The potential use of mitochondrial ribosomal genes (12S and 16S) in DNA barcoding and phylogenetic analysis of trematodes

Abstract

Background Genetic markers like the nuclear ribosomal RNA (rRNA) genes, internal transcribed spacer regions, mitochondrial protein-coding genes, and genomes have been utilized for molecular identification of parasitic trematodes. However, challenges such as the design of broadly applicable primers for the vast number of species within Digenea and the genetic markers’ ability to provide sufficient species-level resolution limited their utility. This study presented novel and broadly applicable primers using the mitochondrial 12S and 16S rRNA genes for Digenea and aimed to show their suitability as alternative genetic markers for molecular identification of orders Plagiorchiida, Echinostomida, and Strigeida. Results Our results revealed that the mitochondrial 12S and 16S rRNA genes are suitable for trematode molecular identification, with sufficient resolution to discriminate closely related species and achieve accurate species identification through phylogenetic placements. Moreover, the robustness of our newly designed primers to amplify medically important parasitic trematodes encompassing three orders was demonstrated through successful amplification. The convenience and applicability of the newly designed primers and adequate genetic variation of the mitochondrial rRNA genes can be useful as complementary markers for trematode molecular-based studies. Conclusions We demonstrated that the mitochondrial rRNA genes could be alternative genetic markers robust for trematode molecular identification and potentially helpful for DNA barcoding where our primers can be widely applied across the major Digenea orders. Furthermore, the potential of the mitochondrial rRNA genes for molecular systematics can be explored, enhancing their appeal for trematode molecular-based studies. The novelty of utilizing the mitochondrial rRNA genes and the designed primers in this study can potentially open avenues for species identification, discovery, and systematics in the future.

Sensitive and accurate DNA metabarcoding of parasitic helminth mock communities using the mitochondrial rRNA genes

“…We showed that parasitic nematodes belonging to the four clades (clades I, III, IV, and V), trematodes belonging to three orders (Plagiorchiida, Echinostomida, and Strigeida), and cestodes in two orders (Cyclophylllidea and Diphyllobothridea) were able to be successfully detected. Our results are congruent with Chan et al (2020, 2022), where the same parasitic nematodes and trematodes were able to be identified via Sanger sequencing using the mitochondrial rRNA gene primers 29 , 30 . Through targeting a broad range of helminth species added in the mock communities, we validated that these primers can be applicable for various types of helminth DNA metabarcoding studies targeting different hosts and environment types.…”

“…Additionally, the 12S and 16S platyhelminth primers recovered a single cercaria of S. mekongi present in the pond water mock helminth community. Our results support the recent findings by Chan et al (2022), where the high sensitivity of the mitochondrial 12S and 16S primers was revealed by successfully amplifying all representative trematode specimens of various life-cycle stages for DNA barcoding 30 . As research on platyhelminth DNA metabarcoding remains relatively unexplored, apart from the study by Douchet et al (2021), having a highly sensitive primer for platyhelminth detection can be helpful 31 .…”

“…Helminths were identified to the species level based on morphological characters and preserved in 70% ethanol at − 20 °C. For the mock communities, helminths that had available partial 12S and 16S rRNA gene sequences from previous studies were selected 29 , 30 . Five types of mock helminth communities were simulated through spiking of helminth communities with environmental samples.…”

“…For the 12S rRNA gene, two PCR reactions with primers targeting each helminth group (12S-platyhelminth and 12S-nematode) were conducted per mock community, while for the 16S rRNA gene, one PCR reaction with primers targeting both platyhelminths and nematodes (16S-helminth) was performed. We used the 12S and 16S locus-specific primers from Chan et al (2020, 2022) 29 , 30 that targeted parasitic nematodes and trematodes. We designed new primers targeting the 12S rRNA gene for cestodes in this study.…”

“…Utilizing the mitochondrial 12S and 16S rRNA genes, primers targeting parasitic nematodes and trematodes were recently developed 29 , 30 . The potential of using the mitochondrial rRNA genes for helminth molecular-based studies was illustrated by its sensitivity to detect various life-cycle stages of parasitic helminths with robust species-level taxonomic resolution and the ability to amplify DNA for a broad range of nematodes and trematodes.…”