利用生物测定引导分馏法鉴定海洋鳗信息素
Summary
在这里, 我们提出了一个协议, 以分离和描述的结构, 嗅觉效力, 和行为反应的推测信息素化合物的海洋鳗。
Abstract
生物测定引导分馏是利用生理和行为生物鉴定的结果来指导活性信息素化合物的分离和鉴定的一种迭代方法。这种方法的成功表征了化学信号的功能作为信息素在广泛的动物物种。海洋鳗依赖嗅觉来检测介导行为或生理反应的费洛蒙。我们利用这一知识的鱼类生物学的假设的功能, 假设的信息素, 并指导的分离和鉴定活性的费洛蒙成分。色谱用于从条件水中提取、浓缩和分离化合物。电 olfactogram (EOG) 记录, 以确定哪些分数引出嗅觉反应。两个选择迷宫行为化验, 然后用来确定是否有任何气味的分数也行为上活跃和诱发偏好。光谱和光谱方法提供了分子量和结构信息, 以协助结构的澄清。通过 EOG 和行为学测定, 证实了纯化合物的生物活性。在迷宫中观察到的行为响应最终应在字段设置中进行验证, 以确认它们在自然流设置中的作用。这些生物鉴定发挥双重作用到 1) 指导分馏过程和 2) 确认并进一步确定分离组分的活性。在这里, 我们报告了一个海洋鳗信息素鉴定的代表性结果, 证明了生物测定指导分馏方法的效用。海洋鳗信息素的识别是特别重要的, 因为它是控制劳伦琴大湖侵入性海鳗的选择之一。该方法可以很容易地适应于广泛的分类群中的化学通信, 并揭示了水化学生态学的特点。
Introduction
信息素是个人发布的特定化学信号, 帮助他们找到食物来源, 检测捕食者, 并调解 conspecifics1的社会互动。昆虫信息素的传播研究已有2;然而, 水生脊椎动物信息素的化学鉴定和生物学功能还没有得到广泛的研究。对所释放的信息素的特性和功能的了解可以应用于促进被威胁物种3、4或控制害虫物种5、6的恢复。这些技术的应用需要对生物活性素成分进行分离和表征。
信息素识别是天然产物化学的一个分支。由于信息素分子本身的性质, 信息素研究的进展受到部分限制。信息素往往不稳定, 很少释放, 只有少数抽样技术存在, 以检测微量的挥发性7,8或水溶性化合物9。识别信息素的方法包括 1) 目标筛选已知化合物, 2) 新陈代谢和 3) 生物测定引导分馏。对已知化合物进行有针对性的筛选, 可以检验被推测为费洛蒙功能的生理过程的商业性代谢副产品。这种方法是有限的, 因为研究人员只能测试已知和可用的化合物。然而, 它导致了在金鱼中的性激素的成功识别功能作为费洛蒙10,11,12。新陈代谢是第二种信息素识别方法, 它区分了生物系统中潜在的小分子代谢产物13。对两组代谢剖面 (即活性与非活性提取物) 进行比较, 可以识别出代谢产物纯化的潜在新陈代谢剖面, 阐明结构, 以及生物活性证实14。复杂配方的添加剂或协同效应更可能被发现与新陈代谢, 因为代谢物被考虑在一起, 而不是作为一系列的分数13。然而, 新陈代谢的实施依赖于合成参照的可用性, 因为由此产生的数据不利于澄清新结构。
生物鉴定-引导分馏是一个综合的, 迭代的方法, 跨越两个领域: 化学和生物学。该方法利用生理和行为生物鉴定的结果来指导活性信息素化合物的分离和鉴定。粗萃取物由化学性质 (即分子量、极性等) 分馏, 并用电 olfactogram (EOG) 记录和/或在生物测定中进行测试。通过重复这些步骤的分馏和 EOGs 和/或生物鉴定, 筛选出活性成分。用光谱和光谱方法阐明了纯活性化合物的结构, 提供了分子量和结构信息, 生成了合成的模板。生物测定引导分馏可以产生不同的代谢物和潜在的新的信息素与独特的化学骨骼, 是不可能预测的生物合成途径。
在这里, 我们描述了用于分离和表征雄性海鳗性信息素化合物活性的生物测定制导分馏协议。海鳗 (Petromyzon marinus) 是研究信息素传播的理想脊椎动物模型, 因为这些鱼类大量依赖化学线索的嗅觉检测, 以调解其溯河产卵生命史, 包括三个不同阶段:幼虫, 幼仔和成年。海洋鳗幼虫钻入淡水溪流的沉积物中, 经历剧烈的蜕变, 转化为迁徙到湖泊或海洋的幼鱼, 它们寄生大的寄主鱼类。从宿主鱼分离后, 成虫迁移回产卵流, 由流驻留幼虫15、16、17、18、19 所释放的迁移信息素引导..成熟雄性上升到产卵场, 释放多组分性信息素吸引配偶, 间歇产卵约一周, 然后死15,20。海洋鳗信息素的识别是很重要的, 因为对劳伦琴大湖21的入侵性海洋鳗的控制, 是一种调控费洛蒙传播系统的方法。
Protocol
这里描述的所有方法都已获得密歇根州立大学机构动物保育和使用委员会 (AUF 03/14-054-00 和 02/17-031-00) 的批准。 1. 海洋鳗条件水的收集和提取 安置性成熟男性海鳗 (15-30 个动物) 在供应与 250 L 的被加气的湖休伦的水被维护在 16-18 °c 的坦克。 每晚从琼到7月收集男性条件的水。注: 海洋鳗产卵后自然死亡。如果一条鱼在它的生命中接近这一点, 用新鲜成熟的雄?…
Representative Results
图 1中概述了生物测定导向分馏的协议中描述的步骤。该协议涉及的步骤, 以分离和特征的结构, 嗅觉能力和行为活动的5假定海鳗信息素 (图 2)。利用质谱和核磁共振数据 (图 3和图 4), 从条件与成熟的雄性海鳗22、29的水中阐明了 petromyzene a-B…
Discussion
鱼类生活在一个充满化合物的化学世界中, 但尚未确定。生物鉴定-引导分馏已证明是必不可少的识别和表征活性分子, 调解许多化学相互作用, 如观察鲑鲑鱼31, 亚洲大象32, 海鳗33, 34,35。生物测定引导分馏是一种有效的方法, 可以准确地从起始提取物到纯化活性化合物中精确地跟踪和定位活性?…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
我们感谢美国地质调查哈蒙德湾生物站的使用他们的研究设施和工作人员的美国鱼类和野生动物服务和渔业和海洋加拿大提供海洋鳗。这项研究得到了大湖渔业委员会给予未明李和科李的赠款的支持。
Materials
| Premium standard wall borosilicate capillaries with filament | Warner Instruments | G150F-4 | recording and reference electrode (OD 1.5 mm, ID 0.86 mm) |
| Pipette puller instrument | Narishige | PC-10 | pulls electrodes for EOGs |
| Diamond-tipped glass cutter | Generic | cut tip of electrodes for EOG | |
| Borosilicate glass capillaries | World Precision Instruments | 1B150-4 | odorant delivery tube for EOG |
| Recording electrode holder E Series straight body with Ag/AgCl pellet for glass capillary OD 1.5 mm | Warner Instruments | ESP-M15N | recording electrode holder |
| Reference electrode holder E Series with handle with Ag/AgCl pellet for glass capillary OD 1.5 mm | Warner Instruments | E45P-F15NH | reference electrode holder |
| 1 mm pin | Warner Instruments | WC1-10 | to bridge reference and recording electrode holders |
| 2 mm pin | Warner Instruments | WC2-5 | to bridge reference and recording electrode holders |
| Agar | Sigma | A1296 | molten agar to fill electrodes |
| Potassium chloride (KCl) | Sigma | P9333 | 3M KCl to fill electrodes and electrode holders |
| Micropipette microfil | World Precision Instruments | MF28G-5 | to fill electrodes and electrode holders |
| L-Arginine | Sigma | A5006 | positive control odorant for EOG |
| Methanol | Sigma | 34860 | |
| Water bath | Custom made | N/A | holds odorants for EOG |
| 3-aminobenzoic acid ethyl ester (MS222) | Syndel USA | Tricaine1G | EOG anesthetic |
| Gallamine triethiodide | Sigma | G8134-5G | EOG paralytic |
| 1 mL syringe | BD Biosciences | 301025 | to administer paralytic |
| Subcutaneous needle 26G 5/8 | BD Biosciences | 305115 | to administer paralytic |
| Roller clamp | World Precision Instruments | 14043-20 | adjust flow rate of anesthic into lamprey's mouth |
| Sodium chloride (NaCl) | J.T. Baker | 3624-05 | for preparation of 0.9% saline |
| V-shaped plastic stand as specimen stage | Custom made | N/A | holds lamprey during EOG |
| Plastic trough | Custom made | N/A | holds V-shaped plastic stand during EOG |
| Scalpel Blades – #11 | Fine Science Tools | 10011-00 | for EOG dissection |
| Scalpel Handle – #3 | Fine Science Tools | 10003-12 | for EOG dissection |
| Straight ultra fine forceps | Fine Science Tools | 11252-00 | for EOG dissection, Dumont #5SF Forceps |
| Curved ultra fine forceps | Fine Science Tools | 11370-42 | for EOG dissection, Moria MC40B |
| Straight pring Scissors | Fine Science Tools | 15003-08 | for EOG dissection |
| Stereomicroscope | Zeiss | Discovery V8 | for EOG dissection |
| Illuminator light | Zeiss | CL 1500 ECO | for EOG dissection |
| Plastic tubing | Generic | to connect re-circulating EOG setup and water baths | |
| Odorant delivery tubing | Custom made | N/A | |
| In line filter and gasket set | Lee Company | TCFA1201035A | |
| Micromanipulators | Narishige | MM-3 | to position electrodes and odorant delivery capillary tube |
| Magnetic holding devices | Kanetec | MB-K | |
| Valve driver | Arduino | custom made | to control the opening of the valve for odor stimulation |
| Electromagnetic valve | Lee Company | LFAA1201618H | valve for odor stimulation |
| NeuroLog AC/DC amplifier | Digitimer Ltd. | NL106 | to increase the amplitude of the elictrical signal |
| NeuroLog DC pre-amplifier with headstage | Digitimer Ltd. | NL102G | to increase the amplitude of the elictrical signal |
| Low-pass 60 Hz filter | Digitimer Ltd. | NL125 | |
| Digitizer | Molecular Devices LLC | Axon Digidata 1440A | |
| Dell computer (OptiPlex 745) running Axoscope data acquistion software | Molecular Devices LLC | AxoScope version 10.4 | |
| Faraday cage | Custom made | N/A | Electromagnetic noise shielding |
| Two-choice maze | Custom made | N/A | waterproofed marine grade plywood covered with plastic liner |
| Trash pump | Honda | WT30XK4A | fills maze with water from nearby river |
| Peristaltic pump with tubing | Cole Parmer | Masterflex 07557-00 | to adminster odorants in maze |
| Inverter Generator | Honda | EU1000i | powers perstaltic pump |
| Release cage | Custom made | N/A | used to acclimate lamprey in the maze |
| Mesh | Generic | used to contain the dimensions of the maze and minimize water turbulance with mesh rollers | |
| Buckets (5 gallon) | Generic | to mix odorants | |
| Flow meter | Marsh-McBirney | Flo-Mate 2000 | to measure discharge |
| XAD 7 HP resin | Dow chemical | 37380-43-1 | for extraction of conditioned water |
| Methanol | Sigma | 34860 | for extraction of conditioned water |
| Water bath | Yamato | BM 200 | for extraction of conditioned water |
| Freeze dryer | Labconco | CentriVap Concentrator | for extraction of conditioned water |
| chloroform | Sigma | CX1050 | for isolation of fraction pools |
| Silica gel 70-230 mesh | Sigma | 112926-00-8 | for isolation of fraction pools |
| Silica gel 230-400 mesh | Sigma | 112926-00-8 | for isolation of fraction pools |
| Pre-coated silica gel TLC plates | Sigma | 99571 | for isolation of fraction pools |
| anisaldehyde | Sigma | A88107 | for isolation of fraction pools |
| Sephadex LH-20 | GE Healthcare | 17-0090-01 | for isolation of fraction pools |
| Amberlite XAD 7 HP resin | Sigma | XAD7HP | for extraction of conditioned water |
| 4, 2.5L capacity glass columns | Ace Glass Inc. | 5820 | for extraction of conditioned water |
| Acetone | Sigma | 650501 | for extraction of conditioned water |
| TQ-S TOF LC Mass spectrometer (or equivalent) | Waters Co. | N/A | for structure elucidation |
| Binary HPLC pump | Waters Co. | 1525 | for isolation of fraction pools/compounds |
| Agilent NMR spectrometer, 900MHz (or equivalent) | Agilent | N/A | for structure elucidation |
| Rotovap drying system | Buchi | RII | for extraction of conditioned water |
| UV lamp (254 nm) | Spectronics Co. | ENF-240C | for thin layer chromatography |
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Citar este artículo
Scott, A. M., Li, K., Li, W. The Identification of Sea Lamprey Pheromones Using Bioassay-Guided Fractionation. J. Vis. Exp. (137), e58059, doi:10.3791/58059 (2018).