Expanding the known haemosporidian parasite diversity in Eurasian Bluethroat (Luscinia svecica) subspecies through amplicon sequencing

Monitoring haemosporidian parasites in birds is essential for comprehending the dynamics of avian malaria, a disease that significantly affects bird populations worldwide. This study concentrated on the prevalence and diversity of haemosporidian parasites in 1989 specimens from two subspecies of the Eurasian bluethroat (Luscinia svecica), aiming to explore the genetic diversity and species richness of haemosporidian lineages fauna across the host populations. By utilizing next-generation (NGS) amplicon high-throughput sequencing, we observed a marked increase in the detection of haemosporidian diversity, revealing cryptic lineages variants and species previously unidentified by Sanger sequencing. A high prevalence of the Plasmodium haplotypes was seen in all studied sites, accompanied by a less frequent Leucocytozoon infection in the red-spotted subspecies and minimal occurrence of Haemoproteus lineages. Both the previously known lineages and new, low-prevalence cryptic lineages variants were detected, underscoring the complexity of haemosporidian infections in avian hosts. The use of species delimitation tools provided a detailed understanding of haemosporidian species diversity, their coexistence within hosts, and their phylogenetic relationships. Despite the varying ecological characteristics of the study sites, no significant difference in haemosporidian alpha diversity among populations was found. However, significant differences in beta diversity were identified, suggesting that habitat characteristics and geographic distance influence parasite distribution. These findings highlight the importance of advanced molecular techniques in revealing the hidden diversity of parasites, offering valuable insights into the ecology and evolution of haemosporidian infections. Given the threatened status of one of the host’s populations, knowledge on local diversity of haemosporidian parasites also has implications for possible conservation strategies.监测鸟类中的血孢子虫（haemosporidian parasites），对于理解禽疟疾（avian malaria）的流行动态至关重要——该疾病在全球范围内对鸟类种群造成显著负面影响。本研究针对1989份采自欧亚蓝喉歌鸲（Luscinia svecica）两个亚种的标本，聚焦血孢子虫的感染率与多样性，旨在探究宿主种群中血孢子虫谱系类群的遗传多样性与物种丰富度。通过运用下一代测序（next-generation sequencing, NGS）扩增子高通量测序技术，本研究实现了血孢子虫多样性检测效能的显著提升，发现了此前桑格（Sanger）测序未能识别的隐秘谱系变异与新物种。所有研究样地均检出高流行率的疟原虫（Plasmodium）单倍型；其中红斑亚种的住白细胞虫（Leucocytozoon）感染率较低，血变原虫（Haemoproteus）谱系的检出率极低。本研究既检出了已知的血孢子虫谱系，也发现了新的低流行率隐秘谱系变异，这凸显了鸟类宿主血孢子虫感染的复杂性。借助物种界定工具，我们得以深入解析血孢子虫的物种多样性、其在宿主内的共存模式以及系统发育关系。尽管各研究样地的生态特征存在差异，但不同宿主种群间的血孢子虫α（alpha）多样性并无显著差异。然而，β（beta）多样性却呈现出显著差异，这表明生境特征与地理距离会影响寄生虫的分布格局。本研究结果凸显了先进分子技术在揭示寄生虫隐秘多样性方面的重要价值，为血孢子虫感染的生态学与进化研究提供了宝贵见解。鉴于其中一个宿主种群处于受威胁状态，掌握当地血孢子虫的多样性信息，也可为潜在的保护策略制定提供参考依据。