Can Environmental DNA Be Used Within Pest Insect Agricultural Biosecurity? Detecting Khapra Beetle Within Stored Rice

Environmental DNA (eDNA) has recently emerged as an effective tool for invasive species biosecurity. We explored the use of eDNA for the detection of khapra beetle (Trogoderma granarium, Everts 1898), an invasive insect of cereal grains and other food products that has a high global economic impact. We developed a novel method for aggregating khapra beetle eDNA deposited in stored grain that entails washing a sample of rice, filtering the sample, and detecting trace beetle DNA using a standard qPCR workflow. To explore the performance of this method, we raised 500 khapra beetle larvae within 500 g of rice over a 14-day period and then removed them. We then used this "spiked" rice to create a range of simulated densities of khapra beetle larvae. This lab approach mimics conditions that are comparable to field densities of similar to 1.4 to 180 beetles per 50 kg of rice (1/8 to 16 spiked rice grains per 100 g sample of clean rice), assuming DNA is uniformly distributed within the rice. We detected khapra beetle eDNA from all density levels tested. Logistic models revealed that eDNA amounts equivalent to what is left by similar to 1 khapra beetle larva in a 50 kg container of rice can be detected with 85% to >97% certainty, depending on the number of qPCR technical replicates run per sample. Based on this model, we estimated that for one 50 kg container of rice where beetle DNA is uniformly distributed, a single sample of 100 g with six technical replicates would be sufficient to be >99% certain that the container was free of khapra beetle eDNA (95% credible intervals: 97.7%-100%). Our results suggest that eDNA surveys may be useful as a cost-effective, first-step detection of khapra beetle in stored grain and provide a means to map the relative magnitude of khapra beetle transport pathways, informing allocation of conventional biosecurity inspection efforts.