Leptotrombidium (Acari: Trombiculidae) mites are carriers of Orientia tsutsugamushi, the bacterial pathogen causing scrub typhus in humans. Classification of Leptotrombidium is vital because limited mite species carry O. tsutsugamushi. Generally, Leptotrombidium at the larval stage (approximately 0.2 mm in size) are used for morphological identification. However, morphological identification is often challenging because it requires considerable skills and taxonomic expertise. In this study, we found that the full-length sequences of the mitochondrial cytochrome c oxidase subunit 1 gene varied among the significant Leptotrombidium. On the basis of these, we modified the canonical deoxyribonucleic acid (DNA) barcoding method for animals by redesigning the primer set to be suitable for Leptotrombidium. Polymerase chain reaction with the redesigned primer set drastically increased the detection sensitivity, especially against Leptotrombidium scutellare (approximately 17% increase), one of the significant mites carrying O. tsutsugamushi. Phylogenetic analysis showed that the samples morphologically classified as L. scutellare and Leptotrombidium pallidum were further split into 3 and 2 distinct subclusters respectively. The mean genetic distance (p-distance) between L. scutellare and L. pallidum was 0.2147, whereas the mean distances within each species were 0.052 and 0.044, respectively. Within L. scutellare, the mean genetic distances between the 3 subclusters were 0.1626–0.1732, whereas the distances within each subcluster were 0.003–0.017. Within L. pallidum, the mean genetic distance between the 2 subclusters was 0.1029, whereas the distances within each subcluster were 0.010–0.013. The DNA barcoding uncovered a broad genetic diversity of Leptotrombidium, especially of L. scutellare and L. pallidum, the notable species carrying O. tsutsugamushi. We conclude that the DNA barcoding using our primers enables precise and detailed classification of Leptotrombidium and implies the existence of a subgenotype in Leptotrombidium that had not been found by morphological identification.

细螨（蜱螨亚纲：螨科）螨是恙虫病东方体的携带者，东方体是引起人类恙虫病的细菌病原体。纤螨的分类至关重要，因为携带恙虫病螨的螨种有限。一般采用幼虫期（大小约0.2毫米）的纤颤虫进行形态学鉴定。然而，形态识别通常具有挑战性，因为它需要大量的技能和分类学专业知识。在这项研究中，我们发现线粒体细胞色素c氧化酶亚基1基因的全长序列在重要的细纤菌属中存在差异。在此基础上，我们通过重新设计适用于细褶菌的引物组，修改了规范的动物脱氧核糖核酸（DNA）条形码方法。使用重新设计的引物组进行的聚合酶链反应极大地提高了检测灵敏度，特别是针对盾片细螨（增加约 17%），这是携带恙虫病的重要螨虫之一。系统发育分析表明，形态学上分类为L. scutellare和Leptotrombidium pallidum的样本分别进一步分为 3 个和 2 个不同的亚簇。L. scutellare和L. pallidum之间的平均遗传距离（p 距离）为 0.2147，而每个物种内的平均距离分别为 0.052 和 0.044。在L. scutellare中，3 个亚簇之间的平均遗传距离为 0.1626-0.1732，而每个亚簇内的距离为 0.003-0.017。在苍白球菌中，2个亚簇之间的平均遗传距离为0.1029，而每个亚簇内的距离为0.010-0.013。DNA条形码揭示了细褶菌广泛的遗传多样性，特别是盾片细褶菌和苍白细褶菌，这两种携带恙虫病的著名物种。我们得出的结论是，使用我们的引物进行的 DNA 条形码能够对细褶菌进行精确和详细的分类，并暗示细褶菌中存在形态学鉴定未发现的亚基因型。