Jonsson Per R, Wrange Anna-Lisa, Lind Ulrika, Abramova Anna, Ogemark Martin, Blomberg Anders
Department of Marine Sciences, University of Gothenburg.
IVL Swedish Environmental Research Institute.
J Vis Exp. 2018 Aug 8(138):57825. doi: 10.3791/57825.
Barnacles are marine crustaceans with a sessile adult and free-swimming, planktonic larvae. The barnacle Balanus (Amphibalanus) improvisus is particularly relevant as a model for the studies of osmoregulatory mechanisms because of its extreme tolerance to low salinity. It is also widely used as a model of settling biology, in particular in relation to antifouling research. However, natural seasonal spawning yields an unpredictable supply of cyprid larvae for studies. A protocol for the all-year-round culturing of B. improvisus has been developed and a detailed description of all steps in the production line is outlined (i.e., the establishment of adult cultures on panels, the collection and rearing of barnacle larvae, and the administration of feed for adults and larvae). The description also provides guidance on troubleshooting and discusses critical parameters (e.g., the removal of contamination, the production of high-quality feed, the manpower needed, and the importance of high-quality seawater). Each batch from the culturing system maximally yields roughly 12,000 nauplii and can deliver four batches in a week, so up to almost 50,000 larvae per week can be produced. The method used to culture B. improvisus is, probably, to a large extent also applicable to other marine invertebrates with free-swimminglarvae. Protocols are presented for the dissection of various tissues from adults as well as the production of high-quality RNA for studies on gene expression. It is also described how cultured adults and reared cyprids can be utilized in a wide array of experimental designs for examining gene expression in relation to external factors. The use of cultured barnacles in gene expression is illustrated with studies of possible osmoregulatory roles of Na/K ATPase and aquaporins.
藤壶是一种海洋甲壳类动物，成年后固着生活，幼虫则自由游动，处于浮游生物阶段。由于对低盐度具有极强的耐受性，即兴藤壶（Amphibalanus improvisus）作为渗透调节机制研究的模型特别有意义。它还被广泛用作附着生物学的模型，尤其是在防污研究方面。然而，自然季节性产卵为研究提供的无节幼体供应不可预测。现已开发出一种全年培养即兴藤壶的方案，并概述了生产线所有步骤的详细描述（即，在面板上建立成年藤壶培养物、收集和饲养藤壶幼虫，以及为成年藤壶和幼虫投喂饲料）。该描述还提供了故障排除指南，并讨论了关键参数（例如，去除污染、生产高质量饲料、所需人力以及优质海水的重要性）。培养系统的每一批次最多可产出约12000个无节幼体，每周可产出四批次，因此每周最多可生产近50000个幼虫。用于培养即兴藤壶的方法在很大程度上可能也适用于其他具有自由游动幼虫的海洋无脊椎动物。文中介绍了从成年藤壶解剖各种组织以及生产用于基因表达研究的高质量RNA的方案。还描述了如何将培养的成年藤壶和饲养的无节幼体用于各种实验设计，以研究与外部因素相关的基因表达。通过对钠钾ATP酶和水通道蛋白可能的渗透调节作用的研究，说明了培养的藤壶在基因表达研究中的应用。
