这项研究是资助部分从收到的美国环境保护局的针对性流域资助计划（项目＃WS97704801）的支持，在美国国家科学基金会的动态，加上自然和人类系统（项目＃DEB1010259-CARD-KLIN），和美国能源部农业研究所，Foodand农业的协调农业项目（项目编号20116800230190-CARD）。

1。准备流域模型，并提供输入数据优化<ol><li>创建一个i_SWAT数据库<ol><li>从多个输入数据库，包括土壤，气候，管理和肥料使用了一项名为“旋转”，建立数据库。 </li><li>另外，现有的SWAT的运行（可能创建与ArcSWAT或AVSWAT）可以被导入i_SWAT.exe中。在这种情况下，程序“swat_rewrite”可以用来取代管理或其他HRU字段级别的数据的基础上的信息的。 </li><li>在这一点上的SWAT模型进行校正和验证。 SWAT模型（2005年版）这的浣熊河流域EA建模框架内成立，最初的最大日负荷总量（TMDL）的研究Jha等人所描述的校准和验证。 （2010年）。进一步的校准和验证的SWAT模型进行支持阿狸河流域总体规划的发展，为所记述床Agren公司（2011年），这是SWAT模型，该模型被用于这项研究。 </li><li>使用的SWAT2005.exe修改后的版本，称为SWAT2005GA.exe。 </li></ol></li><li>准备文件“分水岭presets.csv” - 这是一个文本文件存储许多浣熊流域的设置。它是只读的由GeneticiSWAT和MapSWAT设置多个控制和数据领域的分水岭只要按一下。 </li><li>准备保护做法要素的成本。在这个例子中，这些被存储在表[的实践成本 - 浣熊县]在一个数据库“的实践费用的海盆Josh.mdb”。 </li></ol>一个候选的解决方案的总成本是流域单元（“水文响应单位”，或HRU的）的保护措施的费用的总和。优化程序认为一个单一的HRU的分水岭每农田保护的做法一组特定的保护实践的最佳分配。的sETS保护的做法可能分配给一个HRU被称为等位基因套，。 <ol start="4"><li>建立特警文件夹。此运行，16个CPU，这意味着运行在16份SWAT2005GA.exe 16个单独的文件夹（同样适用于系统，较少的CPU， 例如 ，应建立4个文件夹的“四核”处理器）。 </li></ol> 2。选择优化参数<ol><li>优化控制由程序称为“GeneticISWAT”。要执行优化，开放GeneticISWAT.exe。 <ol><li>转到“文件”，然后“打开”和，选择i_SWAT数据库的“浣熊GA.mdb”。 </li><li>转到“文件”，然后选择“配置”指定的路径SWAT模型的可执行文件（SWAT2005GA.exe）。 </li><li> “执行”，然后在“选择等位基因集”。这一步决定了用于优化组合保护的做法。此运行，等位基因设置＃14，其中有23个组合保护的做法。 AV集存储在ailable等位基因配置文件“Alleles.csv的”。 </li><li>转到“执行”，然后选择“的SPEA2存档基线意识到子集”SPEA2进化算法进行多目标优化。 </li></ol></li></ol><img src="/files/ftp_upload/4009/4009fig1.jpg" alt="图1"/> 图1。优化目标和参数设置。 优化参数可供选择： 预设：选择的分水岭进行优化。单击“应用”，选择从预设文件的“分水岭presets.csv”项目，以填补在此屏幕上的控制值。 输出变量：选择优化的环境目标。由于选择（N电源插座，P出口），这定义了一个3维的目标函数：氮（有机氮+ NO3 + NH4 + NO2）5年平均出口处，磷（2921网卡P +矿物P）5年平均出口处，总成本保护的做法。需要注意的是，这将创建一个3维的权衡前沿。可以选择替代输出变量，多目标的程序是最大限度地减少（输出变量}，总成本）。 人口规模：设置初始的人口规模。这就决定了初始的候选解决方案。当“种子与每个等位基因”选项的选择，候选的解决方案是创建一个统一的应用程序指定的各保护实践中的等位基因，所有农田的分水岭HRU的第一次。剩余的候选解决方案所建立的一个随机分配的等位基因为农田HRU的保护实践。当选择的每个等位基因“选项”种子，需要确保的初始人口规模至少是大等位基因组的等位基因数在本演示中（23）。 数代：设置所需的世代数 ​​（迭代）的优化运行（注意，可以重新启动运行）。 交叉概率：当两个候选方案中选择创建新的候选解决方案，交叉概率指定创建不同的新的解决方案的可能性（此演示设置为1）。 暂住人口的大小：这就决定了一些新的候选解决方案。处理器资源的最有效地使用时，此值是处理器线程的数目的整数倍（16在此演示）。 变异概率：指定HRU分配到另一个保护实践的等位基因组的随机变化的可能性。 （这个演示中，它被设置为0.03）。 数线程：选择所使用的处理器或线程的数量。 16本演示中使用。 曲线号的校正因子：这是从SWAT模型的校准。 保存在文本文件中：人口，这是非常重要的选择，如果希望重新启动后运行指定数量的迭代优化完成。选中此选项，生成一个文本文件，每一个的HRU在每一个幸存的候选方案（个人）的基因值。这可以被读回重新启动，继续运行。 二级优化参数 第一年：必须设置为一年开始的历史气象资料后，不迟于7月底前该数据。 玉米价格估计成本减少化肥的产量损失公式。 <p类=“jove_content”&gt; 评分方法：SPEA2存档。评分确定可能幸存的人是如何选择交叉。 清除方法：占主导地位。更糟糕的个人，在所有3个维度控制和清除。 HUC来源：设置为“指定位置”，意为“7100006”，从下面的字段“流域HUC”是用来发现的的HUC区表中的行。值“07100006”是的八位的HUC代码为浣熊流域。 成本资料来源：设置为“县（HRU位置代码）”，表示在实践中上述费用表以外CRP的成本将由县FIPS代码。 在实践中上述费用表， 费用来源CRP：设置为“1”，到表明，CRP成本，将由县FIPS代码。 <strONG&gt; SWAT版本：SWAT2005 3。代表性的成果 GeneticiSWAT.exe生成一个日志文件，示出了设置和结果可用于所有的候选解决方案（个人），以及一个“保存”的文件，该文件编码的查询结果从最终的算法迭代和可用于重新启动的优化运行。 在这一点上，我们可以可视化的整套帕累托有效的解决方案的权衡（边）境的，按照以下步骤： <ol><li>运行GeneticiSWAT; <ol><li>转到“文件”，然后在“打开”来打开的i_SWAT数据库的“浣熊GA.mdb”。 </li><li> “执行”，然后选择“导出HRU名单”。保存文件为“浣熊等位基因HRU.txt的”。 </li></ol></li><li>制作一个动画通过运行Mapswat.exe，选择“执行”，然后选择“三维动画”。 </li></ol><img src="/files/ftp_upload/4009/4009fig2.jpg" alt="图2"/> 图2。钪reenshot创建3维前沿的可视化的“快照”。 输出是一系列的文件可以呈现一次成图像文件，通过使用POV-RAY计划的，然后选择“渲染”，然后在“文件队列”。这些图像可以被用于对自己或结合到一部电影中示出的算法的进展。 <img src="/files/ftp_upload/4009/4009fig3.jpg" alt="图3"/> 图3。静态的权衡前沿的可视化。 如果需要的话，可以创建一个电影的算法进展由运行“Framescanner.exe”的，按照以下步骤： <ol><li>转到“文件”，然后“新建”，然后单击“文件”，然后选择“导入”，然后选择“PNG文件”。选择静态图像。 </li><li>要创建一个电影，“文件”，然后选择“导出”，然后选择“AVI”。 </li><li>选择编解码器“DIB”创建批次的AVI文件的图像文件。 </li></ol>在边境的每一个点代表一个分水岭的配置（具体分配上的景观保护的做法）。这些配置可以看出，对整个边境的地图，通过执行以下步骤： <ol><li>运行Mapswat.exe，选择“执行”，然后在“地图动画”。 </li><li>从预设列表中选择“浣熊”，然后单击“应用”。 </li><li>选择“布局（浣熊）”从地图布局“列表，然后单击”OK“。 </li></ol><img src="/files/ftp_upload/4009/4009fig4.jpg" alt="图4"/> 图4：在最后的边界创建地图的每一个人。截图。 导出特定流域的配置（个人）的利益。 通常情况下，利益的问题是选择特定的分水岭配置（个人）实现特定的水质目标。例如，可能希望找到一个个人，idual在降低氮30％，磷20％，相对于基线负荷的前沿。 MapSWAT允许一个搜索个人前沿与指定的目标的最小欧几里得距离。这是可以做到通过执行以下操作： <ol><li>打开MapSWAT.exe。选择“执行”|“搜索”。 </li></ol><img src="/files/ftp_upload/4009/4009fig5.jpg" alt="图5"/> 图5。寻找一个具体的个人在边境水质目标的基础上的屏幕截图。 <ol start="2"><li>输入最小和最大的减排目标Tmin和 Tmax的时间间隔以及色调 。也进入从基线的特定百分比减少氮（Nspec）在“％缩小”到N基线的空间，减少百分之在“％减少的的磷（PSPEC）中 ”磷基线。程序产生的输出弹出一个窗口： </li></ol><html><img src="/files/ftp_upload/4009/4009fig6.jpg" alt="图6"/> 搜索输出的屏幕截图如图6所示。 <ol start="3"><li>点击“复制文字”，并粘贴到电子表格中。三个表的产生。在第一个人最近的氮和磷的目标相同％的减少，其范围从TMIN至TMAX色调。就低于这个最接近的单个人的目标（Nspec，PSPEC）出现。二，表给其中P的目标范围TMIN至TMAX而N是保持不变附近Nspec的最近的个人。第三，最近Ň目标，从TMIN至TMAX范围P保持不变附近PSPEC的表给个人。在这种情况下，减少了30％的N最接近的个人ID 8423与N值14,637,279.60。下面是地图显示的空间分布，保护的做法，在权衡前沿的位置，该流域的配置： </li></ol>files/ftp_upload/4009/4009fig7.jpg“ALT =”图7“/&gt; 图7。在边境的样品描述所选择的个别截图。 <a href="http://www.jove.com/files/ftp_upload/4009/4009fig7large.jpg" target="_blank">点击此处查看大图</a> 。 导出地图数据作进一步的分析，可以通过执行以下步骤： <ol><li>运行Mapswat.exe，选择“执行”，然后选择“导出地图数据”。 </li><li>从预设列表中选择“浣熊”，然后单击“应用”。 </li><li>输入ID的的分水岭配置（个人）（8423示出一个），检查“显示等位基因属性”和“显示成本”，然后单击“确定”。这些数据可以用来创建使用GIS程序选定的分水岭配置的自定义地图。 </li></ol><table border="1"><tbody><tr><td> 节目名称 </td><td> 源 </td><td> 描述 </td></tr><tr><td>肩</td><td> CARD </TD&gt; <td>创建数据库，土壤，气象和管理数据的一个分水岭，填充的i_SWAT的。 </td></tr><tr><td> Swat2005GA.exe </td><td>美国农业部草原，土壤和水资源研究实验室</td><td>流域仿真模型</td></tr><tr><td> i_SWAT.exe </td><td> CARD </td><td> SWAT数据库接口</td></tr><tr><td> GeneticISWAT.exe </td><td> CARD </td><td>进化算法SWAT控制器。内置加利布从<a href="http://lancet.mit.edu/ga/" target="_blank">http://lancet.mit.edu/ga/</a> 。 </td></tr><tr><td> MapSWAT.exe </td><td> CARD </td><td> i_SWAT数据库和shapefile读取，生成的图像世代的个人。 </td></tr><tr><td> POV-Ray的</td><td> Povray.org </td><td>视觉光线追踪的持续性。 </td></tr><tr><td> Framescanner.exe </td><td>托德·坎贝尔</td><td> PNG图像到AVI转换器</td></tr><tr><td>的Windows Live Movie Maker中</td><td>微软</td><td>用于压缩的AV我WMV </td></tr></tbody></table> 表1。表所需的程序。 <table border="1"><tbody><tr><td> 文件的名称 </td><td> 类型 </td><td> 描述 </td></tr><tr><td>浣熊GA.mdb </td><td> Access数据库</td><td>结构和管理的描述的浣熊分水岭。阅读由GeneticiSWAT和MapSWAT。 </td></tr><tr><td>分水岭presets.csv </td><td>文本</td><td>设置的GeneticiSWAT.exe和MapSWAT的预设</td></tr><tr><td> Alleles.csv </td><td>文本</td><td>进化算法的等位基因设置列表。 </td></tr><tr><td>浣熊等位基因HRU.txt </td><td>文本</td><td>创建的文件的GeneticISWAT列出这些基因被确定为农田。阅读的MapSWAT。 </td></tr><tr><td>作业成本海盆Josh.mdb </td><td> Access数据库</td><td>成本管理做法和县。 </td></tr><tr><td>露台Zones.mdb </td><td> Access数据库</td><td>表HUC数据]拥有露台和屈服区号码为分水岭。 </td></tr><tr><td> NRI Budgets.mdb </td><td> Access数据库</td><td>阅读GeneticISWAT.exe作物及机械表中不使用此运行。 </td></tr><tr><td> phucrp 2008-12-15.dat </td><td>文本</td><td>植物热单位没有使用的查找表，在此运行。 </td></tr><tr><td> Management.mdb </td><td> Access数据库</td><td>旋转没有使用的查找表，在此运行。 </td></tr><tr><td> 2011-09-28 1313.log浣熊GA，GA浣熊2011-09-29 0732.log，浣熊GA 2011-10-07 0644.log </td><td>文本</td><td>日志文件的GeneticISWAT运行。 </td></tr><tr><td> <a href="http://www.jove.com/4009/Raccoon_GA.wmv">浣熊GA.wmv</a> </td><td>动画</td><td> 3D显示的个人代</td></tr><tr><td> <a href="http://www.jove.com/4009/Subbasin.shp" target="_blank">Subbasin.shp</a> </A&gt; </td><td> ESRI的Shapefile </td><td>纲要的拗陷盆地的分水岭。 </td></tr><tr><td> <a href="http://www.jove.com/4009/Raccoon_Map.wmv" target="_blank">浣熊Map.wmv</a> </td><td>动画</td><td>显示显性等位基因每个海盆每一个人在边境的。 </td></tr></tbody></table> 表2。表所需的样本文件。

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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Least+Cost+Control+of+Agricultural+Nutrient+Contributions+to+the+Gulf+of+Mexico+Hypoxic+Zone.">Least Cost Control of Agricultural Nutrient Contributions to the Gulf of Mexico Hypoxic Zone.</a> Ecol. Appl. 20, 1542-1555 (2010).</li><li>Rabotyagov, S., Jha, M., Campbell, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+crop+rotations+on+optimal+selection+of+conservation+practices+for+water+quality+protection.">Impact of crop rotations on optimal selection of conservation practices for water quality protection.</a> J. Soil. Water Conserv. 65 (6), 369-380 (2010).</li><li>Rabotyagov, S. S., Jha, M., Campbell, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonpoint-source+pollution+for+an+Iowa+watershed:+An+application+of+evolutionary+algorithms.">Nonpoint-source pollution for an Iowa watershed: An application of evolutionary algorithms.</a> Canadian Journal of Agricultural Economics/Revue canadienne d'agroeconomie. , (2010).</li><li>Rabotyagov, S. S., Jha, M., Campbell, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Searching+for+Efficiency:+Least+Cost+Nonpoint+Source+Pollution+Control+with+Multiple+Pollutants,+Practices,+and+Targets.">Searching for Efficiency: Least Cost Nonpoint Source Pollution Control with Multiple Pollutants, Practices, and Targets.</a> J. Nat. and Environ. Sci. 1, 75-90 (2010).</li><li>Ribaudo, M. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Consideration+of+off-site+impacts+in+targeting+soil+conservation+programs.">Consideration of off-site impacts in targeting soil conservation programs.</a> Land Econ. 62, 402-411 (1986).</li><li>Ribaudo, M. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+the+conservation+reserve+program+to+maximize+water+quality+benefits.">Targeting the conservation reserve program to maximize water quality benefits.</a> Land Econ. 65, 320-332 (1989).</li><li>Srivastava, P., Hamlett, J. M., Robillard, P. D., Day, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Watershed+optimization+of+best+management+practices+using+AnnAGNPS+and+a+genetic+algorithm.">Watershed optimization of best management practices using AnnAGNPS and a genetic algorithm.</a> Water Resour. Res. 38 (3), 1-14 (2002).</li><li>Veith, T. L., Wolfe, M. L., Heatwole, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+optimization+procedure+for+cost-effective+BMP+placement.">Development of optimization procedure for cost-effective BMP placement.</a> J. Am. Water Resour. Assoc. 39 (6), 1331-1343 (2003).</li><li>Whittaker, G., Confesor, R., Griffith, S. M., F&#228;re, R., Grosskopf, S., Steiner, J. J., Mueller-Warrant, G. W., Banowetz, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Hybrid+Genetic+Algorithm+for+Multiobjective+Problems+with+Activity+Analysis-based+Local+Search.">A Hybrid Genetic Algorithm for Multiobjective Problems with Activity Analysis-based Local Search.</a> Eur. J. Operat. Res. , (2007).</li><li>Zitzler, E., Laumanns, M., Thiele, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SPEA2:+Improving+the+Strength+Pareto+Evolutionary+Algorithm+for+Multiobjective+Optimization.">SPEA2: Improving the Strength Pareto Evolutionary Algorithm for Multiobjective Optimization.</a> CIMNE. , 95-100 (2002).</li></ol>

没有利益冲突的声明。

我们建立一个综合性的仿真优化框架，以寻找成本最低的组合和位置的农业保护措施，实现了流域范围内的营养物减少的目标，涉及的分水岭配置的帕累托有效的套。提出了在图8的仿真优化系统的概念图。流域模拟，包括模拟的水质影响的农业保护措施的水文模型，SWAT2005，再加上基于Windows的数据库控制系统，i_SWAT 6,8处理。优化组件）SWAT水文响应单元（HRU的，采用的逻辑进...

spatial multiobjective optimization of agricultural conservation practices using a swat model and an evolutionary algorithm

寻找具有成本效益（ 即成本最低）的方式保护实践的投资目标为实现特定的水质目标，在景观在流域管理是最重要的。传统的经济学方法在流域范围内寻找最低成本的解决方案（ 例如 ，5,12,20）认为场外的影响，可以准确地描述为一定比例的现场产生的污染。这种方法是不太可能代表实际的污染过程中的一个分水岭，在污染源的影响往往取决于复杂的生物物理过程。现代物理为基础的，空间分布式水文模拟模型的使用允许更大程度的现实主义的过程表示，但需要开发的模拟优化的模型框架，​​成为优化的一个组成部分。 进化进制算法似乎是一个特别有用的优化工具，能够处理的组合性质的一个分水岭模拟优化问题，并允许使用的水质模型。进化算法处理一个特定的空间分配作为候选的解决方案中的一个分水岭的保护措施，并利用迭代采用随机选择的运营商，重组和突变，以找到改进的优化目标集（群体）候选解决方案。在这种情况下的优化目标是，以尽量减少非点源污染的分水岭，同时最大限度地降低成本，保护的做法。最近一个不断扩大的研究组正在尝试使用类似的方法，将水质模型的广泛定义的进化优化方法3,4,9,10,13-15,17-19,22,23,25。在此应用中，我们展示了程序如下Rabotyagov等。的适当ACH，并集成了现代常用SWAT水质模型多目标进化算法SPEA2 26，和用户指定的一组保护的做法，其成本搜索完整的折衷前沿的成本之间的保护方法和用户指定的水质的目标。的前沿量化所面临的流域管理者，通过提供全方位的各种水质改善的目标与成本的权衡。该计划允许选择的分水岭配置，达到规定的水质改善目标和生产的保护实践的最佳位置地图。

Watch this Scientific Journal Video about Spatial Multiobjective Optimization of Agricultural Conservation Practices using a SWAT Model and an Evolutionary Algorithm at JoVE.com

Spatial Multiobjective Optimization of Agricultural Conservation Practices using a SWAT Model and an Evolutionary Algorithm

这项工作表明水质模型的集成与优化组件，利用进化算法求解最优（最低成本）的水质改善目标指定的一组安置的农业保护措施。的解决方案是使用一个多目标的方法，可以明确的量化的权衡。

空间使用SWAT模型的农业保护的做法和多目标优化的进化算法

Finding the cost-efficient (i.e., lowest-cost) ways of targeting conservation practice investments for the achievement of specific water quality goals ...

spatial-multiobjective-optimization-agricultural-conservation

Research

JoVE Journal

Biology

12.9K 次观看.  University of Washington. 这项工作表明水质模型的集成与优化组件，利用进化算法求解最优（最低成本）的水质改善目标指定的一组安置的农业保护措施。的解决方案是使用一个多目标的方法，可以明确的量化的权衡。

视频: Spatial Multiobjective Optimization of Agricultural Conservation Practices using a SWAT Model and an Evolutionary Algorithm

Predicting the Effectiveness of Population Replacement Strategy Using Mathematical Modeling

Charles Taylor and John Marshall explain the utility of mathematical modeling for evaluating the effectiveness of population replacement strategy.  Insight is given into how computational models can provide information on the population dynamics of mosquitoes and the spread of transposable elements through A. gambiae subspecies.  The ethical considerations of releasing genetically modified mosquitoes into the wild are discussed.

Charles Taylor and John Marshall explain the utility of mathematical modeling for evaluating the effectiveness of population replacement strategy. Insight is given into how computational models can provide information on the population dynamics of mosquitoes and the spread of transposable elements through A. gambiae subspecies. The ethical considerations of releasing genetically modified mosquitoes into the wild are discussed.

利用数学建模预测的人口更替策略的有效性

Charles Taylor and John Marshall explain the utility of mathematical modeling for evaluating the effectiveness of population replacement strategy.  ...

科研

生物学

Non-radioactive in situ Hybridization Protocol Applicable for Norway Spruce and a Range of Plant Species

The high-throughput expression analysis technologies available today give scientists an overflow of expression profiles but their resolution in terms of tissue specific expression is limited because of problems in dissecting individual tissues. Expression data needs to be confirmed and complemented with expression patterns using e.g. in situ hybridization, a technique used to localize cell specific mRNA expression. The in situ hybridization method is laborious, time-consuming and often requires extensive optimization depending on species and tissue. In situ experiments are relatively more difficult to perform in woody species such as the conifer Norway spruce (Picea abies). Here we present a modified DIG in situ hybridization protocol, which is fast and applicable on a wide range of plant species including P. abies. With just a few adjustments, including altered RNase treatment and proteinase K concentration, we could use the protocol to study tissue specific expression of homologous genes in male reproductive organs of one gymnosperm and two angiosperm species; P. abies, Arabidopsis thaliana and Brassica napus. The protocol worked equally well for the species and genes studied. AtAP3 and BnAP3 were observed in second and third whorl floral organs in A. thaliana and B. napus and DAL13 in microsporophylls of male cones from P. abies. For P. abies the proteinase K concentration, used to permeablize the tissues, had to be increased to 3 g/ml instead of 1 g/ml, possibly due to more compact tissues and higher levels of phenolics and polysaccharides. For all species the RNase treatment was removed due to reduced signal strength without a corresponding increase in specificity. By comparing tissue specific expression patterns of homologous genes from both flowering plants and a coniferous tree we demonstrate that the DIG in situ protocol presented here, with only minute adjustments, can be applied to a wide range of plant species. Hence, the protocol avoids both extensive species specific optimization and the laborious use of radioactively labeled probes in favor of DIG labeled probes. We have chosen to illustrate the technically demanding steps of the protocol in our film.
Anna Karlgren and Jenny Carlsson contributed equally to this study.
Corresponding authors: Anna Karlgren at Anna.Karlgren@ebc.uu.se and Jens F. Sundstr&#246;m at Jens.Sundstrom@vbsg.slu.se

Non-radioactive in situ Hybridization Protocol Applicable for Norway Spruce and a Range of Plant Species

We describe a modified DIG in situ hybridization protocol, which is fast and applicable on a wide range of plant species including Norway spruce. With just a few adjustments, including altered RNase treatment and proteinase K concentration, the protocol may be used in studies of different tissues and species.

非放射性原位挪威云杉杂交议定书适用的植物种类范围

The high-throughput expression analysis technologies available today give scientists an overflow of expression profiles but their resolution in terms of ...

An Optimized Protocol for Rearing Fopius arisanus, a Parasitoid of Tephritid Fruit Flies

Fopius arisanus (Sonan) is an important parasitoid of Tephritid fruit flies for at least two reasons. First, it is the one of only three opiine parasitoids known to infect the host during the egg stage1. Second, it has a wide range of potential fruit fly hosts. Perhaps due to its life history, F. arisanus has been a successfully used for biological control of fruit flies in multiple tropical regions2-4. One impediment to the wide use of F. arisanus for fruit fly control is that it is difficult to establish a stable laboratory colony5-9. Despite this difficulty, in the 1990s USDA researchers developed a reliable method to maintain laboratory populations of F. arisanus10-12. There is significant interest in F. arisanus biology13,14, especially regarding its ability to colonize a wide variety of Tephritid hosts14-17; interest is especially driven by the alarming spread of Bactrocera fruit fly pests to new continents in the last decade18. Further research on F. arisanus and additional deployments of this species as a biological control agent will benefit from optimizations and improvements of rearing methods. In this protocol and associated video article we describe an optimized method for rearing F. arisanus based on a previously described approach12. The method we describe here allows rearing of F. arisanus in a small scale without the use of fruit, using materials available in tropical regions around the world and with relatively low manual labor requirements.

An Optimized Protocol for Rearing Fopius arisanus, a Parasitoid of Tephritid Fruit Flies

Fopius arisanus is an egg-larval parasitoid of Tephritid fruit flies that is successfully used in biological control of these important tropical pests. We describe here an optimized protocol for rearing F. arisanus on a small scale using readily available materials.

一个优化的协议饲养 Fopius arisanus Tephritid果寄生蝇

Fopius arisanus (Sonan) is an important parasitoid of Tephritid fruit flies for at least two reasons. First, it is the one of only three opiine ...

Ablation of a Single Cell From Eight-cell Embryos of the Amphipod Crustacean Parhyale hawaiensis

The amphipod Parhyale hawaiensis is a small crustacean found in intertidal marine habitats worldwide. Over the past decade, Parhyale has emerged as a promising model organism for laboratory studies of development, providing a useful outgroup comparison to the well studied arthropod model organism Drosophila melanogaster. In contrast to the syncytial cleavages of Drosophila, the early cleavages of Parhyale are holoblastic. Fate mapping using tracer dyes injected into early blastomeres have shown that all three germ layers and the germ line are established by the eight-cell stage. At this stage, three blastomeres are fated to give rise to the ectoderm, three are fated to give rise to the mesoderm, and the remaining two blastomeres are the precursors of the endoderm and germ line respectively. However, blastomere ablation experiments have shown that Parhyale embryos also possess significant regulatory capabilities, such that the fates of blastomeres ablated at the eight-cell stage can be taken over by the descendants of some of the remaining blastomeres. Blastomere ablation has previously been described by one of two methods: injection and subsequent activation of phototoxic dyes or manual ablation. However, photoablation kills blastomeres but does not remove the dead cell body from the embryo. Complete physical removal of specific blastomeres may therefore be a preferred method of ablation for some applications. Here we present a protocol for manual removal of single blastomeres from the eight-cell stage of Parhyale embryos, illustrating the instruments and manual procedures necessary for complete removal of the cell body while keeping the remaining blastomeres alive and intact. This protocol can be applied to any Parhyale cell at the eight-cell stage, or to blastomeres of other early cleavage stages. In addition, in principle this protocol could be applicable to early cleavage stage embryos of other holoblastically cleaving marine invertebrates.

Ablation of a Single Cell From Eight-cell Embryos of the Amphipod Crustacean Parhyale hawaiensis

The amphipod Parhyale hawaiensis is a promising model organism for studies of crustacean embryology and comparative arthropod development and evolution. This protocol describes a method for manual removal of single blastomeres from early cleavage stage embryos of Parhyale.

一个单细胞消融从甲壳动物端足八细胞胚胎<em&gt; Parhyale hawaiensis</em

The amphipod Parhyale hawaiensis is a small crustacean found in intertidal marine habitats worldwide. Over the past decade, Parhyale has emerged as a ...

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Developmental and environmental cues induce Ca2+ fluctuations in plant cells. Stimulus-specific spatial-temporal Ca2+ patterns are sensed by cellular Ca2+ binding proteins that initiate Ca2+ signaling cascades. However, we still know little about how stimulus specific Ca2+ signals are generated. The specificity of a Ca2+ signal may be attributed to the sophisticated regulation of the activities of Ca2+ channels and/or transporters in response to a given stimulus. To identify these cellular components and understand their functions, it is crucial to use systems that allow a sensitive and robust recording of Ca2+ signals at both the tissue and cellular levels. Genetically encoded Ca2+ indicators that are targeted to different cellular compartments have provided a platform for live cell confocal imaging of cellular Ca2+ signals. Here we describe instructions for the use of two Ca2+ detection systems: aequorin based FAS (film adhesive seedlings) luminescence Ca2+ imaging and case12 based live cell confocal fluorescence Ca2+ imaging. Luminescence imaging using the FAS system provides a simple, robust and sensitive detection of spatial and temporal Ca2+ signals at the tissue level, while live cell confocal imaging using Case12 provides simultaneous detection of cytosolic and nuclear Ca2+ signals at a high resolution.

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Ca2+ signaling regulates diverse biological processes in plants. Here we present approaches for monitoring abiotic stress induced spatial and temporal Ca2+ signals in Arabidopsis cells and tissues using the genetically encoded Ca2+ indicators Aequorin or Case12.

测量时空的Ca<sup&gt; 2 +</sup&gt;在拟南芥信号

Developmental and environmental cues induce Ca2+ fluctuations in plant cells. Stimulus-specific spatial-temporal Ca2+ patterns are sensed by cellular Ca2+ ...

Observation and Quantification of Mating Behavior in the Pinewood Nematode, Bursaphelenchus xylophilus

A method for observing and quantifying the mating behavior of the pinewood nematode, Bursaphelenchus xylophilus, was established under a stereomicroscope. To improve the mating efficiency of B. Xylophilus and to increase the chances of mating observation, virgin adults were cultured and used for the investigation. Eggs were obtained by keeping the nematodes in water and allowing the females to lay eggs for 10 min. The second-stage juveniles (J2) were synchronized by incubating the eggs for 24 h at 25 &#176;C in the dark, and the early J4 were obtained by culturing the J2 with grey mold, Botrytis cinerea, for another 52 h. At this time point, most J4 nematodes could be clearly distinguished as being male or female using their genital morphology. The male and female J4 were collected and cultured separately in two different Petri dishes for 24 h to get virgin adult nematodes. A virgin male and a virgin female were paired in a drop of water in the well of a concave slide. The mating behavior was filmed with a video recorder under a stereomicroscope. The whole period of the mating process was 82.8 &#177;3.91 min (mean &#177;SE) and could be divided into 4 different phases: searching, contacting, copulating, and lingering. The mean minutes of duration were 21.8 &#177; 2.0, 28.0 &#177; 1.9, 25.8 &#177; 0.7 and 7.2 &#177; 0.5, respectively. Eleven sub-behaviors were described: cruising, approaching, encountering, touching, hooping, locating, attaching, ejaculating, separating, quiescence, and roaming. Interestingly, obvious intra-sexual competition was observed when one female was grouped with 3 males or one male with 3 females. This protocol is useful and valuable, not only in investigating the mating behavior of B. xylophilus, but also in acting as a reference for ethological studies of other nematodes.

Observation and Quantification of Mating Behavior in the Pinewood Nematode, Bursaphelenchus xylophilus

A protocol for investigating the mating behavior of the pinewood nematode, Bursaphelenchus xylophilus, is presented. Behavioral features of B. xylophilus are described in the mating process.

观察和交配行为的量化在我国松材线虫，<em&gt;松材线虫</em

A method for observing and quantifying the mating behavior of the pinewood nematode, Bursaphelenchus xylophilus, was established under a stereomicroscope. ...

Transient Expression and Cellular Localization of Recombinant Proteins in Cultured Insect Cells

Heterologous protein expression systems are used for the production of recombinant proteins, the interpretation of cellular trafficking/localization, and the determination of the biochemical function of proteins at the sub-organismal level. Although baculovirus expression systems are increasingly used for protein production in numerous biotechnological, pharmaceutical, and industrial applications, nonlytic systems that do not involve viral infection have clear benefits but are often overlooked and underutilized. Here, we describe a method for generating nonlytic expression vectors and transient recombinant protein expression. This protocol allows for the efficient cellular localization of recombinant proteins and can be used to rapidly discern protein trafficking within the cell. We show the expression of four&#160;recombinant proteins in a commercially available insect cell line, including two aquaporin proteins from the insect Bemisia tabaci, as well as subcellular marker proteins specific for the cell plasma membrane and for intracellular lysosomes. All recombinant proteins were produced as chimeras with fluorescent protein markers at their carboxyl termini, which allows for the direct detection of the recombinant proteins. The double transfection of cells with plasmids harboring constructs for the genes of interest and a known subcellular marker allows for live cell imaging and improved validation of cellular protein localization.

Nonlytic insect cell expression systems are underutilized for production, cellular trafficking/localization, and recombinant protein functional analysis. Here, we describe methods to generate expression vectors and subsequent transient protein expression in commercially available lepidopteran cell lines. The co-localization of Bemisia tabaci aquaporins with subcellular fluorescent marker proteins is also presented.

瞬时表达和重组蛋白的细胞定位在培养的昆虫细胞

Heterologous protein expression systems are used for the production of recombinant proteins, the interpretation of cellular trafficking/localization, and ...

Reproductive Techniques for Ovarian Monitoring and Control in Amphibians

Ovarian control and monitoring in amphibians require a multi-faceted approach. There are several applications that can successfully induce reproductive behaviors and the acquisition of gametes and embryos for physiological or molecular research. Amphibians contribute to one-quarter to one-third of vertebrate research, and of interest in this context is their contribution to the scientific community&#39;s knowledge of reproductive processes and embryological development. However, most of this knowledge is derived from a small number of species. In recent times, the decimation of amphibians across the globe has required increasing intervention by conservationists. The captive recovery and assurance colonies that continue to emerge in response to the extinction risk make existing research and clinical applications invaluable to the survival and reproduction of amphibians held under human care. The success of any captive population is founded on its health and reproduction and the ability to develop viable offspring that carry forward the most diverse genetic representation of their species. For researchers and veterinarians, the ability to monitor and control ovarian development and health is, therefore, imperative. The focus of this article is to highlight the different assisted reproductive techniques that can be used to monitor and, where appropriate or necessary, control ovarian function in amphibians. Ideally, any reproductive and health issues should be reduced through proper captive husbandry, but, as with any animal, issues of health and reproductive pathologies are inevitable. Non-invasive techniques include behavioral assessments, visual inspection and palpation and morphometric measurements for the calculation of body condition indices and ultrasound. Invasive techniques include hormonal injections, blood sampling, and surgery. Ovarian control can be exercised in a number of ways depending on the application required and species of interest.

The study of amphibian biology provides valuable information on the reproductive, physiological, embryological and developmental processes that drive organisms of many taxonomic groups. Here, we present a comprehensive guide on different methodologies that can be used to study ovarian control and monitoring in amphibians.

两栖动物卵巢监测和控制的生殖技术

Ovarian control and monitoring in amphibians require a multi-faceted approach. There are several applications that can successfully induce reproductive ...

Using a Thermal Camera to Measure Heat Loss Through Bird Feather Coats

Feathers are essential to insulation, and therefore to the cost of thermoregulation,&#160;in birds. There is robust literature on the energetic cost of thermoregulation in birds across a variety of ecological circumstances. However, few studies characterize the contribution of the feathers alone to thermoregulation. Several previous studies have established methods for measuring the insulation value of animal pelts, but they require destructive sampling methods that are problematic for birds, whose feathers are not distributed evenly across the skin. More information is needed about 1) how the contribution of feathers to thermoregulation varies both across and within species and 2) how feather coats may change over space and time. Reported here is a method for rapidly and directly measuring the thermal performance of feather coats and the skin using dried whole skin specimens, without the need to destroy the skin specimen. This method isolates and measures the thermal gradient across a feather coat in a way that measurements of heat loss and metabolic cost in live birds, which use behavioral and physiological strategies to thermoregulate, cannot. The method employs a thermal camera, which allows the rapid collection of quantitative thermal data to measure heat loss from a stable source through the skin. This protocol can easily be applied to various research questions, is applicable to any avian taxa, and does not require destruction of the skin specimen. Finally, it will further the understanding of the importance of passive thermoregulation in birds by simplifying and accelerating the collection of quantitative data.

This protocol describes the quantification of heat transmission through a flat skinned avian specimen using a thermal camera and hot water bath. The method allows obtaining of quantitative, comparative data about the thermal performance of feather coats across species using dried flat skin specimens.

使用热像仪测量通过鸟羽毛大衣的热损失

Feathers are essential to insulation, and therefore to the cost of thermoregulation,&#160;in birds. There is robust literature on the energetic cost of ...

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A)

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer progression. How the chromosomal location and function of a centromere (i.e., centromere identity) are determined and participate in accurate chromosome segregation is a fundamental question. CENP-A is proposed to be the non-DNA indicator (epigenetic mark) of centromere identity, and CENP-A ubiquitylation is required for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability.
Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-CENP-A K124R mutant suggesting that ubiquitylation at a different lysine is induced because of the EYFP tagging in the CENP-A K124R mutant protein. Lysine 306 (K306) ubiquitylation in EYFP-CENP-A K124R was successfully identified, which corresponds to lysine 56 (K56) in CENP-A through mass spectrometry analysis. A caveat is discussed in the use of GFP/EYFP or the tagging of high molecular weight protein as a tool to analyze the function of a protein. Current technical limit is also discussed for the detection of ubiquitylated bands, identification of site-specific ubiquitylation(s), and visualization of ubiquitylation in living cells or a specific single cell during the whole cell cycle.
The method of mass spectrometry analysis presented here can be applied to human CENP-A protein with different tags and other centromere-kinetochore proteins. These combinatory methods consisting of several assays/analyses could be recommended for researchers who are interested in identifying functional roles of ubiquitylation.

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A EYFP-CENP-A

CENP-A ubiquitylation is an important requirement for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability. Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A) protein.