Pataradool Thanapat, Siriyasatien Padet, Sukhumavasi Woraporn, Tiwananthagorn Saruda, Suankratay Chusana, Preativatanyou Kanok
Medical Sciences Program, Graduate School, Faculty of Medicine, Chulalongkorn University, Bangkok, 10330, Thailand.
Department of Parasitology, Faculty of Medicine, Chulalongkorn University, Bangkok, 10330, Thailand.
Parasitol Res. 2025 Jun 27;124(6):69. doi: 10.1007/s00436-025-08516-x.
Pentastomiasis, a zoonotic parasitic disease caused by pentastomids, has been increasingly documented in Thailand. Historically, morphological identification has been the only diagnostic method, but it is inadequate due to morphological similarities between larval stages and limited taxonomic keys. To overcome this, molecular diagnosis is required. However, the DNA sequence information of these neglected parasites remains largely unknown. In this study, pentastomid larvae were collected from the greater omentum of a patient in Northern Thailand. The species of pentastomid larvae was molecularly identified as Armillifer moniliformis, using PCRs targeting nuclear and mitochondrial regions, followed by Sanger sequencing. Nanopore sequencing was employed to characterize the complete mitochondrial genome of A. moniliformis, revealing a total length of 16,367 bp with 37 mitochondrial genes, including 13 protein-coding genes, 22 tRNA genes, and two rRNA genes, as well as one non-coding region. The entire mitogenome is highly AT-rich (62.1%), with nucleotide skews indicating compositional bias between strands. The non-coding region (2616 bp) lies between the tRNA-Leu and tRNA-Ser genes and comprises three repetitive segments. The third repetitive segment is the longest (1659 bp) and has the highest A + T content of 72.2%, likely serving as a regulatory element related to DNA replication. Comparative mitogenome analysis revealed cox1 as the most conserved gene, while atp6, atp8, and nad6 exhibit high variability among species. Codon usage analysis indicated that two codon families (Leu1 and Ile) are used more frequently than others, suggesting that these branched-chain hydrophobic amino acids are predominant in transmembrane proteins and underscoring the critical role of the mitochondrion in energy production. Based on single gene (cox1) and concatenated gene-based phylogenetic approaches, A. moniliformis clustered closely with other Armillifer species, with all pentastomids placed taxonomically in the subphylum Crustacea. Mitogenomic phylogenetics in this study demonstrates greater topological precision than cox1 phylogenetics. In conclusion, this study revealed the first complete mitochondrial genome of A. moniliformis, providing us with novel molecular markers that could effectively facilitate species diagnosis, treatment monitoring, and epidemiological surveillance of pentastomiasis.
舌形虫病是一种由舌形虫引起的人畜共患寄生虫病，在泰国的报道日益增多。从历史上看，形态学鉴定一直是唯一的诊断方法，但由于幼虫阶段的形态相似性以及分类学关键特征有限，这种方法并不充分。为了克服这一问题，需要进行分子诊断。然而，这些被忽视的寄生虫的DNA序列信息在很大程度上仍然未知。在本研究中，从泰国北部一名患者的大网膜中收集了舌形虫幼虫。通过针对核区域和线粒体区域的PCR，随后进行Sanger测序，从分子水平上将舌形虫幼虫鉴定为串珠状 Armillifer moniliformis。采用纳米孔测序对串珠状 Armillifer moniliformis 的完整线粒体基因组进行了表征，结果显示其全长为16367 bp，包含37个线粒体基因，其中包括13个蛋白质编码基因、22个tRNA基因和2个rRNA基因，以及一个非编码区。整个线粒体基因组富含AT（62.1%），核苷酸偏斜表明链间存在组成偏差。非编码区（2616 bp）位于tRNA-Leu和tRNA-Ser基因之间，由三个重复片段组成。第三个重复片段最长（1659 bp），A+T含量最高，为72.2%，可能作为与DNA复制相关的调控元件。线粒体基因组比较分析显示cox1是最保守的基因，而atp6、atp8和nad6在物种间表现出高度变异性。密码子使用分析表明，两个密码子家族（Leu1和Ile）的使用频率高于其他家族，这表明这些支链疏水氨基酸在跨膜蛋白中占主导地位，并强调了线粒体在能量产生中的关键作用。基于单基因（cox1）和串联基因的系统发育方法，串珠状 Armillifer moniliformis 与其他 Armillifer 物种紧密聚类，所有舌形虫在分类学上都属于甲壳亚门。本研究中的线粒体基因组系统发育学显示出比cox1系统发育学更高的拓扑精度。总之，本研究揭示了串珠状 Armillifer moniliformis 的首个完整线粒体基因组，为我们提供了新的分子标记，可有效促进舌形虫病的物种诊断、治疗监测和流行病学监测。
