A comparative analysis of multiple loci in hematophagous leech blood meals for wildlife monitoring in southeast asian tropical rainforests

Next-generation sequencing ( NGS ) enables the use invertebrate-derived DNA (iDNA) metabarcoding to enhance wildlife survey efficiency and coverage. However, this approach remains relatively new in Malaysia, and a precise DNA marker suited for the tropical rainforest climate of Southeast Asia has yet to be established. Hematophagous (blood-feeding) leeches inhabit various ecosystems, including tropical rainforests, providing a promising pilot target. Here, we used three primer pairs targeting the cytochrome c oxidase ( COI ), and the 16S and 12S ribosomal DNA loci in over 300 individual leeches collected from forests in central Peninsular Malaysia. We obtained 95 amplicon sequence variants ( ASV s) for COI , 81 ASV s for 16S, and 46 ASV s for 12S. We identified 42 vertebrate species, including greater mousedeer ( Tragulus napu ), Sunda pangolin ( Manis javanica ), Asian elephant ( Elephas maximus ), red junglefowl ( Gallus gallus ), and Malayan field rat ( Rattus tiomanicus ). Additionally, several amphibian and reptile species were also detected, such as Blyth’s river frog ( Limnonectes blythii ), field frog ( Fejervarya limnocharis ) and many-lined sun skink ( Eutropis multifasciata ). The 16S locus identified the highest number of species (28), followed by 12S (25 species) and COI (19 species). Our findings thus support the use of these loci as a complementary suite of DNA markers to enhance wildlife monitoring. Although traditional methods such as camera trapping can detect a larger number of wildlife species, these are considerably more time- and labour-intensive compared to DNA metabarcoding. The application of iDNA metabarcoding is highly recommended as a complementary approach to traditional surveys, particularly in dense tropical forests, to improve wildlife inventory efforts.