The metabarcoding of Grubs: Traditional herbal medicine of Scarabaeidae larvae.

Grubs, called Qicao in China, have a long tradition as herbal medicine in East Asia. These larvae belong to the diverse family Scarabaeidae and are typically harvested from the wild during their immature stage based on morphological characteristics. However, rapid and accurate identification becomes challenging when relying solely on external morphological features, as the lack of clarity on biological sources raises safety concerns for clinical applications. The application of DNA metabarcoding provides a solution by enabling the determination of the biological source of a large sample. In the current study, we collected 19 batches of Grubs, consisting of 11,539 individuals, from the market and analyzed their biological composition through metabarcoding. We identified 49 Amplicon Sequence Variants (ASVs), 21 of which were Grubs. The 21 ASVs were classified into seven Molecular Operational Taxonomic Units (MOTUs) through species delimitation, which revealed that commercially available Grubs are predominantly sourced from Protaetia brevitarsis seulensis, while species of Rutelinae, Anomala, and Holotrichia were also abundant in some commercial batches. Among the identified ASVs, 28 belonged to non-Grub species and indicated adulteration from different animal families; high abundances of these ASVs were detected for Bombycidae, Tabanidae, and Viviparidae. Our findings underscore the complexity of Grubs' species composition and advocate for a deeper understanding of the wildlife sources contributing to herbal products. This research contributes valuable insights into the molecular identification of Grubs, paving the way for enhanced quality assurance in traditional medicine applications to provide safe and effective medicines for humanity.

Qualitative identification of lonicerae japonicae flos in traditional chinese medicine using metabarcoding combined with specific mini-barcodes.

BACKGROUND: Lonicerae japonicae flos, also known as Jinyinhua (JYH), is an important component of traditional Chinese patent medicine (TCPM) products. However, the potential for adulteration and substitution with low-quality materials highlights the need for a reliable and sensitive approach to identify the species composition of TCPM products for consumer safety. METHODS AND RESULTS: We used universal ITS2 primers to amplify TCPMs containing JYH. However, the results were inconclusive, as only one operational taxonomic unit (OTU) was identified as Lonicera sp., which could not be identified at the species level. To confirm the species identification of Lonicera sp. in TCPM, we developed a short mini-barcode primer based on the psbA-trnH region, which, in combination with DNA metabarcoding technology, allowed for qualitative and quantitative analysis of artificially mixed samples. We applied the mini-barcode to distinguish TCPMs containing JYH and demonstrated its relatively accurate quantitative ability in identifying two Lonicera species. CONCLUSIONS: Our study presents a method for qualitative and quantitative identification of JYH, providing a promising application of DNA metabarcoding technology in the quality control of TCPM products.

A novel biological sources consistency evaluation method reveals high level of biodiversity within wild natural medicine: A case study of Amynthas earthworms as "Guang Dilong".

For wild natural medicine, unanticipated biodiversity as species or varieties with similar morphological characteristics and sympatric distribution may co-exist in a single batch of medical materials, which affects the efficacy and safety of clinical medication. DNA barcoding as an effective species identification tool is limited by its low sample throughput nature. In this study, combining DNA mini-barcode, DNA metabarcoding and species delimitation method, a novel biological sources consistency evaluation strategy was proposed, and high level of interspecific and intraspecific variations were observed and validated among 5376 Amynthas samples from 19 sampling points regarded as "Guang Dilong" and 25 batches of proprietary Chinese medicines. Besides Amynthas aspergillum as the authentic source, 8 other Molecular Operational Taxonomic Units (MOTUs) were elucidated. Significantly, even the subgroups within A. aspergillum revealed here differ significantly on chemical compositions and biological activity. Fortunately, this biodiversity could be controlled when the collection was limited to designated areas, as proved by 2796 "decoction pieces" samples. This batch biological identification method should be introduced as a novel concept regarding natural medicine quality control, and to offer guidelines for in-situ conservation and breeding bases construction of wild natural medicine.