Abstract
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.
Highlights
Many insects require a blood meal to complete their gonotrophic cycle
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 and unexpected hosts
A number of PCR-based molecular techniques have been developed to characterize host blood meals ([7] and references within) and determine the blood feeding preference of mosquitoes [8,9,10,11], ticks [12,13,14], sandflies [15,16,17] and Tsetse flies [18, 19]. While these PCR-based approaches enable rigorous identification of the host species, they typically focus on species-specific amplification of putative hosts and are not designed to identify novel, unanticipated host blood sources
Summary
Many insects require a blood meal to complete their gonotrophic cycle. By feeding successively on different hosts, these insects can transmit blood borne pathogens that cause diseases responsible for significant burden on global health [1, 2]. The blood meals of arthropods have traditionally been analyzed using serological techniques such as ELISA or precipitin tests [3,4,5] While these methods have provided valuable information, they have limited taxonomic resolution as they are generally only able to characterize hosts at the order or family levels [6]. The detection of mixed blood meals (i.e., when an insect feeds on more than one host) by these approaches is complicated as the dominant host signal can completely overwhelm signals from other minor hosts These limitations may have biased our understanding of the transmission of many vector-borne diseases and have prevented identification of important disease reservoirs
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