遗传分析对中欧的意外硬蜱的存在提出了挑战：开发一种多重 PCR 来区分意外硬蜱和蓖子硬蜱。
Genetic analysis challenges the presence of Ixodes inopinatus in Central Europe: development of a multiplex PCR to distinguish I. inopinatus from I. ricinus.
机构信息
Department of Chemistry and Biochemistry, Mendel University, Brno, Czech Republic.
Biomedical Center, Faculty of Medicine in Pilsen, Charles University, Plzen, Czech Republic.
出版信息
Parasit Vectors. 2023 Oct 9;16(1):354. doi: 10.1186/s13071-023-05971-2.
BACKGROUND
Ixodes ricinus is an important vector of several pathogens, primarily in Europe. Recently, Ixodes inopinatus was described from Spain, Portugal, and North Africa and then reported from several European countries. In this study, a multiplex polymerase chain reaction (PCR) was developed to distinguish I. ricinus from I. inopinatus and used in the surveillance of I. inopinatus in Algeria (ALG) and three regions in the Czech Republic (CZ).
METHODS
A multiplex PCR on TROSPA and sequencing of several mitochondrial (16S rDNA, COI) and nuclear markers (TROSPA, ITS2, calreticulin) were used to differentiate these two species and for a subsequent phylogenetic analysis.
RESULTS
Sequencing of TROSPA, COI, and ITS2 separated these two species into two subclades, while 16S rDNA and calreticulin could not distinguish I. ricinus from I. inopinatus. Interestingly, 23 nucleotide positions in the TROSPA gene had consistently double peaks in a subset of ticks from CZ. Cloning of these PCR products led to a clear separation of I. ricinus and I. inopinatus indicating hybridization and introgression between these two tick taxa. Based on a multiplex PCR of TROSPA and analysis of sequences of TROSPA, COI, and ITS2, the majority of ticks in CZ were I. ricinus, no I. inopinatus ticks were found, and 10 specimens showed signs of hybridization. In contrast, most ticks in ALG were I. inopinatus, four ticks were I. ricinus, and no signs of hybridization and introgression were detected.
CONCLUSIONS
We developed a multiplex PCR method based on the TROSPA gene to differentiate I. ricinus and I. inopinatus. We demonstrate the lack of evidence for the presence of I. inopinatus in Central Europe and propose that previous studies be re-examined. Mitochondrial markers are not suitable for distinguishing I. inopinatus from I. ricinus. Furthermore, our data indicate that I. inopinatus and I. ricinus can hybridize, and the hybrids can survive in Europe.
背景
硬蜱属（Ixodes ricinus）是几种病原体的重要载体，主要在欧洲。最近，硬蜱属无柄种（Ixodes inopinatus）从西班牙、葡萄牙和北非被描述，随后在几个欧洲国家被报道。在这项研究中，开发了一种多重聚合酶链反应（PCR）来区分硬蜱属无柄种和硬蜱属（Ixodes ricinus），并用于阿尔及利亚（ALG）和捷克共和国三个地区（CZ）的硬蜱属无柄种监测。
方法
使用 TROSPA 的多重 PCR 以及几个线粒体（16S rDNA、COI）和核标记（TROSPA、ITS2、钙网蛋白）的测序来区分这两个物种，并进行随后的系统发育分析。
结果
TROSPA、COI 和 ITS2 的测序将这两个物种分为两个亚群，而 16S rDNA 和钙网蛋白不能将硬蜱属无柄种与硬蜱属区分开来。有趣的是，在 CZ 的一小部分蜱中，TROSPA 基因的 23 个核苷酸位置始终有双峰。这些 PCR 产物的克隆导致硬蜱属无柄种和硬蜱属清晰分离，表明这两个蜱类之间存在杂交和基因渗入。基于 TROSPA 的多重 PCR 和 TROSPA、COI 和 ITS2 序列分析，CZ 的大多数蜱为硬蜱属，未发现硬蜱属无柄种，有 10 个标本显示出杂交的迹象。相比之下，ALG 的大多数蜱为硬蜱属无柄种，有 4 个蜱为硬蜱属，未检测到杂交和基因渗入的迹象。
结论
我们开发了一种基于 TROSPA 基因的多重 PCR 方法来区分硬蜱属无柄种和硬蜱属。我们证明中欧缺乏硬蜱属无柄种存在的证据，并建议重新检查以前的研究。线粒体标记不适合区分硬蜱属无柄种和硬蜱属。此外，我们的数据表明硬蜱属无柄种和硬蜱属可以杂交，杂种可以在欧洲生存。
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