Effects of storage conditions and digestion time on DNA amplification of biting midge (Culicoides) blood meals.

BACKGROUND: Molecular analysis of blood meals is increasingly used to identify the hosts of biting insects such as midges and mosquitoes. Successful host identification depends on the availability of sufficient host DNA template for PCR amplification, making it important to understand how amplification success changes under different storage conditions and with different durations of blood meal digestion within the insect gut before being placed into the storage medium. METHOD: We characterised and compared the digestion profile of two species of Culicoides over a 96-h period using a novel set of general vertebrate primers targeting the 16S rRNA gene. A set number of individuals from each species were killed over 13 time points post-blood feeding and preserved in 95% ethanol. Samples were stored either at ambient room temperature or in a - 20 °C freezer to examine the effect of storage condition on the PCR amplification success of host DNA. RESULTS: We found that amplification success across the 96-h sampling period post-feeding was reduced from 96 to 6% and 96% to 14% for Culicoides nubeculosus and Culicoides sonorensis, respectively. We found no effect of storage condition on PCR amplification success, and storage in 95% ethanol was sufficient to maintain high rates of amplifiable host DNA for at least 9 months, even at room temperature. CONCLUSIONS: These findings highlight the limited time frame during which an individual may contain amplifiable host DNA and demonstrate the importance of timely sample capture and processing post-blood feeding. Moreover, storage in 95% ethanol alone is sufficient to limit host DNA degradation. These results are relevant to the design of studies investigating the biting behaviour and disease transmission potential of Culicoides and other biting Diptera.