细胞死亡（细胞凋亡）区分开来。介绍了用草酸和杀虫剂（杀虫剂和杀螨剂）处理成年蜜蜂的结果以及脑室和HPG中细胞死亡的测定。还讨论了该方法的优缺点。
Introduction
蜜蜂（Apis mellifera L.）是其他野生传粉媒介，也是农业植物最重要的传粉媒介。几千年来，不断变化的环境影响了蜜蜂调整其形态、生理、行为和对几种病原体和寄生虫的耐受性。因此，蜜蜂在全球范围内发展出高度多样化的物种和亚种1。这些结果与先前的发现一致，即蜜蜂的消化道结构存在遗传变异，但也表明中肠的改变是由于环境因素2，3。
蜜蜂的消化道有三个主要部分：前肠、中肠（脑室）和后肠4。脑室是消化花粉和花蜜/蜂蜜的重要器官;在后肠中，渗透控制是通过吸收水和离子2进行的。蜜蜂工人的下咽腺（HPG）位于头部，合成和分泌蜂王浆成分，以喂养育雏，蜂王和蜂群成员。它们的大小随着年龄和任务而变化，并取决于适当的营养（优质花粉）。6至18天的护士进行育雏，HPG的大小增加5，6。在觅食蜂中，HPGs退化并仅分泌对将蜂蜜中的复杂糖转化为简单糖（α-葡萄糖苷酶，亮氨酸芳基酰胺酶，转化酶）很重要的酶7。
蜜蜂暴露于几种生物和非生物应激源8，消化道可能受到几种负面兴奋剂的影响。保护生物体免受病原体侵害的第一道屏障是中肠的周养膜，它由肠粘膜组成，以防止病原体4。HPG 的发育和功能取决于饮食、年龄和菌落条件9，并受到杀虫剂、杀螨剂10 和病原体11、12、13 的影响。由于瓦罗亚控制处理和来自环境的杀虫剂，蜂巢中的杀螨剂残留会影响觅食蜂和护士蜂14，15。对蜜蜂群体的最大威胁是螨虫Varroa破坏者，既是导致蜂群损失的病毒载体16，也是宿主肥胖身体（蜜蜂的重要重要器官）的消费者，因此会影响个体的身体和群体功能17。
然而，集约化的农田栖息地可以为蜜蜂提供短期的食物供应。因此，农业环境计划应提高农业景观中蜜花的供应18.为了评估不同亚种6，19，20，21的形态或这些因素在细胞或组织水平上的亚致死作用，特别是中肠和HPG，组织学和免疫组织化学方法是实用的，并且足够准确，可用于蜜蜂的组织学研究。
Protocol
1. 蜜蜂研究的基础组织学 蜜蜂组织解剖注意：要解剖工蜂，请使用带LED光源的解剖显微镜。最有用的放大倍率是~20倍。操作和解剖小心地用镊子取一只工蜂，并将其放在冰上（或放入-20°C的冰箱）2分钟以固定22。将蜜蜂斜着固定在培养皿上，从左到右和从右到左两次穿过胸部的最上部。 倒入昆虫盐水覆盖身体。将培养皿放在显微镜下…
Representative Results
中肠细胞死亡检测来自卢布尔雅那斯洛文尼亚农业研究所实验养蜂场的新出现的工蜂（Apis mellifera carnica）用3%草酸（OA）单独处理23。OA经常用于养蜂业，用于 瓦罗阿析构函数 控制。治疗后，将工蜂（每组三只）固定在冰上。解剖中肠并将其固定在10%福尔马林中。然后将组织在一系列酒精溶液中脱水，最后包埋在石蜡中。用切片机切成7μm薄片后，制?…
Discussion
在生物体中，细胞死亡被定义为细胞凋亡或坏死25，并可伴有自噬26。凋亡细胞和坏死细胞之间的区别在于，细胞凋亡是程序性细胞死亡的一种形式，出现在正常细胞中，而坏死是由于致命条件（例如，事故，疾病）而发生的27，28。可以使用基于TUNEL技术的检测试剂盒检测细胞凋亡（通过在细胞凋亡过程中产生的双链D…
Declarações
The authors have nothing to disclose.
Acknowledgements
我非常感谢斯洛文尼亚研究机构的支持，批准号P4-133。
Materials
| 2-Propanol | |||
| ApopTag Peroxidase kit (ApopTag Peroxidase In Situ Apoptosis Detection) | Sigma-Aldrich | S7100 | Assay B, https://www.sigmaaldrich.com/SI/en/product/mm/s7100?gclid=CjwKCA jw7vuUBhBUEiwAEdu2pPanI9SE j81ZTl-nLHEoxXAv7ViKwPA_QRx H7fciMRNcYwR7lbPQbhoCqcQQA vD_BwE; Positive controls included in S7101 |
| Covers | |||
| DeadEnd Colorimetric TUNEL system | Promega | G7360 | Assay A, https://worldwide.promega.com/products/cell-health-assays/apoptosis-assays/deadend-colorimetric-tunel-system/?catNum=G7360 |
| Dissecting microscope (for bee dissection) | Zeiss | ||
| Distilled water | |||
| Embedding cassette | |||
| EnVision System alkaline phosphatase kit | Dako | ||
| Eosin Y Solution | Sigma-Aldrich | alcoholic | |
| Ethanol | 95% (or less pure), 90%, 80% | ||
| Faramount mounting medium, aqueous | Dako | mounting medium | |
| Flattening table | Leica | HI1220 | |
| Forceps (for bee dissection) | Fine science tools | 11294-00 | Standard #4 |
| Formalin 10% | Formaldehyde | ||
| Hematoxylin | Sigma-Aldrich | ||
| HistoChoice Clearing Agent | Sigma-Aldrich | clearing agent | |
| Hydrogen peroxidase 3% | |||
| Incubator | BioRad | ||
| Insect pins (for bee dissection) | Entosphinx | 44594 | Insect pins stainless steel – white, size 2 |
| ISCDDK, AP (In Situ Cell Death Detecteion Kit, Alkaline Phosphatase) | Roche | 11684809910 | Assay C, https://www.sigmaaldrich.com/deepweb/assets/sigmaaldrich/product/documents/362/737/11684809910b ul.pdf |
| KH2PO4 | |||
| Lab clock | |||
| Light microscope | Leica | ||
| Microscope slides | Box with the slides must be preserved in a plastic wrap to prevent dust | ||
| Microtome | Leica | ||
| Modular tissue embedding station | Leica | ||
| Na2HPO4 | |||
| NaCl | |||
| Paraformaldehyde 4% | |||
| Paraplast | Leica | ||
| Pasteur pipettes | 1.5 mL; 3 mL | ||
| PBS | |||
| Petri dish (for bee dissection) | Filled with condensation silicon (Xantoprene L blue and Universal liquid plus activator) | ||
| Proteinase K | Merck | 21627 | |
| Ringers' solution (for bee dissection) | 7.5 g NaCL, 2.38 g Na2HPO4, 2.72 g KH2PO4, 1 L distilled water | ||
| Scissors (for bee dissection) | Fine science tools | 1406-09, 14061-09 | Straight and curved, 9 cm |
| Universal liquid plus activator (for bee dissection) | Kulzer | ||
| Watchmaker’s forceps (for bee dissection) | Fine science tools | 91100-12 | |
| Water bath | Leica | ||
| Watercolor brush | 2x | ||
| Xantoprene L blue (for bee dissection) | Kulzer |
Referências
- Ruttner, F. . Naturgeschichte der Honigbienen. , (1992).
- Jordan, R. Kleine Bienenkunde. Österreichischer Agrarverlag Wien München. , 41-45 (1964).
- Snodgrass, R. E. The Anatomy of the Honey Bee. The Hive and the Honey Bee. , 111-113 (1975).
- Snodgrass, R. E. . The Anatomy of the Honey Bee. , (2004).
- Hrassnigg, N., Crailsheim, K. Adaptation of hypopharyngeal gland development to the brood status of honeybee (Apis mellifera L.) colonies. Journal of Insect Physiology. 44 (10), 929-939 (1998).
- Smodiš Škerl, M. I., Gregorc, A. Characteristics of hypopharyngeal glands in honeybees (Apis mellifera carnica) from a nurse colony. Slovenian Veterinary Research. 52 (2), 67-74 (2015).
- Kubo, T. Change in the expression of hypopharyngeal-gland proteins of the worker honeybees (Apis mellifera L.) with age and/or role. Journal of Biochemistry. 119 (2), 291-295 (1996).
- Sammataro, D., Yoder, J. A. . Honey bee colony health: Challenges and sustainable solutions. , 302 (2012).
- Crailsheim, K., Stolberg, E. Influence of diet, age and colony condition upon intestinal proteolytic activity and size of the hypopharyngeal glands in the honeybee (Apis mellifera L.). Journal of Insect Physiology. 35 (8), 595-602 (1998).
- Smodiš Škerl, M. I., Gregorc, A. Heat shock proteins and cell death in situ localisation in hypopharyngeal glands of honeybee (Apis mellifera carnica) workers after imidacloprid or coumaphos treatment. Apidologie. 41 (1), 73-86 (2010).
- Gregorc, A., Bowen, I. D. The histochemical characterisation of cell death in honeybee larvae midgut after treatment with Paenibacillus larvae, Amitraz and Oxytetracycline. Cell Biology International. 24 (5), 319-324 (2000).
- Higes, M., et al. Apoptosis in the pathogenesis of Nosema ceranae (Microsporidia: Nosematidae) in honey bees (Apis mellifera). Environmental Microbiology Reports. 5 (4), 530-536 (2013).
- Kurze, C., et al. Infection dynamics of Nosema ceranae in honey bee midgut and host cell apoptosis. Journal of Invertebrate Pathology. 154, 1-4 (2018).
- Johnson, R. M. Honey bee toxicology. Annual Review of Entomology. 60 (1), 415-434 (2005).
- Gashout, H. A., Guzman-Novoa, E., Goodwin, P. H. Synthetic and natural acaricides impair hygienic and foraging behaviors of honey bees. Apidologie. 51 (6), 1155-1165 (2020).
- McMenamin, A. J., Genersch, E. Honey bee colony losses and associated viruses. Current Opinion in Insect Science. 8, 121-129 (2015).
- Ramsey, S. D., et al. Varroa destructor feeds primarily on honey bee fat body tissue and not hemolymph. Proceedings of the National Academy of Sciences USA. 116 (5), 1792-1801 (2019).
- Requier, F., et al. Honey bee diet in intensive farmland habitats reveals an unexpectedly high flower richness and a major role of weeds. Ecological Applications. 25 (4), 881-890 (2015).
- Santos, C., Serrão, J. Histology of the ileum in bees (Hymenoptera, Apoidea). Brazilian Journal of Morphological Sciences. 23 (3), 405-413 (2006).
- Suwannapong, G., Saichon, C., Benbow, M. Histochemical comparison of the hypopharyngeal gland in Apis cerana Fabricius, 1793 workers and Apis mellifera Linnaeus, 1758 workers. Psyche: A Journal of Entomology. , (2010).
- Ceylan, A., Sevin, S., Özgenç, &. #. 2. 1. 4. ;. Histomorphological and histochemical structure of the midgut and hindgut of the Caucasian honey bee (Apis mellifera caucasia). Turkish Journal of Veterinary and Animal Sciences. 43 (6), 747-753 (2019).
- Human, H., et al. Miscellaneous standard methods for Apis mellifera research. Journal of Apicultural Research. 52 (4), 1-53 (2013).
- Gregorc, A., Smodiš Škerl, M. I. Toxicological and immunohistochemical testing of honeybees after oxalic and rotenone treatments. Apidologie. 38 (3), 296-305 (2007).
- Carreck, N. L., et al. Standard methods for Apis mellifera anatomy and dissection. Journal of Apicultural Research. 52 (4), 1-40 (2013).
- Bowen, I. D., Bowen, S. M., Jones, A. H. . Mitosis and apoptosis: Matters of Life and Death. , (1998).
- Eisenberg-Lerner, A., et al. Life and death partners: apoptosis, autophagy and the cross-talk between them. Cell Death and Differentiation. 16 (7), 966-975 (2009).
- Bowen, I. D., Mullarkey, K., Morgan, S. M. Programmed cell death in the salivary gland of the blow fly Calliphora vomitoria. Microscopy Research Techniques. 34, 202-207 (1996).
- D’Arcy, M. S. Cell death: a review of the major forms of apoptosis, necrosis and autophagy. Cell Biology International. 43 (6), 582-592 (2019).
- Matylevitch, N. P., et al. Apoptosis and accidental cell death in cultured human keratinocytes after thermal injury. American Journal of Pathology. 153 (2), 567-577 (1998).
- Perry, S. W., Epstein, L. G., Gelbard, H. A. Simultaneous in situ detection of apoptosis and necrosis in monolayer cultures by TUNEL and trypan blue staining. BioTechniques. 22 (6), 1102-1106 (1997).
- Cuello-Carrion, F. D., Ciocca, D. Improved detection of apoptotic cells using a modified in situ TUNEL technique. Journal of Histochemistry and Cytochemistry. 47 (6), 837-839 (1999).
- Gregorc, A., Bowen, I. D. Programmed cell death in the honeybee (Apis mellifera L.) larvae midgut. Cell Biology International. 21 (3), 151-158 (1997).
- Gregorc, A., Pogačnik, A., Bowen, I. D. Cell death in honey bee (Apis mellifera) larvae treated with oxalic or formic acid. Apidologie. 35 (5), 453-460 (2004).
Citar este artigo
Smodiš Škerl, M. I. Histology Basics and Cell Death Detection in Honeybee Tissue. J. Vis. Exp. (185), e64141, doi:10.3791/64141 (2022).