The phylum Perkinsozoa is an aquatic parasite lineage that has devastating effects on commercial and natural mollusc populations, and also comprises parasites of algae, fish and amphibians. They are related to dinoflagellates and apicomplexans and thus offer excellent genetic models for both parasitological and evolutionary studies. Genetic transformation was previously achieved for Perkinsus spp. but with few tools for transgene expression and limited selection efficacy. We sought to expand the power of experimental genetic tools for Perkinsus using P. marinus as a model. We constructed a modular plasmid assembly system for expression of multiple genes simultaneously. We developed efficient selection systems for three drugs, puromycin, bleomycin and blasticidin, that are effective in as little as three weeks. We developed eleven new promoters of variable expression strength. Furthermore, we identified that genomic integration of transgenes is predominantly via non-homologous recombination but with transgene fragmentation including deletion of some elements. To counter these dynamic processes, we show that bi-cistronic transcripts using the viral 2A peptides can couple selection to the maintenance of the expression of a transgene of interest. Collectively, these new tools and insights provide great new capacity to genetically modify and study Perkinsus as an aquatic parasite and evolutionary model.

Perkinsozoa 门是一种水生寄生虫谱系，对商业和天然软体动物种群具有破坏性影响，还包括藻类、鱼类和两栖动物的寄生虫。它们与甲藻和顶复门有关，因此为寄生虫学和进化研究提供了极好的遗传模型。Perkinsus spp先前已实现遗传转化。但很少有用于转基因表达的工具和有限的选择功效。我们试图使用P. marinus扩大Perkinsus实验性遗传工具的能力作为模特。我们构建了一个模块化质粒组装系统，用于同时表达多个基因。我们为三种药物（嘌呤霉素、博来霉素和杀稻瘟菌素）开发了有效的选择系统，这些系统在短短三周内即可见效。我们开发了 11 个具有可变表达强度的新启动子。此外，我们发现转基因的基因组整合主要是通过非同源重组，但转基因片段化包括一些元件的缺失。为了应对这些动态过程，我们表明使用病毒 2A 肽的双顺反子转录物可以将选择与维持感兴趣的转基因的表达相结合。总的来说，这些新工具和见解为基因改造和研究Perkinsus提供了巨大的新能力 作为一种水生寄生虫和进化模型。