DNA metabarcoding diet analysis in a generalist omnivore: feeding trials reveal the efficacy of extraction kits and a multi-locus approach for identifying diverse diets.

Metabarcoding-based diet analysis is a valuable tool for understanding the feeding behavior of a wide range of species. However, many studies using these methods for wild animals assume accuracy and precision without experimental evaluation with known positive control food items. Here, we conducted a feeding trial experiment with a positive control community in pasture-raised chickens and assessed the efficacy of several commonly used DNA extraction kits and primer sets. We hand-fed 22 known food items, including insects and plants, to six backyard laying hens and collected their excreta for eight h. We evaluated the efficacy of three DNA extraction kits, three primer sets for plant identification (targeting rbcL, trnL, and internal transcribed spacer 2 [ITS2]), and three primer sets for arthropod identification (targeting cytochrome oxidase subunit I [COI]). The detection success rate of our positive control food items was highly variable, ranging from 2.04% to 93.88% for all kit/primer combinations and averaging 37.35% and 43.57% for the most effective kit/primer combination for plants and insects, respectively. Extraction kits using bead-based homogenization positively affected the recovery proportion of plant and insect DNA in excreta samples. The minimum time to detect known food items was 44 min post-feeding. Two COI primer sets significantly outperformed the third, and both recovery proportion and taxonomic resolution from ITS2 were significantly higher than those from rbcL and trnL. Taken together, these results display the potential variability that can be inherently present in DNA-based diet analyses and highlight the utility of experimental feeding trials in validating such approaches, particularly for omnivores with diverse diets.

A new cost-effective and fast direct PCR protocol for insects based on PBS buffer.

Insect DNA barcoding is a species identification technique used in biodiversity assessment and ecological studies. However, DNA extraction can result in the loss of up to 70% of DNA. Recent research has reported that direct PCR can overcome this issue. However, the success rates could still be improved, and tissues used for direct PCR could not be reused for further genetic studies. Here, we developed a direct PCR workflow that incorporates a 2-min sample preparation in PBS-buffer step for fast and effective universal insect species identification. The developed protocol achieved 100% success rates for amplification in six orders: Mantodea, Phasmatodea, Neuroptera, Odonata, Blattodea and Orthoptera. High and moderate success rates were obtained for five other species: Lepidoptera (97.3%), Coleoptera (93.8%), Diptera (90.5%), Hemiptera (81.8%) and Hymenoptera (75.0%). High-quality sequencing data were also obtained from these amplifiable products, allowing confidence in species identification. The method was sensitive down to 1/4th of a 1-mm fragment of leg or body and its success rates with oven-dried, ethanol-preserved, food, bat guano and museum specimens were 100%, 98.6%, 90.0%, 84.0% and 30.0%, respectively. In addition, the pre-PCR solution (PBS with insect tissues) could be used for further DNA extraction if needed. The workflow will be beneficial in the fields of insect taxonomy and ecological studies due to its low cost, simplicity and applicability to highly degraded specimens.

Mini-SNaPshot multiplex assays authenticate elephant ivory and simultaneously identify the species origin.

Illegal trading of ivory is mainly responsible for the dramatic decline in elephant populations. Thailand is one of the largest laundering hotspots for African ivory, as the domestic Asian elephant ivory can be legally traded. So, to help combat ivory poaching and smuggling, an efficient method is needed to identify the elephant species from its ivory and ivory products. In this study, a mini-SNaPshot® multiplex assay was developed and fully validated for the identification of confiscated ivory and low DNA template ivory products. Elephantid- and elephant species-specific mitochondrial single nucleotide polymorphisms (SNPs) were identified from 207 mammalian and 1705 elephant/mammoth cytochrome b sequence alignments. Seven informative SNPs were used for assay development. The assay unambiguously and accurately identified authentic elephant ivory and its species of origin on the basis of peak size and color observed in the haplotype profile. The assay was highly efficient for analysis of confiscated ivory and low-template ivory products with a 99.29% success rate (N=140). It was highly reproducible, exhibited no cross-reaction with eight other mammalian DNA; and had 100% identification accuracy. In addition, nested and direct PCR amplification were also compatible with the developed assay. This efficient assay should benefit wildlife forensic laboratories and aid in the prosecution of elephant-related crimes.