Unbiased Characterization of Anopheles Mosquito Blood Meals by Targeted High-Throughput Sequencing.
Unbiased Characterization of Anopheles Mosquito Blood Meals by Targeted High-Throughput Sequencing.
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DOI：
10.1371/journal.pntd.0004512
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发表时间：
2016-03
影响因子：
3.8
通讯作者：
Serre D
中科院分区：
文献类型：
--
作者：
Logue K;Keven JB;Cannon MV;Reimer L;Siba P;Walker ED;Zimmerman PA;Serre D
了解蚊子的宿主选择对于评估病媒能力或确定疾病宿主非常重要。不幸的是，用于全面评估昆虫血粉组成的无偏方法的可用性非常有限，因为大多数当前的分子测定仅测试少数预选物种的存在。这些方法也具有有限的能力，以确定在一个单一的血餐中存在多个哺乳动物宿主。在这里，我们描述了一种新的高通量测序方法，可以同时分析96只蚊子，并对每种血粉的组成提供全面和定量的观点。我们通过计算机模拟验证了靶向哺乳动物线粒体16S核糖体RNA基因（16S rRNA）的通用引物应扩增NCBI核苷酸数据库中存在的95%以上的哺乳动物16S rRNA序列。我们将此方法应用于442只雌性斑点按蚊。L.在巴布亚新几内亚（PNG）收集的蚊子。虽然人类（52.9%），狗（15.8%）和猪（29.2%）是我们研究中发现的最常见的宿主，但我们还检测到来自小鼠，一种有袋动物和两种蝙蝠的DNA。我们的分析还显示，16.3%的蚊子以一种以上的宿主为食。对102份人类血餐中人类线粒体高变区I的分析显示，5只（4.9%）蚊子明确地以一个以上的人为食。总体而言，对巴布亚新几内亚蚊子的分析说明了这种方法在识别未知宿主和表征混合血餐方面的潜力，并展示了如何调整这种方法来评估人类血餐中的个体间差异。此外，这种方法可以适用于任何疾病传播的节肢动物，可以很容易地定制调查非哺乳动物宿主来源。雌蚊需要吸血来获得产卵所需的营养。在以宿主物种为食的同时，蚊子可以传播导致包括疟疾、淋巴丝虫病和登革热在内的多种疾病的病原体。了解蚊子的宿主选择对于更好地实施控制策略以减少蚊子数量并因此减少疾病传播非常重要。目前，大多数用于评估宿主物种的方法仅测试预先选择的预期宿主的存在。在这里，我们描述了一个公正的分析，结合任何哺乳动物DNA的扩增与高通量测序，全面表征蚊子血粉的组成。我们将这种方法应用于在巴布亚新几内亚收集的按蚊，并观察到它们以预期的（人，狗和猪）和意想不到的宿主（小鼠，蝙蝠，有袋动物）为食。此外，我们发现16.3%的蚊子以来自相同或不同物种的多个宿主为食。总的来说，这种方法能够公正地表征蚊子血餐，并且可以很容易地应用于显着提高我们对任何传播疾病昆虫的进食行为的了解。
Understanding mosquito host choice is important for assessing vector competence or identifying disease reservoirs. Unfortunately, the availability of an unbiased method for comprehensively evaluating the composition of insect blood meals is very limited, as most current molecular assays only test for the presence of a few pre-selected species. These approaches also have limited ability to identify the presence of multiple mammalian hosts in a single blood meal. Here, we describe a novel high-throughput sequencing method that enables analysis of 96 mosquitoes simultaneously and provides a comprehensive and quantitative perspective on the composition of each blood meal. We validated in silico that universal primers targeting the mammalian mitochondrial 16S ribosomal RNA genes (16S rRNA) should amplify more than 95% of the mammalian 16S rRNA sequences present in the NCBI nucleotide database. We applied this method to 442 female Anopheles punctulatus s. l. mosquitoes collected in Papua New Guinea (PNG). While human (52.9%), dog (15.8%) and pig (29.2%) were the most common hosts identified in our study, we also detected DNA from mice, one marsupial species and two bat species. Our analyses also revealed that 16.3% of the mosquitoes fed on more than one host. Analysis of the human mitochondrial hypervariable region I in 102 human blood meals showed that 5 (4.9%) of the mosquitoes unambiguously fed on more than one person. Overall, analysis of PNG mosquitoes illustrates the potential of this approach to identify unsuspected hosts and characterize mixed blood meals, and shows how this approach can be adapted to evaluate inter-individual variations among human blood meals. Furthermore, this approach can be applied to any disease-transmitting arthropod and can be easily customized to investigate non-mammalian host sources. Female mosquitoes require a blood meal to acquire the nutrients necessary for egg production. While feeding on host species, mosquitoes can transmit pathogens that cause several diseases including malaria, lymphatic filariasis and dengue. Understanding the mosquito host choice is important to better implement control strategies to reduce mosquito populations and therefore transmission of disease. Currently, the majority of methods for evaluating host species only test for the presence of pre-selected, expected hosts. Here, we describe an unbiased assay that combines amplification of any mammalian DNA with high-throughput sequencing to comprehensively characterize the composition of mosquito blood meals. We applied this approach to Anopheles mosquitoes collected in Papua New Guinea and observed that they fed on expected (humans, dogs and pigs) and unexpected hosts (mice, bats, marsupials). In addition, we show that 16.3% of mosquitoes fed on multiple hosts, from the same or different species. Overall, this approach enables unbiased characterization of mosquito blood meals and can be easily applied to significantly improve our understanding of the feeding behavior of any disease-transmitting insect.