Molecular methods have advanced the knowledge of host selection behavior by allowing the identification of the bloodmeal host to the species level. The microsphere assay described herein adds to available methodologies by allowing for the rapid identification of the most commonly fed upon species in an area. For future work in other geographies and environments, further probes for a suite of relevant hosts could be easily added to or substituted into the current method.
This assay has major benefits in rapidly analyzing large numbers of bloodmeals. The assay is easy to interpret, because the output is numeric and no analysis of electrophoretic gels is required. It is more robust and flexible than real-time PCR with up to100 possible unique microspheres and fewer complications with multiplexing. It is less expensive and time intensive than DNA sequencing. With current prices at the UC Davis CGF, purification and sequencing of PCR products cost approximately $3.75 per sample. Using the 15-probe set described here, the cost of the microsphere assay is approximately $2.50 per sample. These prices do not include the initial PCR steps that are equivalent for both methods and, for laboratories without current access to a Luminex® system, the price does not include the initial purchase of the instrument
Further cost savings and a major benefit of the assay come from its flexibility. Because the probe mixture is made on a per plate basis, probes can be added or removed depending on host availability in a given area. For the current example, probes can be used in any combination from 1-bead set all the way up to the complete 15-bead set. In most bloodmeal identification studies, a small number of species have accounted for a large percentage of the bloodmeals. For example, in Shelby County, Tennessee, 4 species accounted for 77% of the avian feeds by the Culex pipiens
complex (Savage et al. 2007
), and in Weld County, Colorado, 6 host species accounted for just over 90% of the bloodmeals in July 2007 (Kent et al. 2009a
). In the latter example, a 6-bead set plus 10% sequencing would cost approximately $1.22, less than 1/3 the cost of sequencing an entire plate. Because the same biotin-labeled PCR product used for the microsphere assay can be purified and sequenced, there would be no extra cost or time for additional PCR steps.
In addition to the supply cost savings, much less time is required to analyze the output data from this microsphere assay. Sequence analysis and identification can take 2–3 hours for each 96-well plate, whereas less than 15 min was required for analysis of the microsphere assay. Further benefits over sequencing include the ability to easily identify mixed bloodmeals without an additional cloning step, and the ability to quantify single or mixed bloodmeals in studies where the amount of blood taken by the vector may play an important role.
Although this assay proved cost-effective and high-throughput, there were minor drawbacks. Probe melting temperature, and thus probe length and composition, was limited in the direct hybridization assay analyzed on the Luminex 200®. The available streptavidin-RPE reporters were not thermostable at high temperatures for long time periods and high temperatures led to evaporation of the sample. Therefore, with long probes and corresponding high hybridization temperatures, background fluorescence at the end of the run was sometimes too high for analysis. The current protocol, with a hybridization temperature of 58°C and a run length of approximately 50 min, was near the limit of thermostability. Issues resulting from high hybridization temperature might be mitigated or avoided with one or more of the following adjustments: an increased volume of reporter solution could be added to extend evaporation time; on instruments with a piercing sample probe, the plate can be covered to reduce evaporation; a more thermostable reporter could be used; and/or Luminex xTAG® protocols could be used rather than the direct hybridization (xMAP®) protocols used here.
Similar to other non-sequencing methods, an inherent difficulty was that it was impossible to test our probes against all possible vertebrate host species. We evaluated our probes against all common species within our study areas, but there is always a chance for misidentification with an unexpected host. Therefore in the development stages, 10% of microsphere identifications from all study areas were sequenced to confirm that they were a true match and not a rare or unexpected host. In this manner, the current probes were tested against nearly 130 possible hosts collected in our study sites, but there may be closely related hosts in other areas that result in false positives with the current probe set.
Despite these minor drawbacks, we feel that we have provided a new and useful tool to identify blood meal hosts fed upon by Culex mosquitoes that also should be useful for other bloodfeeding arthropods. It is likely not practical to develop microsphere capture probes to identify every possible host species in a given area. However, when used in conjunction with sequencing, the assay is as robust as sequencing while being higher throughput, more efficient to interpret and less expensive than sequencing alone. Bloodmeal identification studies may still be limited by the ability to find and collect large numbers of bloodfed mosquitoes, but with using this assay, less time and money will be required to analyze those samples once they are collected.