SUMMARY OF THESIS
Echinostomiasis is caused by intestinal flukes (or echinostomes) from the trematode family Echinostomatidae and the suborder Echinostomata (class Trematoda, phylum Platyhelminthes). The majority of these parasite infections are zoonotic and can be found anywhere in the world, although they are particularly prevalent in Asian communities such as India, Indonesia, the Philippines, China, Malaysia, Singapore, Korea, Japan, Thailand, Myanmar, Laos, Cambodia, and Vietnam. It is estimated that tens of millions of people have become infected, with hundreds of millions more at risk. Humans can become infected by consuming raw or undercooked mollusks, fish, crabs, or amphibians that carry the infective encysted metacercariae. In addition to morphological examination, accurate diagnosis and timely treatment of echinostomiasis necessitate the use of molecular techniques like as genetic markers generated from the mitochondrial genome (mtDNA) and the ribosomal transcription unit (rTU). The mtDNA and rTU of trematodes and echinostome intestinal flukes (class Trematoda) are important genetic sources, providing genetic markers for identification, diagnosis, and detection, species differentiation, phylogenetic, origination, and evolution studies, molecular epidemiology, and population genetics, respectively. In studies on the evolution of biological species and taxonomy, there is a great need to use genetic markers and molecular techniques to clarify taxonomic conditions and rankings, especially when dealing with “cryptic,” “synonymous,” “polymorphic,” “sister,” or “hybrid” species, and many developmental forms of parasites present in the same host. Accurate identification of each species, genus, or family is critical for effective global parasite prevention and control strategies, particularly for echinostomes. Novel technological investigations are essential for species identification, differentiation, and pathogenicity assessment. As a result, it is both required and urgent to gather and compile genomic data (mtDNAs and rTUs) for the relevant species, genera, families, superfamilies, suborders, and closely related trematodes, such as the family Echinostomatidae (superfamily Echinostomatoidea and suborder Echinostomata).
This study was undertaken under the title “Study on the genomics of the mitochondrial genome and ribosomal transcription units of some intestinal flukes in the family Echinostomatidae of the suborder Echinostomata”. The primary goal of this study was to obtain complete or nearly complete mitochondrial and nuclear ribosomal transcription unit sequences for
several species of echinostomes in the family Echinostomatidae and then use their annotated data for comparative genomic analysis to investigate the evolution and phylogeny of echinostomes and their congeners in the Trematoda class.
In this study, the mitochondrial genome (mtDNA) of four Thai echinostome species belonging to the family Echinostomatidae, including Echinostoma revolutum (strain MSD15), Artyfechinostomum malayanum (strain EMI3), Echinostoma miyagawai (strain RED11) and Hypoderaeum conoideum (strain RED42), as well as the ribosomal transcription unit (rTU) of the four strains/species aforementioned and additional strain EjPT of the species Echinochasmus japonicus (family Echinochasmidae) of Vietnam were obtained. The mitochondrial genes of Eca. revolutum and Eca./A. malayanum were obtained by PCR/LPCR and sequenced by the Sanger method, with the exception of the non-coding region (NCR) by NGS (next-generation sequencing); the entire mtDNA of Eca. miyagawai and H. conoideum by NGS; and all rTUs of all five species, including Ecs. japonicum, by LPCR and automated Sanger sequencing.
The mitogenomic research
The full-length mitochondrial genomes of four echinostome species have been fully sequenced. These mtDNA sequences include: i) Eca. revolutum (Fröhlich, 1802) Rudolphi, 1809, strain MSD15 (Thailand), with a total length of 17,030 bp with a fully sequenced NCR of 3,549 bp (GenBank accession number MN496162); ii) A. malayanum Leiper, 1911, strain EMI3 (Thailand) (formerly known as Eca. malayanum), with a total length of 17,030 bp with a fully sequenced NCR of 3,622 bp (GenBank: OK509083); iii) Eca. miyagawai Ishii, 1932, strain RED11 (Thailand), with a total length of 19,417 bp with a full NCR of 5,935 bp (GenBank: OP326312); and iv) H. conoideum Dietz, 1909, strain RED42 (Thailand), which has a total length of 18,011 bp with a full NCR of 4,475 bp (GenBank: PP110501). Notably, the mtDNA of Eca. miyagawai (19,417 bp) appears to be the longest of all Echinostomatidae species that have been fully sequenced to date, with the exception of Eca. paraensei/(GenBank: KT008005), which has an estimated length of 20,298 bp but has not been fully sequenced. All four Echinostomatidae species have a circular mtDNA molecule consisting of 12 protein-coding genes (protein-coding genes, PCGs) (cox1−3, cob, nad1−6, nad4L, atp6), two mitoribosomal RNA genes (MRGs) (16S or rrnL and 12S or rrnS),, 22 transfer RNA genes (tRNA or trn), and a non-coding region (NCR) with various numbers and length (long and short) of tandem repeat units. The mtDNA gene arrangement (or gene order) is similar to that of most trematode species and is highly conserved
among closely related species, as follows: 5’-cox3-H-cob-nad4L-nad4-QFM-atp6-nad2-VAD-nad1-NPIK-nad3-S1W-cox1-T-rrnL-C-rrnS-cox2-nad6-YL1S2L2R-nad5-G-E-NCR[LRUs#]-[SRUs]-3’ (mtDNA of Eca. revolutum; Eca. miyagawai; and H. conoideum), and 5’-cox3-H-cob-nad4L-nad4-QFM-atp6-nad2-VAD-nad1-NPIK-nad3-S1W-cox1-T-rrnL-C-rrnS-cox2-nad6-YL1S2L2R-nad5-G-NCR[LRUs#]-E-[SRUs]-3’ (of A. malayanum). The atp8 gene was absent and the nad4L and nad4 genes were overlapped by a 40 bp, which is common in mtDNAs of many trematodes.
Twenty-two transfer RNAs, ranging from 60 to 71 nucleotides, were identified. Twenty had a “cloverleaf” structure and folded into secondary structures with four complete arms (i.e., AA arm, DHU arm, AC arm, and TpsiC arm), but two Serine-specifying tRNAs (tRNAS1(AGN and tRNAS2(UCN)) had a special form, lacking the DHU arm. The mitogenomic NCRs of four species were successfully obtained, and all possessed long and short tandem repeat units ((LRUs and SRUs), with different lengths and numbers. These units were in Eca. revolutum with 7.6 LRUs/317 bp/each and 5.3 SRUs/207 bp/each; in A. malayanum with 5.5 LRUs/336 bp/each and 7.5 SRUs/207 bp/each; in Eca. miyagawai with 15.3 LRUs/319 bp/each and 4.8 SRUs/213 bp/each; and in H. conoideum with 13 LRUs/241 bp/each and 9.7 SRUs/111 bp/each. To date, in addition to the mtDNAs in this study, the family Echinostomatidae (suborder Echinostomata) has 15 mitochondrial genomes from 15 strains of 12 echinostome species sequenced and their data annotated.
The mitogenomes of 15 strains of 12 echinostome species, including four newly collected echinostome genera/species were analyzed for gene/genome characteristics, such as gene comparison, base composition, base usage, pattern of usage, skew/skewness, PCG codons and codon usage, and codon bias, genetic distance and genomic polymorphism.
As result, among all 12 PCGs and two MRGs, the divergence rate of nad6 gene was the highest (64.68–75.84%) while the lowest was seen in cytB/cob gene (13.67–27.08%) and cox1 gene (14.14–28.61%). The four nucleotides (A, T, G, C) appeared to be unevenly used in mtDNA of all echinostome species (family Echinostomatidae) with T being more used than G, G being more used than A, and A being more used than C, forming the usage pattern of “T > G > A > C”. Since T is more used than A and G is more used than C, the AT-skew usage was negative and the GC-skew was positive, which is also a common feature of most trematode mtDNAs analyzed to date. For PCGs, the start codon was mainly ATG, more than GTG, and the stop codon was mainly TAG,
more than TAA. It is noteworthy that TGA (a stop codon in vertebrates) was used to code for tryptophan. The codon usage frequency in PCGs of the four species in this study as well as of all echinostomes (15 strains/12 species) of the family Echinostomatidae was most abundant in phenyalanine (TTT), leucine (TTG), valine (GTT), and least in arginine (CGC). The number of codons with 2 or 3 Ts (thymine) accounts for 24–25% (~10% TTT/Phe, ~8% TTG/Leu, and ~7% GTT/Val) in PCGs, and in many cases, there were over 800 Phe/Leu/Val codons, up to nearly 40% of the 3,359–3,371 codons of 12 echinostome PCGs.
Genetic distances (GD) estimated among 15 strains/12 species of Echinostomatidae based on amino acid identity comparison of 12 PCGs showed that GD was very low among strains/species belonging to the “revolutum” group (group with “37-collar spine”), higher between Artyfechinostomum and Echinostoma species (21–22%) and highest among Echinostoma/Artyfechinostomum species with H. conoideum (23–24%). Intraspecific KCDT (strains of the same species) was very low, with 0.43% for strains in Eca. revolutum, 0.40–0.52% for the Eca. miyagawai strains and 0.62% for the H. conoideum strains.
The NCR polymorphism, which was identified in various numbers of LRUs and SRUs, as well as the regulatory role of their promoter-motifs in the mtDNA of Eca. miyagawai and other echinostome species, is a significant finding in this study. Polymorphism in LRUs and SRUs may also be seen in the palindrome-embedded hairpin and loop structures, and has been analyzed and discussed in the thesis.
For utility of the mtDNA datasets, phylogenetic relationships were determined based on comparative analysis of concatenated amino acid sequences of 12 PCGs of 57 strains belonging to 41 trematode species of five families from the suborder Echinostomata (Echinostomatidae, Cyclocoelidae, Echinochasmidae, Fasciolidae and Himasthlidae), two families from the suborder Opisthorchiata (Opisthorchiidae and Heterophyidae), two families from the suborder Xiphidiata (Paragonimidae and Dicrocoeliidae) and one species from the family Schistosomatidae (the species Schistosoma haematobium) as an outgroup. The ML phylogenetic tree revealed four distinct groups with high bootstrap-support: Echinostomata, Opisthorchiata, and two families, Paragonimidae and Dicrocoeliidae from Xiphidiata. The Opisthorchiata is a well-supported monophyletic group with two “sister” families (Opisthorchiidae and Heterophyidae), but the Xiphidiata suborder is substantially polyphyletic, with two families positioned independently. The topology demonstrated the monophyletic status of Echinostomata, with the Echinostomatidae
family classified as a “sister” group to the Fasciolidae. Six Echinostoma strains of Eca. miyagawai, Eca. revolutum, Eca. caproni, and Eca. paraensei were placed into one subcluster and are a “sister” to the Artyfechinostomum subclade (comprising A. malayanum and A. sufrartyfex species), whereas two strains of H. conoideum (one from Thailand and another from China) formed a subgroup, monophyletic to the two Echinostomatidae spp. of the United States. The [Echinochasmidae+Himasthlidae] form a monophyletic group with the [Echinostomatidae+Fasciolidae], however the Cyclocoelidae are paraphyletic. The interesting phylogenetic relationships of the Echinostomata suborder, particularly the Echinostoma genus, and the genetic subdivision of the newly discovered “cryptic” species within, need to be further investigated.
The ribosomal transcription unit research
The nuclear ribosomal transcription unit (rTU) sequences of five echinostomes and their implications are analyzed and the study of the inter- and intrafamilial relationships of these and other families in the suborder Echinostomata are also presented. The sequences obtained are the complete rTU of A. malayanum (9,499 bp), the near-complete rTU of H. conoideum (8,076 bp), and the coding regions (from 5’-terminus of 18S to 3’-terminus of 28S rRNA gene) in Eca. revolutum (6,856 bp), Eca. miyagawai (6,854 bp), and Ecs. japonicus (7,150 bp). Except for the longer first internal transcribed spacer (ITS1) in Ecs. japonicus, all genes and spacers were almost identical in length. PhyML was used to generate comprehensive maximum-likelihood phylogenies. The datasets consisted of either the concatenated 28S + 18S rDNA sequences (5.7–5.8 kb) from 60 complete rTUs of 19 families or entire 28S sequences (about 3.8–3.9 kb) from 70 strains or species of 22 families. The phylogenetic trees confirmed that the superfamily Echinostomatoidea (suborder Echinostomata) is monophyletic. A phylogeny of 169 28S D1−D3 rDNA sequences (1.1-1.3 kb) from 98 species, 50 genera, and 10 families, including 154 echinostomatoid sequences (85 species/42 genera), revealed generic relationships within Echinostomatidae and Echinochasmidae, as well as family relationships within Echinostomata and other suborders. Within Echinostomatidae, Echinostoma, Artyfechinostomum, and Hypoderaeum appeared to be monophyletic, whereas Echinochasmus (Echinochasmidae) was polyphyletic. The Echinochasmidae are a sister group to the Psilostomidae. The datasets provided here will be useful for taxonomic reappraisal as well as studies of evolutionary and population genetics in the superfamily Echinostomatoidea, the sole superfamily in the suborder Echinostomata.
Contributions of the thesis
Comparison of mitogenome (mtDNA) and ribosomal transcription unit (rTU) sequences can provide insights into genetic/genomic characteristics of trematodes, including echinostomes. Comparative datasets can also reveal a number of disputed issues about evolutionary relationships and histories among species from important groups, such as the suborder Echinostomata of the phylum Platyhelminthes. Sequences from mtDNA and rTU were previously and are currently being obtained in species and strains of a number of important genera in the suborder Echinostomata of the phylum Platyhelminthes, primarily Echinostoma, Artyfechinostomum, Hypoderaeum, and Echinochasmus, in an attempt to improve molecular characterization, classification, and taxonomic groupings. The research reported in this thesis yielded extremely rich datasets encompassing the (near) full mtDNAs and rTUs of multiple species of significant zoonotic echinostomes. Gene order and structural description of mitogenes/mitogenomes, including the polymorphic non-coding regions and tRNAs as well as the rTUs of echinostomes were summarized. The individual mtDNAs and rTUs of each species, as well as 15 strains of 12 echinostomes, were compared. These characteristics included the base/nucleotide usage for construction of sequences and genes (skewness); the arrangement of genes; pairwise comparisons among protein-encoding genes, regions, and genomes; elucidation of the structures of transfer RNAs and mito- and nuclear ribosomal RNAs; a description of some features of unassigned and repetitive sequences included in the non-coding regions; and a reassessment of the mitogenetic code involved in the Echinostomatidae family. The most notable findings of the thesis are updated phylogenetic analyses of the intergeneric, interfamilial, and intersubordinal relationships within and between the family Echinostomatidae/suborder Echinostomata and other families and suborders in the class Trematoda.