Protocol for bloodmeal identification in ticks using retrotransposon-targeted real time PCR

Tick-borne infections are a global public health burden. Our ability to develop effective control measures relies on understanding the natural transmission cycles as well as the mode of exposure to humans. Our current knowledge of the relative importance of the hosts upon which tick vectors feed is based on indirect measurements, such as infestation indices. Bloodmeal identification can determine the host upon which a questing tick had fed in the previous life stage and is an unbiased method for incriminating reservoir hosts. Although bloodmeal identification has been utilized for mosquito ecology since the 1950s, the extended life-cycle of ticks and complete intracellular digestion of the bloodmeal make such analyses for ticks more complicated. We have recently developed a highly sensitive assay for identification of bloodmeals in ticks based upon real-time PCR amplification of mammalian retrotransposons. Although other PCR-based methods for bloodmeal analysis in ticks have been published previously, they have an inadequate success rate with field-collected ticks and have not been adopted for general use. We describe our methods in detail in order to make this assay widely accessible for use in other laboratories.

Nanopore adaptive sampling for mitogenome sequencing and bloodmeal identification in hematophagous insects

Blood-feeding insects are important vectors for an array of zoonotic pathogens. Despite significant research focused on well-documented insect vectors of One Health importance, resources for molecular species identification of a large number of hematophagous arthropods are limited. Advancements in next-generation sequencing technologies provide opportunities for targeting mitochondrial genomes of blood-feeding insects, as well as their bloodmeal hosts. This dual approach holds great promise for elucidating complex disease transmission pathways and enhancing the molecular resources for the identification of cryptic insect species. To this end, we leveraged the newly developed Oxford Nanopore Adaptive Sampling (NAS) pipeline to dually sequence the mitogenomes of hematophagous insects and their bloodmeals. Using NAS, we sequenced the entire mitogenomes of Aedes vexans, Culex restuans, Culex territans, and Chrysops niger and successfully identified bloodmeal hosts of Chrysops niger, Culex restuans, and Aedes trivittatus. We show that NAS has the utility to simultaneously molecularly identify blood-feeding insects and characterize disease transmission pathways through bloodmeal host identification. Moreover, our data indicate NAS can facilitate a wide array of molecular systematic studies through novel 'phylogenetic capture' methods. We conclude the NAS approach has great potential for informing global One Health initiatives centered on the mitigation of vector-borne disease through dual vector and bloodmeal identification.

Modernizing the Toolkit for Arthropod Bloodmeal Identification

Understanding vertebrate–vector interactions is vitally important for understanding the transmission dynamics of arthropod-vectored pathogens and depends on the ability to accurately identify the vertebrate source of blood-engorged arthropods in field collections using molecular methods. A decade ago, molecular techniques being applied to arthropod blood meal identification were thoroughly reviewed, but there have been significant advancements in the techniques and technologies available since that time. This review highlights the available diagnostic markers in mitochondrial and nuclear DNA and discusses their benefits and shortcomings for use in molecular identification assays. Advances in real-time PCR, high resolution melting analysis, digital PCR, next generation sequencing, microsphere assays, mass spectrometry, and stable isotope analysis each offer novel approaches and advantages to bloodmeal analysis that have gained traction in the field. New, field-forward technologies and platforms have also come into use that offer promising solutions for point-of-care and remote field deployment for rapid bloodmeal source identification. Some of the lessons learned over the last decade, particularly in the fields of DNA barcoding and sequence analysis, are discussed. Though many advancements have been made, technical challenges remain concerning the prevention of sample degradation both by the arthropod before the sample has been obtained and during storage. This review provides a roadmap and guide for those considering modern techniques for arthropod bloodmeal identification and reviews how advances in molecular technology over the past decade have been applied in this unique biomedical context.