Endosymbionts and intracellular parasites are common in arthropods and other invertebrate hosts. As a consequence, (co)amplification of untargeted bacterial sequences has been occasionally reported as a common problem in DNA barcoding. The bacterial genus Rickettsia belongs to the order Rickettsiales and consists of two lineages: one including diverse pathogens infecting arthropod hosts, the other consisting of non-pathogenic species with a broader host taxonomic range. While discriminating among amphipod species with universal primers for the COI region, we unexpectedly detected rickettsial endosymbionts belonging to the Torix group. To map the distribution and diversity of Rickettsia among amphipods hosts, we conducted a nationwide molecular screening of seven families of freshwater amphipods collected throughout New Zealand. In addition to uncovering a diversity of Torix group Rickettsia across multiple amphipod populations from three different families, our research indicates that 1) detecting Torix Rickettsia with universal primers is not uncommon, 2) obtaining 'Rickettsia COI sequences' from many host individuals is highly likely when a population is infected, and 3) obtaining ′host COI′ may not be possible with a conventional PCR if an individual is infected. Because Rickettsia COI is highly conserved across diverse host taxa, we were able to design blocking primers that can be used in a wide range of host species infected with Torix Rickettsia. We propose the use of blocking primers to circumvent problems caused by unwanted amplification of Rickettsia and to obtain targeted host COI sequences for DNA barcoding, population genetics, and phylogeographic studies.

中文翻译：

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Torix组立克次体在新西兰淡水两栖动物中很普遍：使用封闭引物拯救宿主COI序列

内共生菌和细胞内寄生虫在节肢动物和其他无脊椎动物宿主中很常见。结果，偶然地报道了未靶向细菌序列的（共）扩增是DNA条形码中的常见问题。立克次体细菌属属于立克次体，由两个谱系组成：一个包括感染节肢动物宿主的多种病原体，另一个包括宿主分类学范围更广的非致病性物种。当使用COI区域通用引物区分两栖动物时，我们意外地发现了属于Torix组的立克次体内共生体。绘制立克次体的分布和多样性在两栖类动物寄主中，我们对新西兰各地收集的七个淡水两栖类动物进行了全国性分子筛查。我们的研究表明，除了发现来自三个不同科的多个两栖动物种群中的Torix立克次体多样性外，我们还发现1）用通用引物检测Torix立克次体并不少见； 2）从许多宿主个体中获得“立克次体COI序列”的可能性很高。 3）如果感染了个体，则无法通过常规PCR获得“宿主COI”。因为立克次体COI在不同的宿主类群中高度保守，我们能够设计封闭引物，这些引物可用于感染了Torix Rickettsia的多种宿主物种。我们建议使用封闭引物来规避由立克次体的无意扩增引起的问题，并获得针对目标的宿主COI序列，用于DNA条形码，种群遗传学和系统地理学研究。