Department of Parasitology, Faculty of Science, Charles University, Prague, Czech Republic.
USDA-ARS, European Biological Control Laboratory, Thessaloniki, Greece.
PLoS Negl Trop Dis. 2019 Sep 9;13(9):e0007669. doi: 10.1371/journal.pntd.0007669. eCollection 2019 Sep.
Identification of blood sources of hematophagous arthropods is crucial for understanding the transmission cycles of vector-borne diseases. Many different approaches towards host determination were proposed, including precipitin test, ELISA, DNA- and mass spectrometry-based methods; yet all face certain complications and limitations, mostly related to blood degradation. This study presents a novel method for blood meal identification, peptide mass mapping (PMM) analysis of host-specific hemoglobin peptides using MALDI-TOF mass spectrometry.
METHODOLOGY/PRINCIPAL FINDINGS: To identify blood meal source, proteins from abdomens of engorged sand fly females were extracted, cleaved by trypsin and peptide fragments of host hemoglobin were sequenced using MALDI-TOF MS. The method provided correct host identification of 100% experimentally fed sand flies until 36h post blood meal (PBM) and for 80% samples even 48h PBM. In females fed on two hosts, both blood meal sources were correctly assigned for 60% of specimens until 36h PBM. In a validation study on field-collected females, the method yielded unambiguous host determination for 96% of specimens. The suitability of PMM-based MALDI-TOF MS was proven experimentally also on lab-reared Culex mosquitoes.
CONCLUSIONS/SIGNIFICANCE: PMM-based MALDI-TOF MS analysis targeting host specific hemoglobin peptides represents a sensitive and cost-effective method with a fast and simple preparation protocol. As demonstrated here on phlebotomine sand flies and mosquitoes, it allows reliable and rapid blood source determination even 48h PBM with minimal material input and provides more robust and specific results than other currently used methods. This approach was also successfully tested on field-caught engorged females and proved to be a promising useful tool for large-scale screening of host preferences studies. Unlike other methods including MALDI-TOF protein profiling, it allows correct identification of mixed blood meals as was demonstrated on both experimentally fed and field-collected sand flies.
鉴定吸血节肢动物的血液来源对于了解媒介传播疾病的传播周期至关重要。已经提出了许多不同的宿主鉴定方法，包括沉淀试验、ELISA、DNA 和质谱法；然而，所有这些方法都面临着一定的并发症和限制，主要与血液降解有关。本研究提出了一种新的血液餐鉴定方法，即使用 MALDI-TOF 质谱对宿主特异性血红蛋白肽进行肽质量图谱（PMM）分析。
方法/主要发现：为了鉴定血液来源，从饱血的沙蝇雌蝇腹部提取蛋白质，用胰蛋白酶酶切，然后使用 MALDI-TOF MS 对宿主血红蛋白的肽段进行测序。该方法能够正确识别 100%经实验喂养的沙蝇，直到饱血后 36 小时（PBM），甚至在 48 小时 PBM 时，80%的样本也能正确识别。在喂养两种宿主的雌性中，60%的样本能够正确分配两种血液来源，直到 36 小时 PBM。在对野外采集的雌性进行的验证研究中，该方法对 96%的样本进行了明确的宿主鉴定。实验还证明，基于 PMM 的 MALDI-TOF MS 对实验室饲养的库蚊也是一种敏感且具有成本效益的方法，具有快速简单的制备方案。正如本文在白蛉和蚊子上所证明的那样，即使在 PBM 后 48 小时，它也可以使用最少的材料输入进行可靠和快速的血液来源鉴定，并提供比其他当前使用的方法更稳健和更特异的结果。这种方法还在野外采集的饱血雌性中进行了测试，证明是宿主偏好研究大规模筛选的一种很有前途的有用工具。与包括 MALDI-TOF 蛋白质谱分析在内的其他方法不同，它可以正确鉴定混合的血液餐，这在实验喂养和野外采集的白蛉中都得到了证明。
