Mosquito tagging using DNA-barcoded nanoporous protein microcrystals

Abstract

Conventional mosquito marking technology for mark-release-recapture (MRR) is quite limited in terms of information capacity and efficacy. To overcome both challenges, we have engineered, lab-tested, and field-evaluated a new class of marker particles, in which synthetic, short DNA oligonucleotides (DNA barcodes) are adsorbed and protected within tough, crosslinked porous protein microcrystals. Mosquitoes self-mark through ingestion of microcrystals in their larval habitat. Barcoded microcrystals persist transstadially through mosquito development if ingested by larvae, do not significantly affect adult mosquito survivorship, and individual barcoded mosquitoes are detectable in pools of up to at least 20 mosquitoes. We have also demonstrated crystal persistence following adult mosquito ingestion. Barcode sequences can be recovered by qPCR and next-generation sequencing (NGS) without detectable amplification of native mosquito DNA. These DNA-laden protein microcrystals have the potential to radically increase the amount of information obtained from future MRR studies compared to previous studies employing conventional mosquito marking materials.

Porous protein crystals: synthesis and applications

“…It was previously demonstrated that CJ crystals were able to adsorb DNA to the crystal interior, 185 so Stuart et al from our group synthesized CJ microcrystals loaded with a synthetic DNA marker; after saturating these microcrystals with DNA, they were deposited in tubs of stagnant water and allowed to incubate for several days alongside mosquito larvae. 185 Emerging adult mosquitoes were subsequently captured and reared in an insectary, whereupon they were sacrificed, and the original barcode sequence was recovered from the adult mosquitoes via qPCR-based detection and next-generation sequencing. 185 This study demonstrates a novel application of LPCs for marking and detection of mosquitoes, which have traditionally relied on fluorescent powders, 195 paints/dyes, or radioisotopes.…”

“…185 Emerging adult mosquitoes were subsequently captured and reared in an insectary, whereupon they were sacrificed, and the original barcode sequence was recovered from the adult mosquitoes via qPCR-based detection and next-generation sequencing. 185 This study demonstrates a novel application of LPCs for marking and detection of mosquitoes, which have traditionally relied on fluorescent powders, 195 paints/dyes, or radioisotopes. 196…”

“…In addition to the aforementioned applications, LPC scaffolds have been utilized for a variety of niche applications, the majority of which are dependent on the unique properties of the protein itself. For instance, the CJ crystals that our lab routinely works with are able to rapidly adsorb nucleic acids to their interior, 32,185 and this property is currently being exploited by our group across multiple projects.…”

Leveraging insect-specific viruses to elucidate mosquito population structure and dynamics

“…Mark-release-recapture (MRR) methods (e.g., fluorescent dye) are logistically straightforward and commonly used but have low recapture rates resulting in low confidence in dispersal estimates obtained [ 20 , 24 , 125 , 126 ]. This has led to the development of additional methods for evaluating dispersal and local population structure, including the use of stable isotope markers [ 22 , 127 ], microcrystals [ 128 ], landscape genomics [ 129 ], and close-kin mark recapture (CKMR) [ 10 , 23 , 130 ]. However, each of these have major limitations when estimating population structure.…”

Tuning Chemical DNA Ligation within DNA Crystals and Protein–DNA Cocrystals

“…Crystals made from protein and DNA are versatile materials that precisely order molecules, self-assemble, and have tunable growth. − Porous crystals have been shown to act as molecular sieves − and to host molecules for structure determination, − enhance enzymatic activity, , and information storage via synthetic DNA sequences . Engineered crystals with DNA building blocks, from pure DNA crystals , to hybrid protein–DNA crystals, have been designed to serve as scaffolds for DNA-binding molecules. − However, the broad utility of crystals held together by DNA is restricted by their ability to survive in varied solution conditions.…”