Mitochondrial ribosomal genes as novel genetic markers for discrimination of closely related species in the Angiostrongylus cantonensis lineage

Saralamba

et al. 2022

BMC Genomics

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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.

Section: Discussionsupporting

confidence: 91%

Section: Introductionmentioning

confidence: 99%

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

Saralamba

et al. 2022

BMC Genomics

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“…Comparing the four genetic markers tested, the mitochondrial COI and 16S rRNA genes had the highest classification accuracy, at 90% (nine out of ten datapoints). This finding is especially important because the mitochondrial COI and 16S rRNA genes have proven to be useful genetic markers for molecular identification due to their high levels of sequence variation [29, 32, 36, 37].…”

Section: Resultsmentioning

confidence: 99%

“…21 rRNA genes have proven to be useful genetic markers for molecular identification due to their high levels of sequence variation [29,32,36,37]. Incongruences between the actual and predicted results can be attributed to the proportion of sequences available in reference databases.…”

Section: Classification Accuracy With Actual Specimensmentioning

confidence: 99%

Applying taxonomic boundaries for species identification (ABIapp): A convenient and accurate application for species delimitation of parasitic helminths

Thaenkham

Wichaita

et al. 2022

Preprint

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Background: Parasitic helminths are highly diverse and ubiquitously distributed in various environments and hosts. Their vast species diversity renders morphological and molecular species delimitation challenging, due to phenotypic and genotypic variations. Currently used approaches to species delimitation are generally computationally intensive. Here, using genetic distances, we developed and validated a simple and easy-to-use application, Applying taxonomic Boundaries for species Identification (ABIapp), to aid in helminth species delimitation. Methodology/Principal Findings: ABIapp uses a database of cut-off genetic distances obtained using the K-means algorithm to determine helminth taxonomic boundaries for ten genetic markers: The nuclear 18S and 28S rRNA genes, ITS1 and 2 regions, and the mitochondrial 12S and 16S rRNA, COI , COII, cytB, and ND1 genes. ABIapp was written in R, and the Shiny framework was used to produce an interactive and user-friendly interface. ABIapp requires just three types of input (genetic distance, genetic marker, helminth group) that are easily generated through basic morphological and molecular analysis. To validate ABIapp’s accuracy and robustness for use, validation was performed both in silico and with actual specimens. Prior to validation, ABIapp’s database of genetic distances and species used was increased to broaden the app’s applicability. In silico validation was conducted by obtaining 534 genetic distances from 91 publications and inputting these into ABIapp. Using confusion matrices, an overall classification accuracy of 79% was achieved, demonstrating the robustness and accuracy of ABIapp. Using sequences of the 12S, 16S, COI, and 18S rRNA genes obtained from ten representative helminth specimens, an overall classification accuracy of 75% was achieved. Conclusions/Significance: Our results demonstrate the applicability and robustness of ABIapp for helminth species delimitation using ten common genetic markers. With a user-friendly interface, coupled with minimal and simple data input and robust classification accuracy, ABIapp provides helminth researchers with a convenient tool for helminth species delimitation.

“…Also, in an assessment of ten genetic markers commonly used for helminth molecular-based studies, the nuclear 18S rRNA gene had the lowest sequence variation across taxonomic categories. In contrast, the mitochondrial rRNA genes had sufficient sequence variation to differentiate between closely related species for nematodes, trematodes, and cestodes 42 , 43 . As DNA metabarcoding requires short diagnostic sequences (≤ 300 base pairs) to accommodate for Illumina sequencing and degraded eDNA present in environmental samples, the high sequence conservation of the 18S rRNA gene can limit its use as there might not be sufficient informative sites for accurate species identification 24 , 44 .…”

Section: Discussionmentioning

confidence: 97%

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

Saralamba

et al. 2022

Sci Rep

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Next-generation sequencing technologies have accelerated the pace of helminth DNA metabarcoding research, enabling species detection in bulk community samples. However, finding suitable genetic markers with robust species-level resolution and primers targeting a broad species range among parasitic helminths are some of the challenges faced. This study aimed to demonstrate the potential use of the mitochondrial 12S and 16S rRNA genes for parasitic helminth (nematodes, trematodes, cestodes) DNA metabarcoding. To demonstrate the robustness of the 12S and 16S rRNA genes for DNA metabarcoding, we determined the proportion of species successfully recovered using mock helminth communities without environment matrix and mock helminth communities artificially spiked with environmental matrices. The environmental matrices are human fecal material, garden soil, tissue, and pond water. Our results revealed the robustness of the mitochondrial rRNA genes, through the high sensitivity of the 12S rRNA gene, and the effectiveness of the 12S and 16S primers targeting platyhelminths. With the mitochondrial rRNA genes, a broad range of parasitc helminths were successfully detected to the species level. The potential of the mitochondrial rRNA genes for helminth DNA metabarcoding was demonstrated, providing a valuable gateway for future helminth DNA metabarcoding applications like helminth detection and biodiversity studies.