低容量通气培养系统的初始开发部分得到了美国国家科学基金会（CRI-OA-1416690 给迪金森学院）的支持。Lauren Mullineaux 博士慷慨地为伍兹霍尔海洋研究所提供了实验室设施，在那里收集了该系统提供的数据（图 4）。

1. 建立和维持 C. fornicata 幼虫培养物的常规操作
注意:这种方法从一加仑（3.8 升）罐装海水开始，其中含有刚刚释放育雏的 Veliger 幼虫的成年 C. fornicata 。成虫可以实地采集或从 材料表中 给出的供应商处获得。成虫是原生雄性雌雄同体，生活在交配堆中，无柄育雏雌性生活在堆底。不要拆散成人堆栈。繁殖的季节性和调节成虫以在非季节产卵的方法之前已经描述过17。幼虫最好在释放后 2-3 小时内收集，此时它们具有很强的地理负性，并且会集中在罐子表面附近。
<ol><li>切下 400 mL 三角一次性塑料烧杯的底部，然后粘在 236 μm 尼龙网板上，制成筛子。为此，特别是对于 mesocosm 结构（如下），请使用对聚乙烯具有良好的粘合力的热熔胶。</li>
	<li>从成虫罐中取出曝气物，静置 15-20 分钟，以便碎屑沉淀，幼虫可以轻松游到水面。</li>
	<li>通过固定在 60-80 厘米长的软塑料水族箱管末端的短 （15-20 cm） 5 mm 玻璃管将幼虫虹吸到如上所述制备的筛子中，悬浮在 600 mL 玻璃烧杯中，使多余的海水溢出玻璃烧杯，而幼虫保留在筛子上。将筛子底部保持在水下，避免搁浅幼虫。</li>
	<li>将筛子短暂地从烧杯中取出，并使用过滤海水 （FSW） 的喷瓶将幼虫冲洗到一个玻璃碗中，该玻璃碗的大小便于在低倍解剖显微镜下进行操作。 注意:C. fornicata 的幼虫在盐度接近 30 ppt 的过滤天然海水或 20-25 °C17,18 的盐度高达 32 ppt. 的 Instant Ocean 人工海水中茁壮成长。< 1 μm 的过滤足以消除可能在实验室培养中引起问题的污染流行性原生生物。</li>
	<li>使用巴斯德移液管手动将所需数量的幼虫转移到培养罐中并计数。为获得最佳结果，请使用 1 只幼虫/5 mL 培养体积的 C. fornicata。数一数幼虫，避免估计数量的诱惑。</li>
	<li>当培养开始时，用所需口粮的微藻喂养幼虫，当更换培养水时，每隔一天更换一次微藻。 注意:1 x 105 个细胞/mL 等黄藻 （菌株 T-ISO）的饮食是一种很好的标准饮食，可产生接近最高的实验室生长速率。食物配给和 T-ISO 的培养方法在早期出版物中提供17。</li>
	<li>如上面的步骤 1.3 所示，将培养物的内容物倒入烧杯内的筛子中，每隔一天更换一次培养水。取出筛子，用 FSW 喷瓶将幼虫冲洗到装有新食物的新鲜 FSW 培养罐中。 注意:最佳做法是将用于幼虫培养的任何玻璃器皿分开，并避免该玻璃器皿与固定剂、可溶性重金属盐或清洁剂接触。日常清洁最好使用小苏打糊 （NaHCO3） 和尼龙擦洗垫进行，而矿物质沉积物可以用白醋或稀释的 HCl 进行温和的酸洗去除。</li>
</ol>2. C. fornicata 幼虫通风培养物的构建
注意:推荐的玻璃罐（材料表）有一个聚丙烯盖，该盖对海水呈惰性，并且具有适合固定管道倒钩入口的厚度，用于通风气流。
<ol><li>在每个培养瓶（包括盖子的内衬）的盖子上钻两个 5 毫米的孔，每个孔距边缘约 1.5 厘米，并且彼此相对。（13/64 英寸英制或 #7 ASME 标准机械师的钻头也将制作合适的间隙孔）。</li>
	<li>使用 10-32 尼龙螺母在每个孔中安装一个 1/8 英寸 x 10-32 螺纹尼龙管适配器，管子倒钩位于盖子的外表面。</li>
	<li>在其中一个管道适配器的螺纹内部的孔上涂抹少许硅橡胶水族密封剂，然后从内部将 20 厘米长的 2 毫米（外径）聚乙烯管推入孔中，使其突出几毫米超出管道倒钩的外孔。</li>
	<li>现在，管材的末端将有一小块水族密封剂塞，从管材倒钩中伸出，但在倒钩末端之外可以看到透明管材。让密封剂固化，然后修剪聚乙烯管的突出端，使其与管倒钩的末端齐平。</li>
	<li>用 FSW 填充培养瓶，使其距离罐顶部肩部 1 cm 以内，拧下盖子，然后将通风空气或实验气体混合物供应连接到固定聚乙烯通气管的管道倒钩上。修剪通风管的长度，使其末端整齐地位于罐子的底部。</li>
	<li>调整通风空气或气体混合物的流量，以产生缓慢的气泡流（图 1）。使用带有普通水族箱联阀的水族箱气泵为环境空气供应通风，或将质量流量控制器用于其他实验性大气气体混合物14。</li>
</ol>3. 构建 C. fornicata 幼虫的现场可部署中宇宙培养物
<ol><li>在标准 7 加仑聚乙烯桶的侧面切 4 个均匀分布的矩形开口，每个开口 25 厘米 x 14.5 厘米。将每个开口的底部放置在距离桶内底部约 3.5 厘米的位置（图 2A）。带有细齿刀片的手持式电刀锯是实现此目的的理想选择。保留切出的废料，用台锯将它们纵向切成 2 厘米宽的条状。</li>
	<li>剪下 4 块 236 μm 尼龙网，每块至少 30 cm x 20 cm，以便舒适地重叠（至少 2 cm）桶中的切口开口。使用小弹簧夹或衣夹将网面板的一个边缘暂时固定在其切口的长边上。</li>
	<li>用专为聚乙烯配制的热熔胶将网板的边缘粘到桶口上。固定第一个边缘后，在保持张力的同时粘合其他边缘，以在网格面板上获得拉紧的表面。</li>
	<li>修剪步骤 3.1 中桶废料条的长度，使加强条整齐地覆盖网板与桶开口重叠的胶合区域。用大量的热熔胶将每个覆盖条压入到位，在胶水硬化之前快速将每个覆盖条压到位。</li>
	<li>用从桶外部安装的尼龙盲铆钉固定每个条带的末端。用实用剃须刀从加固条的外边缘修剪掉多余的胶水和网眼。 注意:每个铆钉都需要一个 15/64 英寸的导向孔。在 图 2B 中，铆钉的内部（盲）部分在成品 mesocosm 的内部可见。</li>
	<li>将 mesocosm 部署在浮动机架中，该浮动机架将桶的顶部保持在水面以上（图 2C）。所示的浮动机架由 30 厘米的 2 英寸 Schedule 40 PVC 管段制成，与弯头和三通接头粘合在一起，形成一个气密结构，可以容纳一排 4 个复制中宇宙。</li>
	<li>根据当地情况，网板可能会在 1-2 天内结垢，从而阻碍海水通过中层的交换。在这种情况下，通过将污损的中胚层从水中拉出 2/3rd ，并用 2 L 水罐反复从其底部捞入新鲜的中胚层，将幼虫转移到新鲜的中层中。</li>
	<li>执行 20-25 次迭代以转移大部分幼虫，或直到在被污染的中宇宙中不再观察到幼虫。用装有小苏打糊 （NaHCO3） 的软海绵轻轻擦洗网板，然后用自来水冲洗，以清洁被污染的 mesocosm。</li>
</ol>

在实验室和田间条件下培养 Crepidula fornicata 的幼虫

<ol><li>Conklin, E. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+embryology+of+Crepidula%3A+a+contribution+to+the+cell+lineage+and+early+development+of+some+marine+gastropods%5BTitle%5D)+AND+%22Gasteropods%22%5BJournal%5D)">The embryology of Crepidula: a contribution to the cell lineage and early development of some marine gastropods.</a> Gasteropods. , (1897).</li>
<li>Henry, J. J., Collin, R., Perry, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+slipper+snail%2C+Crepidula%3A+an+emerging+lophotrochozoan+model+system%5BTitle%5D)+AND+%22Biol+Bull%22%5BJournal%5D)">The slipper snail, Crepidula: an emerging lophotrochozoan model system.</a> Biol Bull. 218 (3), 211-229 (2010).</li>
<li>Lyons, D. C., Henry, J. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Slipper+snail+tales%3A+how+Crepidula+fornicata+and+Crepidula+atrasolea+became+model+molluscs%5BTitle%5D)+AND+%22Curr+Top+Dev+Biol%22%5BJournal%5D)">Slipper snail tales: how Crepidula fornicata and Crepidula atrasolea became model molluscs.</a> Curr Top Dev Biol. 147, 375-399 (2022).</li>
<li>Blanchard, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Spread+of+the+slipper+limpet+Crepidula+fornicata+%28L.+1758%29+in+Europe.+Current+state+and+consequences%5BTitle%5D)+AND+%22ScientiaMarina%22%5BJournal%5D)">Spread of the slipper limpet Crepidula fornicata (L. 1758) in Europe. Current state and consequences.</a> ScientiaMarina. 61 (Suppl 2), 109-118 (1997).</li>
<li>Beninger, P., Valdizan, A., Decottigies, P., Cognie, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Field+reproductive+dynamics+of+the+invasive+slipper+limpet%2C+Crepidula+fornicata%5BTitle%5D)+AND+%22J+Exp+Mar+Biol+Ecol%22%5BJournal%5D)">Field reproductive dynamics of the invasive slipper limpet, Crepidula fornicata.</a> J Exp Mar Biol Ecol. 390 (2), 179-187 (2010).</li>
<li>Pechenik, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+relationship+between+temperature%2C+growth+rate%2C+and+duration+of+planktonic+life+for+larvae+of+the+gastropod+Crepidula+fornicata+%28L.%29%5BTitle%5D)+AND+%22J+Exp+Mar+Biol+Ecol%22%5BJournal%5D)">The relationship between temperature, growth rate, and duration of planktonic life for larvae of the gastropod Crepidula fornicata (L.).</a> J Exp Mar Biol Ecol. 74 (3), 241-257 (1984).</li>
<li>Pechenik, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Latent+effects%3A+surprising+consequences+of+embryonic+and+larval+experience+on+life+after+metamorphosis%5BTitle%5D)+AND+%22Evol+Ecol+Marine+Invertebrate+Larvae%22%5BJournal%5D)">Latent effects: surprising consequences of embryonic and larval experience on life after metamorphosis.</a> Evol Ecol Marine Invertebrate Larvae. , 208-225 (2018).</li>
<li>Pechenik, J. A., Gee, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Onset+of+metamorphic+competence+in+larvae+of+the+gastropod+Crepidula+fornicata+%28L.%29%2C+judged+by+a+natural+and+an+artificial+cue%5BTitle%5D)+AND+%22J+Exp+Mar+Biol+Ecol%22%5BJournal%5D)">Onset of metamorphic competence in larvae of the gastropod Crepidula fornicata (L.), judged by a natural and an artificial cue.</a> J Exp Mar Biol Ecol. 167 (1), 59-72 (1993).</li>
<li>Taris, M., Comtet, T., Viard, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Inhibitory+function+of+nitric+oxide+on+the+onset+of+metamorphosis+in+competent+larvae+of+Crepidula+fornicata%3A+A+transcriptional+perspective%5BTitle%5D)+AND+%22Mar+Genomics%22%5BJournal%5D)">Inhibitory function of nitric oxide on the onset of metamorphosis in competent larvae of Crepidula fornicata: A transcriptional perspective.</a> Mar Genomics. 2 (3-4), 161-167 (2009).</li>
<li>Penniman, J. R., Doll, M. K., Pires, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neural+correlates+of+settlement+in+veliger+larvae+of+the+gastropod%2C+Crepidula+fornicata%5BTitle%5D)+AND+%22Invertebrate+Biol%22%5BJournal%5D)">Neural correlates of settlement in veliger larvae of the gastropod, Crepidula fornicata.</a> Invertebrate Biol. 132 (1), 14-26 (2013).</li>
<li>Meyer-Kaiser, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Carryover+effects+of+brooding+conditions+on+larvae+in+the+slipper+limpet+Crepidula+fornicata%5BTitle%5D)+AND+%22Mar+Ecol+Prog+Ser%22%5BJournal%5D)">Carryover effects of brooding conditions on larvae in the slipper limpet Crepidula fornicata.</a> Mar Ecol Prog Ser. 643, 87-97 (2020).</li>
<li>Noisette, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Does+encapsulation+protect+embryos+from+the+effects+of+ocean+acidification%3F+The+example+of+Crepidula+fornicata%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Does encapsulation protect embryos from the effects of ocean acidification? The example of Crepidula fornicata.</a> PLoS One. 9 (3), e93021(2014).</li>
<li>Kriefall, N. G., Pechenik, J. A., Pires, A., Davies, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Resilience+of+Atlantic+slippersnail+Crepidula+fornicata+larvae+in+the+face+of+severe+coastal+acidification%5BTitle%5D)+AND+%22Front+in+Mar+Sci%22%5BJournal%5D)">Resilience of Atlantic slippersnail Crepidula fornicata larvae in the face of severe coastal acidification.</a> Front in Mar Sci. 5, 312(2018).</li>
<li>Bogan, S. N., McMahon, J. B., Pechenik, J. A., Pires, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Legacy+of+multiple+stressors%3A+Responses+of+gastropod+larvae+and+juveniles+to+ocean+acidification+and+nutrition%5BTitle%5D)+AND+%22Biol+Bull%22%5BJournal%5D)">Legacy of multiple stressors: Responses of gastropod larvae and juveniles to ocean acidification and nutrition.</a> Biol Bull. 236 (3), 159-173 (2019).</li>
<li>Pechenik, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Impact+of+ocean+acidification+on+growth%2C+onset+of+competence%2C+and+perception+of+cues+for+metamorphosis+in+larvae+of+the+slippershell+snail%2C+Crepidula+fornicata%5BTitle%5D)+AND+%22Mar+Biol%22%5BJournal%5D)">Impact of ocean acidification on growth, onset of competence, and perception of cues for metamorphosis in larvae of the slippershell snail, Crepidula fornicata.</a> Mar Biol. 166, 128(2019).</li>
<li>Reyes, C. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+marine+gastropod+Crepidula+fornicata+remains+resilient+to+ocean+acidification+across+two+life+history+stages%5BTitle%5D)+AND+%22Front+Physiol%22%5BJournal%5D)">The marine gastropod Crepidula fornicata remains resilient to ocean acidification across two life history stages.</a> Front Physiol. 12, 702864(2021).</li>
<li>Pires, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Artificial+seawater+culture+of+the+gastropod+Crepidula+fornicata+for+studies+of+larval+settlement+and+metamorphosis.+In%3A+Carroll%2C+D.%2C+Stricker%2C+S.+%28eds%29+Developmental+biology+of+the+sea+urchin+and+other+marine+invertebrates%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">Artificial seawater culture of the gastropod Crepidula fornicata for studies of larval settlement and metamorphosis. In: Carroll, D., Stricker, S. (eds) Developmental biology of the sea urchin and other marine invertebrates.</a> Methods Mol Biol. 1128, 35-44 (2014).</li>
<li>Bashevkin, S. M., Pechenik, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+interactive+influence+of+temperature+and+salinity+on+larval+and+juvenile+growth+in+the+gastropod+Crepidula+fornicata+%28L.%29%5BTitle%5D)+AND+%22J+Exp+Mar+Biol+Ecol%22%5BJournal%5D)">The interactive influence of temperature and salinity on larval and juvenile growth in the gastropod Crepidula fornicata (L.).</a> J Exp Mar Biol Ecol. 470, 78-91 (2015).</li>
<li>Strathmann, M. F. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aStrathmann%2C+MF+%22Reproduction+and+Development+of+Marine+Invertebrates+of+the+Northern+Pacific+Coast%3A+Data+and+Methods+for+the+Study+of+Eggs%2C+Embryos%2C+and+Larvae%22">Reproduction and Development of Marine Invertebrates of the Northern Pacific Coast: Data and Methods for the Study of Eggs, Embryos, and Larvae</a>. , University of Washington Press. (1987).</li>
<li>Clark, H. R., Gobler, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Diurnal+fluctuations+in+CO2+and+dissolved+oxygen+concentrations+do+not+provide+a+refuge+from+hypoxia+and+acidification+for+early-life-stage+bivalves%5BTitle%5D)+AND+%22Mar+Ecol+Prog+Ser%22%5BJournal%5D)">Diurnal fluctuations in CO2 and dissolved oxygen concentrations do not provide a refuge from hypoxia and acidification for early-life-stage bivalves.</a> Mar Ecol Prog Ser. 558, 43114(2016).</li>
<li>Gobler, C. J., Baumann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hypoxia+and+acidification+in+ocean+ecosystems%3A+coupled+dynamics+and+effects+on+marine+life%5BTitle%5D)+AND+%22Biol+Lett%22%5BJournal%5D)">Hypoxia and acidification in ocean ecosystems: coupled dynamics and effects on marine life.</a> Biol Lett. 12 (5), 20150976(2016).</li>
<li>Pechenik, J. A., Hilbish, T. J., Eyster, L. S., Marshall, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Relationship+between+larval+and+juvenile+growth+rates+in+two+marine+gastropods%2C+Crepidula+plana+and+C%5BTitle%5D)+AND+%22fornicata.+Mar+Biol%22%5BJournal%5D)">Relationship between larval and juvenile growth rates in two marine gastropods, Crepidula plana and C.</a> fornicata. Mar Biol. 125, 119-127 (1996).</li>
<li>Padilla, D. K., McCann, M. J., McCarty Glenn, M., Hooks, A. P., Shumway, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effect+of+food+on+metamorphic+competence+in+the+model+system+Crepidula+fornicata%5BTitle%5D)+AND+%22Biol+Bull%22%5BJournal%5D)">Effect of food on metamorphic competence in the model system Crepidula fornicata.</a> Biol Bull. 227 (3), 242-251 (2014).</li>
<li>Wallace, R. B., Baumann, H., Grear, J. S., Aller, R. C., Gobler, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Coastal+ocean+acidification%3A+the+other+eutrophication+problem%5BTitle%5D)+AND+%22Estuarine%2C+Coast+Shelf+Sci%22%5BJournal%5D)">Coastal ocean acidification: the other eutrophication problem.</a> Estuarine, Coast Shelf Sci. 148, 1-13 (2014).</li>
</ol>

没有需要报告的利益冲突。

尽管与其他浮游营养海洋幼虫相比，C. fornicata 的幼虫相对容易养殖，但关注良好养殖规范的基本原则仍然是必不可少的17,19。健康的幼虫应在孵化后立即开始摄食。这在孵化后的第二天通过使用具有透射功能的解剖显微镜观察它们充满藻类细胞的完整肠道，很容易验证。健康幼虫的壳应保持清洁、明亮，并且没有茎纤毛原生生物或无柄羽状硅藻?...

拖鞋帽贝 Crepidula fornicata （腹足纲:Calyptraeidae） 在当前和历史研究文献中得到了很好的体现，因为它作为一种发展模式的实用性以及它作为入侵物种的广泛影响。它是实验胚胎学经典时代螺旋发育的基础例子1，并且随着现代成像和基因组工具的应用来剖析 Lophotrochozoan 早期发育的机制，它经历了兴趣的重生 2,3。在其生命周期历史的另一端，其他研究集中在这种生态系统工程师的成年种群对温带沿海海洋环境的影响，这些海洋环境远非其在北美东部的原始分布 4,5。在胚胎和成虫之间，该物种的绒毛幼虫一直是幼虫发育和生态学的大量研究的主题，特别是影响生长和获得能力的因素，介导幼虫定居的内部和外部线索，以及幼虫经历对幼虫性能的影响 6,7,8,9,10,11 .最近的研究揭示了 C. fornicata 的幼虫和幼虫对海洋酸化的适应能力，这是这种动物的另一种生产性研究利用途径 12,13,14,15,16。
C. fornicata 用于海洋幼虫生物学研究的一个优点是，它在实验室中相对容易在天然或人工海水中以鞭毛状 Isochrysis galbana 的单一饮食生长。作者在早期以方法为重点的印刷出版物中详细介绍了培养方法17。目前做出贡献的原因有两个。首先，建立和照顾培养物所涉及的日常物理操作在概念上非常简单，但如果没有动手或视频演示，很难正确执行。其次，描述了先前描述的培养方法的两种变体，它们特别适用于对环境压力源（如海洋酸化、富营养化和氧气消耗）的反应的实验室和现场研究。其中第一个是小容量 （800 mL） 培养系统，适用于通过少量气泡气体控制海水中的 pH 值和溶解氧，第二个是较大容量 （15 L） 的中层系统，可以放置在田间并允许自由交换环境海水。

<table><tbody><tr><td>Bucket, Polyethylene, 7 gallon</td><td>US Plastic</td><td>16916</td><td>for mesocosm</td></tr><tr><td>&lt;em&gt;Crepidula fornicata&lt;/em&gt;</td><td>Marine Biological Laboratory, Marine Resources Center</td><td>760</td><td>adult broodstock</td></tr><tr><td>Hotmelt glue, Infinity Supertac 500</td><td>Hotmelt.com</td><td>INFINITY IM-SUPERTAC-500-12-1LB</td><td>good for bonding polyethylene</td></tr><tr><td>Jar, glass, 32 oz, with polypropylene lid</td><td>Uline</td><td>S-19316P-W</td><td>for 800 mL ventilated cultures</td></tr><tr><td>Nitex mesh, 236 &amp;micro;m</td><td>Dynamic Aqua Supply Ltd.</td><td>NTX236-136</td><td>for mesocosm</td></tr><tr><td>Nut, hex, nylon, 10-32 thread</td><td>Home Depot</td><td>1004554441</td><td>for fastening tubing barbs</td></tr><tr><td>Rivets, nylon, blind, 15/64&quot; diameter, 5/32&quot;-5/16&quot; grip range, pack of 8</td><td>NAPA auto parts</td><td>BK 6652844</td><td>4 packs needed per mesocosm</td></tr><tr><td>Tubing barb 1/8&quot; x 10-32 thread</td><td>US Plastic</td><td>65593</td><td>2 needed per culture jar</td></tr><tr><td>Tubing, polyethylene, 2.08 mm OD</td><td>Fisher Scientific</td><td>14-170-11G</td><td>for ventilating gas stream inside culture jar</td></tr><tr><td>Tubing, Tygon, 1/8&quot;x3/16&quot;x1/32&quot;</td><td>US Plastic</td><td>57810</td><td>fits barbs for ventilating cultures</td></tr></tbody></table>

laboratory and field culture of larvae of the slipper limpet, crepidula fornicata

蛾蛾腹足类软体动物 Crepidula fornicata 已广泛用于幼虫发育生物学、生理学和生态学的研究。该物种的育雏绒幼虫在成虫自然释放后通过虹吸到筛子上收集，以 200/L 的密度分配到培养物中，并以 1 x 105 个细胞/mL 的密度喂食 Isochrysis galbana（菌株 T-ISO）。记录了在通风的 800 mL 培养物中饲养的兄弟幼虫的壳生长和能力的获得，这些培养物旨在平衡与环境空气或确定的大气气体混合物。与这些实验室培养条件形成对比;还收集了在位于繁殖成虫田间种群中的 15 L 流过环境海水中层饲养的幼虫的生长和能力数据。实验室培养物中能力的生长速率和时间与先前发表的研究中报道的相似。在田间中饲养的幼虫生长得比任何实验室研究报道的要快得多，也早得多。总之，这些方法适用于在实验室中预定的受控条件下以及在田间自然发生的条件下探索幼虫发育。

在 800 mL 通气培养物的 4 个同时重复中测量幼虫生长和能力的获得，每个重复包含 160 只幼虫，来自从单个卵块孵化的兄弟姐妹批次的幼虫，并以 1 x 105 个细胞/mL 的密度喂食 Isochrysis galbana。pH 值为 7.9-8.0，温度为 20-21 °C，盐度为 30-31 ppt。在美国马萨诸塞州秃鹰湾部署的 15 L 中层环境中，还用不同兄弟批次的幼虫测定了生长和，该试验中含有 600 只幼虫，在 pH 值、温度和盐度与实验?...

Read this Scientific Article Protocol about Laboratory and Field Culture of Larvae of The Slipper Limpet, Crepidula fornicata at JoVE.com

作者聚焦:弥合幼虫生态学研究中现场观察和实验室操作之间的差距

本文介绍了在小容量实验室规模系统和可在现场部署的环境海水中宇宙系统中培养腹足类 Crepidula fornicata 幼虫的方法。

鹦鹉幼虫 Crepidula fornicata 幼虫的实验室和田间培养

The calyptraeid gastropod mollusk, Crepidula fornicata, has been widely used for studies of larval developmental biology, physiology, and ecology. Brooded ...

我对海洋幼虫的发育如何受到多种环境因素和压力源的影响以及幼虫神经系统如何整合来自环境的信息来调节幼虫的定居、为底栖幼虫以及招募到成年种群感兴趣。一些海洋物种的幼虫，如拖鞋帽贝 （Crepidula Fornicata），对海洋酸化具有很强的适应能力，但酸化与其他因素（如营养和盐度）之间存在重要的相互作用。我们对幼虫在自然界中的生长、发育和行为知之甚少。野外中宇宙观测可以填补其中一些空白，并提出假设以通过实验室操作进行测试。我们可以开始在神经行为、发育和转录层面上询问幼虫如何在特别容易受到人类影响的生产性沿海环境中处理短时间变化的压力源。

laboratory-field-culture-larvae-slipper-limpet-crepidula

Research

JoVE Journal

Biology

Laboratory and Field Culture of Larvae of The Slipper Limpet, Crepidula fornicata

414 次观看.  Dickinson College. 我对海洋幼虫的发育如何受到多种环境因素和压力源的影响以及幼虫神经系统如何整合来自环境的信息来调节幼虫的定居、为底栖幼虫以及招募到成年种群感兴趣。一些海洋物种的幼虫，如拖鞋帽贝 （Crepidula Fornicata），对海洋酸化具有很强的适应能力，但酸化与其他因素（如营养和盐度）之间存在重要的相互作用。我们对幼虫在自然界中的生长、发育和行为知之甚少。

视频: 作者聚焦:弥合幼虫生态学研究中现场观察和实验室操作之间的差距

Delivery of Nucleic Acids through Embryo Microinjection in the Worldwide Agricultural Pest Insect, Ceratitis capitata

The Mediterranean fruit fly (medfly) Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) is a pest species with extremely high agricultural relevance. This is due to its reproductive behavior: females damage the external surface of fruits and vegetables when they lay eggs and the hatched larvae feed on their pulp. Wild C. capitata populations are traditionally controlled through insecticide spraying and/or eco-friendly approaches, the most successful being the Sterile Insect Technique (SIT). The SIT relies on mass-rearing, radiation-based sterilization and field release of males that retain their capacity to mate but are not able to generate fertile progeny. The advent and the subsequent rapid development of biotechnological tools, together with the availability of the medfly genome sequence, has greatly boosted our understanding of the biology of this species. This favored the proliferation of new strategies for genome manipulation, which can be applied to population control.
In this context, embryo microinjection plays a dual role in expanding the toolbox for medfly control. The ability to interfere with the function of genes that regulate key biological processes, indeed, expands our understanding of the molecular machinery underlying medfly invasiveness. Furthermore, the ability to achieve germ-line transformation facilitates the production of multiple transgenic strains that can be tested for future field applications in novel SIT settings. Indeed, genetic manipulation can be used to confer desirable traits that can, for example, be used to monitor sterile male performance in the field, or that can result in early life-stage lethality. Here we describe a method to microinject nucleic acids into medfly embryos to achieve these two main goals.

Delivery of Nucleic Acids through Embryo Microinjection in the Worldwide Agricultural Pest Insect, Ceratitis capitata

The Mediterranean fruit fly (medfly) Ceratitis capitata (Diptera: Tephritidae) is a worldwide pest of agriculture. A deeper understanding of its biology is key to control medfly populations and thus reduce economic impact. Embryo microinjection is a fundamental tool allowing both germ-line transformation and reverse genetics studies in this species.

核酸的传递通过显微注射胚胎在世界范围内的农业害虫，<em&gt;地中海实蝇</em

The Mediterranean fruit fly (medfly) Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) is a pest species with extremely high agricultural relevance. ...

科研

遗传学

Use of Chironomidae (Diptera) Surface-Floating Pupal Exuviae as a Rapid Bioassessment Protocol for Water Bodies

Rapid bioassessment protocols using benthic macroinvertebrate assemblages have been successfully used to assess human impacts on water quality. Unfortunately, traditional benthic larval sampling methods, such as the dip-net, can be time-consuming and expensive. An alternative protocol involves collection of Chironomidae surface-floating pupal exuviae (SFPE). Chironomidae is a species-rich family of flies (Diptera) whose immature stages typically occur in aquatic habitats. Adult chironomids emerge from the water, leaving their pupal skins, or exuviae, floating on the water&#8217;s surface. Exuviae often accumulate along banks or behind obstructions by action of the wind or water current, where they can be collected to assess chironomid diversity and richness. Chironomids can be used as important biological indicators, since some species are more tolerant to pollution than others. Therefore, the relative abundance and species composition of collected SFPE reflect changes in water quality. Here, methods associated with field collection, laboratory processing, slide mounting, and identification of chironomid SFPE are described in detail. Advantages of the SFPE method include minimal disturbance at a sampling area, efficient and economical sample collection and laboratory processing, ease of identification, applicability in nearly all aquatic environments, and a potentially more sensitive measure of ecosystem stress. Limitations include the inability to determine larval microhabitat use and inability to identify pupal exuviae to species if they have not been associated with adult males.

Use of Chironomidae Diptera Surface-Floating Pupal Exuviae as a Rapid Bioassessment Protocol for Water Bodies

Rapid bioassessment protocols using benthic macroinvertebrates are often used to monitor and assess water quality. An efficient protocol involves collections of Chironomidae surface-floating pupal exuviae (SFPE). Here, techniques for field collection, laboratory processing, slide mounting, and identification of Chironomidae SFPE are described.

使用摇蚊科（双翅目）的表面浮蛹蜕为快速生物议定书水体

Rapid bioassessment protocols using benthic macroinvertebrate assemblages have been successfully used to assess human impacts on water quality. ...

环境科学

Individual Culturing of Tigriopus Copepods and Quantitative Analysis of Their Mate-guarding Behavior

Copepods of the genus Tigriopus, which are common zooplankton in rocky tide pools, show precopulatory mate-guarding behavior where a male clasps a potential mate to form a pair. While this phenomenon has attracted interest of researchers, methods for its analysis have not been well described. Here we describe procedures for: 1) individual culturing and staging of Tigriopus juveniles and adults, and 2) video-based analysis of their mate-guarding behavior. The culturing method enables experimental control of paring experience of animals as well as the ability to track their development before behavioral tests. The analysis method allows quantitative evaluation of several aspects of the mate-guarding behavior, including capturing attempts by males and swimming trajectory of mate-guarding pairs. Although these methods were originally established for ethological studies on Tigriopus, with proper modifications they can also be applied to studies of other zooplankton in different research fields, such as physiology, toxicology, and ecological genetics.

Individual Culturing of Tigriopus Copepods and Quantitative Analysis of Their Mate-guarding Behavior

Mate-guarding behavior plays an important role in reproduction of intertidal copepods of the genus Tigriopus. However, methods for studying this behavior have not been well described. Here we describe methods for: 1) individual culture of virgin Tigriopus animals, and 2) quantitative analysis of their mate-guarding behavior.

Tigriopus桡足的个体培养及其交配行为的定量分析

Copepods of the genus Tigriopus, which are common zooplankton in rocky tide pools, show precopulatory mate-guarding behavior where a male clasps a ...

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires&#39; disease, is an important human pathogen that infects and replicates within alveolar macrophages. Its virulence depends on the Dot/Icm type IV secretion system (T4SS), which is essential to establish a replication permissive vacuole known as the Legionella containing vacuole (LCV). L. pneumophila infection can be modeled in mice however most mouse strains are not permissive, leading to the search for novel infection models. We have recently shown that the larvae of the wax moth Galleria mellonella are suitable for investigation of L. pneumophila infection. G. mellonella is increasingly used as an infection model for human pathogens and a good correlation exists between virulence of several bacterial species in the insect and in mammalian models. A key component of the larvae&#39;s immune defenses are hemocytes, professional phagocytes, which take up and destroy invaders. L. pneumophila is able to infect, form a LCV and replicate within these cells. Here we demonstrate protocols for analyzing L. pneumophila virulence in the G. mellonella model, including how to grow infectious L. pneumophila, pretreat the larvae with inhibitors, infect the larvae and how to extract infected cells for quantification and immunofluorescence microscopy. We also describe how to quantify bacterial replication and fitness in competition assays. These approaches allow for the rapid screening of mutants to determine factors important in L. pneumophila virulence, describing a new tool to aid our understanding of this complex pathogen.

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

The larva of the wax moth Galleria mellonella was recently established as an in vivo model to study Legionella pneumophila infection. Here, we demonstrate fundamental techniques to characterize the pathogenesis of Legionella in the larvae, including inoculation, measurement of bacterial virulence and replication as well as extraction and analysis of infected hemocytes.

使用<em&gt;大蜡螟</em&gt;作为模式生物来研究<em&gt;嗜肺军团菌</em&gt;感染

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires&#39; disease, is an important human pathogen that infects and ...

免疫与感染

Exploring Life History Choices: Using Temperature and Substrate Type as Interacting Factors for Blowfly Larval and Female Preferences

Blowflies (Diptera: Calliphoridae) present a wide range of larval lifestyles, typically classified as obligate parasitism, facultative parasitism, and complete sapro-necrophagy. Several parasitic species, both obligate and facultative, are considered to be of sanitary and economic importance, as their larvae can cause myiasis (maggot infestation in live tissue). However, it is noteworthy that the adult female plays a decisive role as she chooses the oviposition site, and, therefore, largely determines the feeding habit and developmental conditions of the larvae. In this study, two protocols are proposed to test larval feeding preference and female oviposition site preference considering two interacting factors: meat substrate type and temperature. The setups presented here allowed to test Lucilia cuprina larvae and gravid females in a four-choice assay with two temperatures (33 &plusmn; 2 &#176;C and 25 &plusmn; 2 &#176;C) and two types of meat substrates (fresh meat supplemented with blood and 5-day-old rotten meat). Larvae or gravid females can choose to burrow or lay their eggs, respectively, in either of the following: rotten meat at 25 &#176;C (simulating a necrophagous species condition), fresh meat supplemented with blood at 33 &#176;C (simulating a parasitic species condition), and two controls, rotten meat at 33 &#176;C, or fresh meat supplemented with blood at 25 &#176;C. The preference is assessed by counting the number of larvae or eggs laid in each option for each replicate. Comparing the observed results to a random distribution allowed for the estimation of the statistical significance of the preference. The results indicated that L. cuprina larvae have a strong preference for the rotten substrate at 25 &#176;C. Conversely, oviposition-site preference by females was more varied for the meat type. This methodology can be adapted to test the preference of other insect species of similar size. Other questions can also be explored by using alternative conditions.

Herein, two protocols for assessing food source and oviposition preferences in larvae and females of blowflies are detailed. These comprise four choices with two interacting factors: substrate type and temperature. The assays enable the determination of the food source preference of the larvae and the oviposition site preference for the females.

探索生活史选择:使用温度和基质类型作为吹蝇幼虫和雌性偏好的相互作用因素

Blowflies (Diptera: Calliphoridae) present a wide range of larval lifestyles, typically classified as obligate parasitism, facultative parasitism, and ...

行为学

The Galleria mellonella Waxworm Infection Model for Disseminated Candidiasis

Candida species are common fungal commensals of humans colonizing the skin, mucosal surfaces, and gastrointestinal tract. Under certain conditions, Candida can overgrow their natural niches resulting in debilitating mucosal infections as well as life-threatening systemic infections, which are a major focus of investigation due to their associated high mortality rates. Animal models of disseminated infection exist for studying disease progression and dissecting the characteristics of Candida pathogenicity. Of these, the Galleria mellonella waxworm infection model provides a cost-effective experimental tool for high-throughput investigations of systemic virulence. Many other bacterial and eukaryotic infectious agents have been effectively studied in G. mellonella to understand pathogenicity, making it a widely accepted model system. Yet, variation in the method used to infect G. mellonella can alter phenotypic outcomes and complicate interpretation of the results. Here, we outline the benefits and drawbacks of the waxworm model to study systemic Candida pathogenesis and detail an approach to improve reproducibility. Our results highlight the range of mortality kinetics in G. mellonella and describe the variables which can modulate these kinetics. Ultimately, this method stands as an ethical, rapid, and cost-effective approach to study virulence in a model of disseminated candidiasis.

The Galleria mellonella Waxworm Infection Model for Disseminated Candidiasis

Galleria mellonella serves as an invertebrate model for disseminated candidiasis. Here, we detail the infection protocol and provide supporting data for the model&#39;s effectiveness.

弥散性念珠菌感染模型

Candida species are common fungal commensals of humans colonizing the skin, mucosal surfaces, and gastrointestinal tract. Under certain conditions, ...

Laboratory Rearing of Stable Flies and Other Muscoid Diptera

Stable flies, Stomoxys calcitrans, are serious pests of livestock, humans, companion animals and wildlife worldwide. During the last 20+ years, changes in agronomic practices resulted in serious outbreaks of stable flies in several countries. These outbreaks disrupted livestock production and human recreation resulting in public demands for increasing research and management efforts for this pest. A simple and inexpensive procedure for rearing stable flies for laboratory studies is presented. The procedure uses locally available diet components, equipment and supplies. The procedure can be adapted for rearing other muscoid flies including face fly (Musca autumnalis), horn fly (Haematobia irritans), and house fly (Musca domestica). The procedure produces stable fly puparia averaging 12.5 mg and ~35% egg to adult survival. Approximately 3000 flies are produced in each pan.

A procedure for rearing stable flies (Stomoxys calcitrans) is presented. The procedure uses locally available materials for diet components, equipment and supplies.

稳定蝇及其他 Muscoid 双翅目的实验室饲养

Stable flies, Stomoxys calcitrans, are serious pests of livestock, humans, companion animals and wildlife worldwide. During the last 20+ years, changes in ...

生物学

At-Risk Butterfly Captive Propagation Programs to Enhance Life History Knowledge and Effective Ex Situ Conservation Techniques

Improving knowledge of ex situ best practices for at-risk butterflies is important for generating successful conservation and recovery program outcomes. Research on such captive populations can also yield valuable data to address key information gaps about the behavior, life history, and ecology of the target taxa. We describe a protocol for captive propagation of the federally endangered Cyclargus thomasi bethunebakeri that can be used as a model for other at-risk butterfly ex situ programs, especially those in the family Lycaenidae. We further provide a simple and straightforward protocol for recording various life history metrics that can be useful for informing ex situ methodologies as well as adapted for laboratory studies of other lepidoptera.

Here, we present protocols for 1) the laboratory captive propagation of the federally endangered Miami blue butterfly (Cyclargus thomasi bethunebakeri), and 2) assessing basic life history information such as immature development time and number of larval stadia. Both methods can be adapted for use with other ex situ conservation programs.

风险蝴蝶捕获繁殖计划，以提高生命历史知识和有效的异地保护技术

Improving knowledge of ex situ best practices for at-risk butterflies is important for generating successful conservation and recovery program outcomes. ...

Preparing and Rearing Axenic Insects with Tissue Cultured Seedlings for Host-Gut Microbiota Interaction Studies of the Leaf Beetle

Insect guts are colonized by diverse bacteria that can profoundly impact the host&#39;s physiological traits. Introducing a particular bacterial strain into an axenic insect is a powerful method to verify gut microbial function and elucidate the mechanisms underlying gut microbe-host interactions. Administering antibiotics or sterilizing egg surfaces are two commonly used methods to remove gut bacteria from insects. However, in addition to the potential adverse effects of antibiotics on insects, previous studies indicated that feeding antibiotics could not eliminate gut bacteria. Thus, germ-free artificial diets are generally employed to maintain axenic insects, which is a tedious and labor-intensive process that cannot fully resemble nutritional components in natural food. Described here is an efficient and simple protocol to prepare and maintain axenic larvae of a leaf beetle&#160;(Plagiodera versicolora). Specifically, surfaces of the beetle eggs were sterilized, following which germ-free poplar leaves were used to rear axenic larvae. The axenic status of the insects was further confirmed via culture-dependent and culture-independent assays. Collectively, by combining egg disinfection and germ-free cultivation, an efficient and convenient method was developed to obtain axenic P. versicolora, providing a readily transferable tool for other leaf-eating insects.

To obtain an axenic insect, its egg surface is sterilized, and the hatched larva is subsequently reared using axenic leaves. This method provides an efficient way for axenic insect preparation without administering antibiotics or developing an artificial diet, which can also be applied to other leaf-eating insects.

用组织培养幼苗制备和饲养轴性昆虫用于叶甲虫的宿主 - 肠道微生物群相互作用研究

Insect guts are colonized by diverse bacteria that can profoundly impact the host&#39;s physiological traits. Introducing a particular bacterial strain ...

Maintaining Laboratory Cultures of Gryllus bimaculatus, a Versatile Orthopteran Model for Insect Agriculture and Invertebrate Physiology

Gryllus bimaculatus (De Geer)&#160;is a large-bodied cricket distributed throughout Africa and Southern Eurasia where it is often wild-harvested as human food. Outside its native range, culturing G. bimaculatus is feasible due to its dietary plasticity, rapid reproductive cycle, lack of diapause requirement, tolerance for high-density rearing, and robustness against pathogens. Thus, G. bimaculatus can be&#160;a versatile model for studies of insect physiology, behavior, embryology, or genetics.
Cultural parameters, such as stocking density, within-cage refugia, photoperiod, temperature, relative humidity, and diet, all impact cricket growth, behavior, and gene expression and should be standardized. In the burgeoning literature on farming insects for human consumption, these crickets are frequently employed to evaluate candidate feed admixtures derived from crop residues, food-processing byproducts, and other low-cost waste streams.
To support ongoing experiments evaluating G. bimaculatus growth performance and nutritional quality in response to variable feed substrates, a comprehensive set of standard protocols for breeding, upkeep, handling, measurement, and euthanasia in the laboratory was developed and is presented here. An industry-standard cricket feed has proven nutritionally adequate and functionally appropriate for the long-term maintenance of cricket breeding stocks, as well as for use as an experimental control feed. Rearing these crickets at a density of 0.005 crickets/cm3 in screen-topped 29.3 L polyethylene cages at an average temperature of 27 &#176;C on a 12 light (L)/12 dark (D) photoperiod, with moistened coconut coir serving both as hydration source and oviposition medium has successfully sustained healthy crickets over a 2-year span. Following these methods, crickets in a controlled experiment yielded an average mass of 0.724 g 0.190 g at harvest, with 89% survivorship and 68.2% sexual maturation between stocking (22 days) and harvest (65 days).

Maintaining Laboratory Cultures of Gryllus bimaculatus, a Versatile Orthopteran Model for Insect Agriculture and Invertebrate Physiology

This paper outlines basic methods to standardize important factors such as density, feed availability, hydration source, and environmental controls for the long-term rearing of laboratory cultures of the edible cricket, Gryllus bimaculatus.

维持 双斑蝽螈的实验室培养物，这是一种用于昆虫农业和无脊椎动物生理学的多功能正翅目动物模型

Gryllus bimaculatus (De Geer)&#160;is a large-bodied cricket distributed throughout Africa and Southern Eurasia where it is often wild-harvested as human ...

鹦鹉幼虫 Crepidula fornicata 幼虫的实验室和田间培养

副本

摘要

探索更多视频

核酸的传递通过显微注射胚胎在世界范围内的农业害虫，

使用摇蚊科（双翅目）的表面浮蛹蜕为快速生物议定书水体

Tigriopus桡足的个体培养及其交配行为的定量分析

使用

探索生活史选择:使用温度和基质类型作为吹蝇幼虫和雌性偏好的相互作用因素

弥散性念珠菌感染模型

稳定蝇及其他 Muscoid 双翅目的实验室饲养

风险蝴蝶捕获繁殖计划，以提高生命历史知识和有效的异地保护技术

用组织培养幼苗制备和饲养轴性昆虫用于叶甲虫的宿主 - 肠道微生物群相互作用研究

维持双斑蝽螈的实验室培养物，这是一种用于昆虫农业和无脊椎动物生理学的多功能正翅目动物模型

鹦鹉幼虫 Crepidula fornicata 幼虫的实验室和田间培养

副本

摘要

探索更多视频

核酸的传递通过显微注射胚胎在世界范围内的农业害虫，

使用摇蚊科（双翅目）的表面浮蛹蜕为快速生物议定书水体

Tigriopus桡足的个体培养及其交配行为的定量分析

使用

探索生活史选择:使用温度和基质类型作为吹蝇幼虫和雌性偏好的相互作用因素

弥散性念珠菌感染模型

稳定蝇及其他 Muscoid 双翅目的实验室饲养

风险蝴蝶捕获繁殖计划，以提高生命历史知识和有效的异地保护技术

用组织培养幼苗制备和饲养轴性昆虫用于叶甲虫的宿主 - 肠道微生物群相互作用研究

维持 双斑蝽螈的实验室培养物，这是一种用于昆虫农业和无脊椎动物生理学的多功能正翅目动物模型

维持双斑蝽螈的实验室培养物，这是一种用于昆虫农业和无脊椎动物生理学的多功能正翅目动物模型