Department of Biology, University of Oxford, 11a Mansfield Road, Oxford, OX1 3SZ, UK.
The Pirbright Institute, Ash Road, Pirbright, Surrey, GU24 0NF, UK.
Parasit Vectors. 2023 Jan 13;16(1):13. doi: 10.1186/s13071-022-05607-x.
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.
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.
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.
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.
血液餐的分子分析越来越多地用于鉴定吸血昆虫（如蠓和蚊子）的宿主。成功的宿主鉴定取决于是否有足够的用于 PCR 扩增的宿主 DNA 模板，因此了解在将昆虫放入储存介质之前，在不同的储存条件下以及在昆虫肠道中消化不同时间的血液餐时，扩增成功率如何变化非常重要。
我们使用一组针对 16S rRNA 基因的新型通用脊椎动物引物，在 96 小时的时间内对两种库蠓进行了消化情况的描述和比较。每一种库蠓都在吸血后经过 13 个时间点杀死并保存在 95%乙醇中。将样本储存在室温下或-20°C 的冰箱中，以检查储存条件对宿主 DNA PCR 扩增成功率的影响。
我们发现，从喂食后 96 小时的采样期开始，Culicoides nubeculosus 和 Culicoides sonorensis 的扩增成功率分别从 96%降至 6%和 96%降至 14%。我们发现储存条件对 PCR 扩增成功率没有影响，在 95%乙醇中储存至少 9 个月甚至在室温下也足以保持高比例可扩增的宿主 DNA。
这些发现强调了个体可能包含可扩增宿主 DNA 的时间有限，并证明了及时捕获和处理喂食后样本的重要性。此外，单独使用 95%乙醇储存足以限制宿主 DNA 的降解。这些结果与研究库蠓和其他吸血双翅目昆虫的叮咬行为和疾病传播潜力的研究设计有关。
