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Article

First Report of Isolapotamon sp. as a Potential Intermediate Host of Paragonimus westermani in Davao Oriental, Philippines

by
Diadem R. Ricarte
1,
Joshua M. Cambronero
1,
Carmela H. Lorico
1,
Herbert J. Santos
2,
Nestor S. Arce, Jr.
3,4 and
Aleyla E. de Cadiz
1,5,*
1
Philippine Genome Center Mindanao, University of the Philippines Mindanao, Davao City 8000, Philippines
2
Department of Biomedical Chemistry, Graduate School of Medicine, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
3
College of Medicine, Jose Maria College Foundation, Inc., Philippine-Japan Friendship Highway, Davao City 8000, Philippines
4
Department of Clinical Tropical Medicine, Faculty of Tropical Medicine, Mahidol University, Bangkok 10400, Thailand
5
Department of Biological Sciences and Environmental Studies, College of Science and Mathematics, University of the Philippines Mindanao, Davao City 8000, Philippines
*
Author to whom correspondence should be addressed.
Parasitologia 2025, 5(4), 67; https://doi.org/10.3390/parasitologia5040067
Submission received: 21 August 2025 / Revised: 11 November 2025 / Accepted: 1 December 2025 / Published: 11 December 2025
Browse Figures
Versions Notes

Abstract

Paragonimus westermani is a food-borne zoonotic trematode transmitted to humans through the consumption of undercooked crustaceans. Freshwater crabs act as the second intermediate host for the encysted metacercariae stage. However, accurate identification of intermediate hosts remains a challenge. Here, we aimed to detect and identify P. westermani in randomly collected freshwater crabs and determine the species of infected crabs in Davao Oriental through molecular methods. Specifically, Sanger and next-generation sequencing were conducted for species identification through BLASTn, followed by phylogenetic analyses to understand geographic and taxonomic relationships. Results showed P. westermani DNA was detected in five out of eleven crab samples and these sequences were closely grouped to the Philippine reference sequence. Through a similar approach, the infected crabs showed high sequence similarity and formed tight clustering to Isolapotamon sp. Overall, the results provided evidence that P. westermani DNA was detected in Isolapotamon sp., a genus endemic to Mindanao, and can be a potential intermediate host. This expands our current understanding of transmission ecology beyond the only known intermediate host in the Philippines, Sundathelphusa philippina.

1. Introduction

Paragonimus westermani, the Oriental lung fluke, is a food-borne zoonotic trematode (FZT) that causes human paragonimiasis, along with more than 30 species of the genus Paragonimus have been reported [1]. It is transmitted through the consumption of improperly cooked crustaceans [2]. Globally, the disease affects around 23 million people, including approximately five million severe cases, with more than 240 reported deaths in 2010 [3]. In the Philippines, there are 12 endemic provinces identified, including Davao Oriental, with a reported prevalence of 6.6% [4,5].
The parasite’s complex life cycle, which occurs in freshwater environments, involves three host taxa: freshwater snail as first immediate host (IH), freshwater crab or crayfish as second IH, and mammals, including humans, as definitive hosts [6]. The parasite’s eggs are expelled in sputum and/or feces and embryonate in water. The miracidia hatch and penetrate snails, and the parasite progresses to the sporocyst, redia, and cercaria stages. The cercariae then leave the snails, penetrate crabs or crayfish, and encyst as metacercariae [6,7]. Humans acquire infection by consuming raw or undercooked infected crustaceans, after which larvae excyst in the gut and migrate to the lungs, maturing into hermaphroditic adults that typically reside in pairs within cystic cavities. Clinical features include chronic cough, fever, and hemoptysis, often mimicking tuberculosis, and in some cases, ectopic infections involving the brain or spinal cord occur [8,9].
Conventional methods, such as morphological identification of metacercariae in intermediate hosts and microscopic analysis of eggs in stool or sputum samples, are used for diagnosing paragonimiasis [10]. However, these procedures are labor-intensive and prone to misdiagnosis, which limits their effectiveness for surveillance use [11]. To add to this burden, species variation as well as the possibility of co-infection between species present a challenge in identifying intermediate hosts. To mitigate these limitations, studies have utilized molecular techniques alongside their traditional processes, which offer rapid and sensitive detection, such as loop-mediated isothermal amplification (LAMP), ELISA, and immunodiagnostic tests [12,13,14,15,16,17]. Moreover, genomic advances, including whole-genome sequencing and gene typing, as well as transcriptomics, have further enhanced understanding of strain diversity, host–parasite relationships, and epidemiological patterns, thereby providing more precise diagnostics and targeted control strategies [15,18,19,20].
The objectives of this study were to molecularly detect P. westermani in freshwater crabs from Davao Oriental, identify the species of infected crabs through NGS, and understand the phylogenetic relationships of the P. westermani in crab hosts, and crabs known to be IH. The molecular identification of the parasite and the infected freshwater crabs, as well as the genetic relationships are discussed herein.

2. Materials and Methods

2.1. Sample Collection

A total of eleven freshwater crabs were randomly collected by the community health workers from Barangay Cayawan, Manay, Davao Oriental, which is located in the southeastern part of the Philippines. Samples were transported to the Philippine Genome Center Mindanao (PGCMin), University of the Philippines Mindanao, for molecular identification. Crab samples were individually preserved in absolute ethanol and stored in a 50 mL conical tube at 4 °C refrigerator at the Molecular Biology Laboratory of PGCMin.

2.2. DNA Extraction

Two distinct sample types were utilized, although both are obtained from the same source. For the detection of P. westermani, crab’s branchiae were utilized while muscle tissue from the appendages was intended for crab species identification. A 25 mg sample was weighed for both sample types for all eleven crab specimens. Genomic DNA was isolated from the freshwater crabs using Qiagen DNEasy Blood and Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The quality, concentration, and purity of the genomic DNA were determined through agarose gel electrophoresis and a MultiSkan SkyHigh spectrophotometer (ThermoFisher, Waltham, MA, USA).

2.3. Polymerase Chain Reaction

DNA amplification was performed using a 10 µL total reaction volume containing 5 µL 2X Promega GoTaq Master Mix (Promega, Madison, WI, USA), 0.4 µL of 10 µM forward and reverse primers, 2.2 µL Nuclease Free Water, and 2 µL of 30 ng/µL genomic DNA. The 16S mitochondrial rRNA primer pairs T7-1 (Forward: 5-ATT TAC ATC AGT GGG CCG TC-3) with SP6-1 (Reverse: 5-GAT CCA AA A GCA TGT GAA AC-3) was used to amplify the partial region of the COX1, full region of tRNA-Thr, and partial region of 16S rRNA with the following conditions: initial denaturation at 94 °C for 5 min, followed by 35 cycles of denaturation at 95 °C for 30 s, annealing temperatures between 55 °C and 60 °C for 30 s, and extension at 72 °C for 30 s, and final extension at 72 °C for 10 min [21]. Optimized annealing temperature of crab samples CRB01, CRB02, CRB03, CRB04, CRB05, CRB07, CRB09, and CRB11 was 60 °C while CRB08 and CRB10 was at 55 °C for this primer. Additionally, the amplification of the COX1 region using primers LCO1490 (5′-GGT CAA CAA ATC ATA AAG ATA TTG G-3′) and HC02198 (5′-TAA ACT TCA GGG TGA CCA AAA AAT CA-3′) includes initial denaturation at 95 °C for 5 min, followed by 35 cycles of denaturation at 95 °C for 30 s, annealing at 52 °C for 30 s, and extension at 72 °C for 1 min and 30 s, and final extension at 72 °C for 5 min [22]. Amplicons of both primers were gel-purified using Vivantis GF-1 Purification kit following the product’s protocol.

2.4. Sequencing

2.4.1. Next-Generation Sequencing

Library preparation was performed following the Illumina kit manufacturer’s instructions (Illumina, San Diego, CA, USA) and was quantified on Qubit 4 Fluorometer (ThermoFisher Scientific, Wilmington, DE, USA), and controlled on the Agilent Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA). Libraries were sequenced in 2 × 101 bp paired-end reads on an Illumina NextSeq 1000 platform at the Philippine Genome Center Mindanao.

2.4.2. Sanger Sequencing

Purified DNA samples were processed using Big Dye terminator cycle sequencing kit V3.1 manufacturer’s protocol (PE Applied Biosystems, Foster City, CA, USA) and sequenced in ABI SeqStudio Genetic Analyzer (PE Applied Biosystems, Foster City, CA, USA).

2.5. Bioinformatics Analysis

Sequence data from Illumina NextSeq 1000 were extracted in compressed FASTQ format, while the Sanger Sequencing data were in ABI format chromatogram file.

2.5.1. Next-Generation Sequencing Data Processing

Illumina paired-end reads were assessed for quality using FastQC v0.12.1 [23] and trimmed with Trimmomatic v0.39 using default parameters to remove low-quality reads and Nextera adapters [24,25]. Cutadapt v5.0 was then used to remove sample-specific primers [26]. Cleaned reads were assembled de novo using SPAdes v4.2.0, resulting in a set of contigs [27]. Contigs were filtered by mapping against the complete mitochondrial reference of P. westermani (MN412706.1) using BWA v0.7.17 [28], and alignments were processed with SAMtools [29]. Taxonomic identification was performed by querying contigs to BLASTn (NCBI, core_nt database, https://blast.ncbi.nlm.nih.gov/Blast.cgi (accessed on 21 July 2025)) [30]. For crab samples, where the expected identity was unknown, BLASTn searches were performed on contigs with reasonable length (>200 bp). The most frequently occurring taxonomic assignment was considered valid, and the contig with the highest percent identity (>95%) was selected as the representative sequence. Final sequences were extracted from the set of contigs, renamed, and saved in FASTA format.

2.5.2. Sanger Sequencing Data Processing

For raw sequence data generated, FinchTV 1.4.0 was used to visually assess sequence quality, reject low-quality reads, and trim end regions with erratic or ambiguous signals [31]. The trimmed sequences were then aligned using the CAP contig alignment tool within BioEdit v7.2, producing a consensus sequence [32]. To resolve ambiguous base calls, the aligned reads were inspected in MEGA v7 (Molecular Evolutionary Genetics Analysis) by comparing paired read chromatogram signals and nucleotide base quality scores, as viewed in FinchTV [33]. Final sequences were saved in FASTA format and subjected to BLASTn analysis for identity confirmation.

2.5.3. Intraspecific Sequence Comparison

The degree of similarity among the sequences of this study was assessed by estimating pairwise evolutionary divergence using the Maximum Composite Likelihood model implemented in MEGA v12 [34,35]. Because the sequences varied in amplicon length, MUSCLE alignment was applied, and non-aligned regions were subsequently trimmed prior to analysis to account for differences in amplicon size.

2.5.4. Phylogenetic Tree Construction

Reference sequences were retrieved from the NCBI GenBank database, based on taxonomically verified entries and relevant publications (Tables S1 and S2). For P. westermani samples, the targeted gene included tRNA-Thr in addition to the 16S rRNA (rrnL or large ribosomal subunit), as most available reference sequences mapping to our samples contained these adjacent genes (Table S1).
The selection of sequences was further refined to include only sequences originating from natural intermediate hosts, in order to preserve ecological relevance and ensure accurate representation of naturally occurring host–parasite relationships. An exception was made for the official reference sequence and a Philippine-derived reference, both of which were included despite lacking host information, to support geographic mapping within the phylogenetic context.
Multiple sequence alignment was performed in MEGA v12 using the MUSCLE algorithm. The alignments were trimmed to a uniform length of 463 bp for P. westermani and 528 bp for Isolapotamon sp. A model test using the “use all sites” option was conducted to determine the best-fit nucleotide substitution model for each dataset: GTR + G + I for the crabs, and HKY for P. westermani. Midpoint-rooted phylogenetic trees were constructed using the Maximum Likelihood method with 1,000 bootstrap replicates, also implemented in MEGA. The Newick files of each tree were exported to R studio to improve the visualizations of the phylogenetic tree.

2.5.5. Sequence Data Submission

The sequence data that passed quality control and were used for the analysis were annotated, curated, and submitted in the GenBank submissions portal through BankIt (Tables S1 and S2).

3. Results

3.1. Molecular Identification of P. westermani Targeting 16S Mitochondrial rRNA Gene from Freshwater Crabs Using BLASTn

Out of eleven samples, ten were successfully amplified and proceeded to next-generation sequencing. To confirm the presence of P. westermani, raw sequence data generated were subjected to BLASTn analysis and revealed that five samples, specifically CRB02, CRB03, CRB04, CRB09, and CRB11, were identified as P. westermani with high percent similarity (ranging from 99.47% to 99.87%) to the partial mitochondrial sequence of the Philippine isolate (Paragonimus westermani tRNA-Thr gene and large subunit ribosomal RNA gene; Accession No. AY190064.1) [36]. The detailed BLASTn results are presented in Appendix A Table A1. For CRB01, CRB05, CRB06, CRB07, CRB08, and CRB10, different organisms were identified (Appendix A Table A2).

3.2. Phylogenetic Analysis of P. westermani 16S Mitochondrial rRNA Gene from Freshwater Crabs and Other Hosts

Phylogenetic analysis was performed to determine the placement of P. westermani sequences identified from our samples using 16S rRNA primers, relative to reference sequences in the NCBI database containing host metadata and both tRNA-Thr and 16s rRNA (rrnL) genes.
As shown in Figure 1, our P. westermani sequences—identified from Isolapotamon sp. crab hosts—form a distinct clade, separate from those of other hosts, with 100% bootstrap support. The figure also shows that P. westermani sequences from the same host species and country tend to cluster together with a high bootstrap value, suggesting host- and geography-specific genetic patterns. Sequences from Vietopotamon aluoiense and Indochinamon tannanti (Vietnam) and Eriocheir japonica and Geothelphusa dehaani (Japan) cluster separately by country and host, each with high bootstrap values.
The sequence similarity was further assessed among P. westermani samples by calculating the pairwise genetic distances (Table A5). All sequences were identical with an exception to CRB11 differing slightly (0.005 distance, approximately three nucleotide differences) from the others. Nevertheless, this still confirms their close genetic relatedness and supports the phylogenetic clustering observed in Figure 1.

3.3. COX1-Gene-Based Identification of the Examined Freshwater Crabs Using BLASTn

NCBI BLASTn results from assembled sequences obtained from next-generation sequencing indicated that COX1 amplified freshwater crabs (CRB02, CRB03, CRB04, CRB09, and CRB11) carrying P. westermani were most closely related to Isolapotamon sp. with a sequence similarity of 99% sequence similarity to the Isolapotamon sp. COX1 gene (cytochrome c oxidase subunit I, partial cds; mitochondrial; Accession No. MT514316.1 in Table A3) [37]. To confirm these findings, Sanger sequencing was performed, revealing similar results (Table A4).

3.4. Phylogenetic Analysis of Crabs as a Potential Intermediate Host for P. westermani

Based on Figure 2 shown below, all identified Isolapotamon sp. formed a monophyletic group (100% bootstrap) under the reference, Isolapotamon sp. (MT514316.1) [37]. Specifically, Isolapotamon sp. (MT514316.1), CRB02, CRB03, and CRB04 formed a tight subclade with 98% bootstrap support. Moreover, all identified crab hosts of P. westermani belong to the order Decapoda, with most classified under the infraorder Brachyura. Only Lithodes nintokuae belongs to the infraorder Anomura.
Pairwise comparison of crab sequences (Table A6) revealed no divergence (0.000 distance), indicating identical haplotypes among the Isolapotamon sp. carrying P westermani.

4. Discussion

In this paper, we detected DNA of P. westermani in freshwater crab Isolapotamon sp. (Decapoda: Brachyura: Potamidae) in the province of Davao Oriental through the application of molecular methods. Our study presents the first report suggesting that Isolapotamon sp. could serve as a potential IH aside from the only known key IH of P. westermani in the Philippines, the Sundathelphusa philippina [38].
The P. westermani sequences amplified from the freshwater crab samples were highly similar, representing a single haplotype despite minor variation in BLASTn percent identities (Table A1). Detection of other organisms in some samples reflects the broad-binding nature of the 16S rRNA gene, which can amplify conserved regions across taxa [39]; however, this does not affect the identification of P. westermani. Phylogenetic analysis further supported these findings, showing that all P. westermani sequences from our samples cluster together with high bootstrap support. Similarly, the COX1 sequences of the freshwater crab samples showed no intraspecific variation, forming a monophyletic clade in the phylogenetic tree, indicating that all analyzed individuals represent the same haplotype of Isolapotamon sp. Most of the previously reported crab intermediate hosts of P. westermani were under the infraorder Brachyura which aligns with the study by Yeo et al. (2008), particularly identified freshwater crabs (Brachyura) as intermediate hosts of Paragonimus, thereby underscoring their role in the transmission of paragonimiasis [40]. Moreover, within this infraorder, two distinct superfamilies can be recognized. The first family comprises Isolapotamon species and other representatives of the superfamily Potamoidae while the second encompasses the genus Eriocheir under the superfamily Grapsoidea. Phylogenetic analysis revealed that Isolapotamon sp.-associated sequences form a distinct Philippine clade (Figure 1), suggesting a locally adapted lineage. This geographic structuring is consistent with the separation of Vietnamese sequences (V. aluoiense and I. tannanti) and Japanese sequences (E. japonica and G. dehaani) into their respective clades. While host-specific clustering was also observed, geographic origin played a more dominant role, as sequences from different hosts but the same country tend to cluster together, as shown in the Japan group (Figure 1). This observation supports previous findings that geography strongly influences the genetic differentiation of Paragonimus complex species, even at the regional level [12,41].
Comparable studies from Vietnam [12], Japan [42], and Laos [43,44] have reported P. westermani in other crab genera such as V. aluoiense, I. tannanti, E. japonica, G. dehaani, Indochinamon ou, and Potamon lipkei, respectively. Our findings added to this existing knowledge that Paragonimus populations may utilize more than one crab taxa as an intermediate host. Incorporating parasite sequences from multiple host species is therefore critical for phylogenetic analysis, as it captures broader genetic diversity and provides a clearer view of transmission networks. Different crab hosts may harbor Paragonimus within the same locality [12,41,43], revealing the parasite’s capacity to persist and adapt when suitable hosts are available. However, the scarcity of reference sequences from diverse hosts and regions limits a more comprehensive interpretation of potential inter-host transmission dynamics, which is expected since Paragonimus distribution is constrained by the limited mobility of its intermediate hosts.
Decapoda freshwater crabs undergo direct development and posthatching brood care which bypasses the free-swimming larval stage. These reproductive traits contribute to its restricted ability to disperse from their original habitat [40]. Cumberlidge and Ng (2009) stated that, if this occurs, it is typically confined to short distances and remains localized [45]. However, these are constrained due to several factors, including large bodies of saltwater [45]. This supports the fact that genus Isolapotamon sp. is endemic to Mindanao, the second largest island located in the southern part of the Philippine archipelago that is surrounded by four major bodies of saltwater comprising the Philippine Sea, Celebes Sea, Sulu Sea, and Bohol Sea; moreover, the sampling site, Davao Oriental, located on the eastern coast of Mindanao, directly faces the Philippine Sea [46]. The parasite’s life cycle is linked with the distribution of its intermediate hosts. Paragonimus spp., including P. westermani, are known to co-exist with their host species, and the limited dispersal of hosts influence the host–parasite co-divergence, genetic differentiation, and even cryptic speciation within both the parasite and host lineages [4]. Taken together, the life cycles of both the parasite and the host, along with the geographic restriction of their distribution supports the localization of P. westermani in this crab species. Moreover, this explains the observed similarity in the COX1 region (ranging from 90.23% to 99.53%) with Isolapotamon sp., which suggests presence of regionally adapted populations or potentially undescribed species.
In the Philippines, the genus Isolapotamon sp. currently comprises five known species—I. mindanaoense, I. sinuatifrons, I. spatha, I. danielae, and I. maranao—all of which have been identified based on morphological characteristics [47,48,49,50,51]. Here, we present our findings derived from sequencing analyses, and to the best of our knowledge, this is the first study to utilize this technique in the country in identifying P. westermani in Isolapotamon sp. As mentioned above, S. philippina is the known key IH of P. westermani in the Philippines, particularly in Bicol Peninsula, Samar, and Leyte [38]. However, this study expands our current understanding of its transmission ecology as multiple suitable IH play a critical role in the transmission dynamics of Paragonimus spp., resulting in its geographic and ecological range expansion, increases the probability of successful life cycle completion, and provides resilience against environmental changes or host population declines. These factors contribute to the persistence and stability of parasite populations, particularly in environments where host availability fluctuates [52].
Although molecular methods provide specific and accurate species identification, we acknowledge key limitations of this study such as the lack of morphological examination of the freshwater crabs for the presence of metacercariae which could have further supported our findings. While employing molecular techniques is expensive, they remain the most reliable approach for confirming the presence of P. westermani in freshwater crab populations [53]. To ensure a cost-effective approach, especially in limited-resource settings, screening P. westermani-positive samples prior to proceeding to molecular techniques would be an efficient and practical public health strategy. Additionally, as this study only utilizes a small number of samples, employing large-scale surveillance would help understand the epidemiological distribution of P. westermani across Mindanao.
In conclusion, this study presented significant findings based on molecular evidence that Isolapotamon sp. is a potential IH for P. westermani in Manay, Davao Oriental, and yielded insights into limited existing data, particularly in Mindanao. Our initial findings will expand the scope of national and local health governments efforts in monitoring disease transmission through surveillance of freshwater crabs, including Isolapotamon sp., which is consumed by humans. Furthermore, the study demonstrated the value of utilizing molecular approaches in detection and control of paragonimiasis.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/parasitologia5040067/s1, Table S1: List of freshwater crab species identified as secondary intermediate hosts of P. westermani included in the phylogenetic analysis; Table S2: Reference sequences of Paragonimus westermani used in phylogenetic analysis; Figure S1 Ventral and dorsal view of freshwater crab, Isolapotamon sp. [54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86].

Author Contributions

Conceptualization, D.R.R. and A.E.d.C.; methodology, D.R.R., J.M.C. and C.H.L.; formal analysis, J.M.C. and C.H.L.; investigation, D.R.R. and C.H.L.; resources, N.S.A.J.; data curation, J.M.C.; writing—original draft preparation, D.R.R., J.M.C., C.H.L., H.J.S. and N.S.A.J.; writing—review and editing, D.R.R., H.J.S. and A.E.d.C.; visualization, J.M.C. and C.H.L.; supervision, D.R.R.; project administration, D.R.R. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article/Supplementary Materials. Further inquiries can be directed to the corresponding authors.

Acknowledgments

We thank the community health workers in Barangay Cayawan, Manay, Davao Oriental, for the collection of freshwater crabs. We also thank Michael G. Bacus for the recommendations on the phylogenetic analysis. During data analysis and visualization, the authors utilized ChatGPT (GPT-4.1) for script optimization of phylogenetic tree visualization. The authors have reviewed and edited the content as necessary and take full responsibility for the final outputs of the script.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
FZTFood-borne zoonotic trematode
NCBINational Center for Biotechnology Information
BLASTBasic Local Alignment Search Tool
IHIntermediate Host
MLMaximum Likelihood
NGSNext-generation Sequencing

Appendix A

Appendix A.1

Detailed BLASTn results for the molecular identification of P. westermani detected in freshwater crab samples (Table A1). Also, the non-P. westermani sequences subjected to BLASTn were also shown in Table A2.
Table A1. NCBI BLASTn results of P. westermani DNA detected in freshwater crabs using sequence data generated from Illumina next-generation sequencing targeting 16S mitochondrial rRNA.
Table A1. NCBI BLASTn results of P. westermani DNA detected in freshwater crabs using sequence data generated from Illumina next-generation sequencing targeting 16S mitochondrial rRNA.
Sample IDAccessionSequence LengthPercent IdentityMost Significant Hit Description
CRB02PX00082683499.87%Paragonimus westermani from the Philippines tRNA-Thr gene, partial sequence; and large subunit ribosomal RNA gene, partial sequence; mitochondrial genes for mitochondrial products
(AY190064.1) [36]
CRB03PX00082787499.87%
CRB04PX00082850099.78%
CRB09PX00082982599.87%
CRB11PX00083082899.47%
Table A2. NCBI BLASTn results of different organisms detected in freshwater crabs using sequence data generated from Illumina next-generation sequencing targeting 16S mitochondrial rRNA gene.
Table A2. NCBI BLASTn results of different organisms detected in freshwater crabs using sequence data generated from Illumina next-generation sequencing targeting 16S mitochondrial rRNA gene.
Sample IDPercent IdentityMost Significant Hit DescriptionAccession of the Reference
CRB01100%Pseudomonas aeruginosa strain CS_182 16S ribosomal RNA gene, partial sequenceJQ433551.1 [87]
CRB05100%Pseudomonas aeruginosa strain GH01 16S ribosomal RNA gene, partial sequencePQ350402.1 [88]
CRB0770.14%Hydrogenophilus thermoluteolus TH-1 DNA, complete genomeAP018558.1 [89]
CRB08100%Pseudomonas aeruginosa strain GH01 16S ribosomal RNA gene, partial sequencePQ350402.1 [88]
CRB10100.00%Pseudomonas sp. strain Lac818 16S ribosomal RNA gene, partial sequenceMZ827028.1 [90]
Note: These sequences were not subjected to further downstream analysis nor submitted to GenBank.

Appendix A.2

We explored the potential of Next-generation Sequencing (NGS) for the detection and identification of the FZT and its hosts. While NGS, particularly Illumina platforms, has revolutionized genetics with its high throughput, the lack of clear guidelines for processing and interpreting data necessitates caution. To mitigate risks associated with potential false positives, we employed Sanger sequencing as a supplementary method validation [91,92]. This allows for confirmation of NGS results as Sanger sequencing is recognized as the gold standard in molecular identification, thereby enhancing the overall reliability of our findings. And we found out that sequence data of freshwater crab samples produced from Sanger has the same species identification with the NGS sequence data.
For phylogenetic analysis of the five freshwater crab sequences confirmed to be positive for P. westermani, two Illumina-derived assemblies under 400 bp were substituted with the corresponding Sanger sequences as they provided longer and more representative coverage of the target region. In cases where both sources produced comparable-length sequences, the Illumina data were retained. Accession ID is listed to identify the specific sequence used in the phylogenetic analysis.
Table A3. NCBI BLASTn analysis from Illumina next-generation sequencing data of P. westermani-positive freshwater crabs targeting the COX1 gene.
Table A3. NCBI BLASTn analysis from Illumina next-generation sequencing data of P. westermani-positive freshwater crabs targeting the COX1 gene.
Sample IDAccessionSequence Length Percent IdentityMost Significant Hit Description
CRB02PV98380763799.24%Isolapotamon sp. DBS 045 cytochrome c oxidase subunit I (COX1) gene, partial cds; mitochondrial
(MT514316.1) [37]
CRB03PV98380863799.24%
CRB04PV98380963799.24%
CRB09PV983809258 199.53%
CRB11PV983811354 198.98%
1 Under 400 bp.
Table A4. NCBI BLASTn analysis from Sanger sequence data of P. westermani-positive freshwater crabs targeting the COX1 gene.
Table A4. NCBI BLASTn analysis from Sanger sequence data of P. westermani-positive freshwater crabs targeting the COX1 gene.
Sample IDAccessionSequence LengthPercent IdentityMost Significant Hit Description
CRB02PV98451465599.24%Isolapotamon sp. DBS 045 cytochrome c oxidase subunit I (COX1) gene, partial cds; mitochondrial (MT514316.1) [37]
CRB03PV98451556899.23%
CRB04PV98451667299.24%
CRB09PV984517677 199.24%
CRB11PV98451861799.24%
1 Sequence used in phylogenetic analysis as substitute as it has longer sequence length.

Appendix A.3

Pairwise genetic distances were computed among P. westermani sequences to assess sequence similarity. The data in Table A5 show that all sequences are nearly identical, except for CRB11, which exhibits a genetic distance of 0.006 (approximately three nucleotide differences) from the others. The COX1 sequences of the examined Isolapotamon sp. showed no divergence (Table A6), indicating identical sequences across samples.
Table A5. Pairwise genetic distances between P. westermani 16s rRNA sequences based on the Maximum Composite Likelihood model.
Table A5. Pairwise genetic distances between P. westermani 16s rRNA sequences based on the Maximum Composite Likelihood model.
CRB11CRB09CRB04CRB03CRB02
CRB11
CRB090.006
CRB040.0060.000
CRB030.0060.0000.000
CRB020.0060.0000.0000.000
Note: Estimates are based on a 500 bp trimmed alignment.
Table A6. Pairwise genetic distances among the COX1 sequences of the examined Isolapotamon sp., based on the maximum composite likelihood model.
Table A6. Pairwise genetic distances among the COX1 sequences of the examined Isolapotamon sp., based on the maximum composite likelihood model.
CRB04CRB11CRB09CRB03CRB02
CRB04
CRB110.000
CRB090.0000.000
CRB030.0000.0000.000
CRB020.0000.0000.0000.000
Note: Estimates are based on a 521 bp trimmed alignment.

Appendix A.4

The Illumina sequences generated in this study primarily cover most of the 16S rRNA (rrnL) gene, the complete tRNA-Thr, and the 3′ end of the COX1 gene (Table A7). Nucleotide positions are contiguous and non-overlapping, as verified by BLASTn alignment against the complete P. westermani mitochondrial reference genome (Accession No. NC_002354.2) [93].
Most publicly available P. westermani mitochondrial sequences include these three regions. Accordingly, reference sequences for phylogenetic analysis were restricted to those containing all three loci to ensure consistent alignment and reliable comparative inference.
Table A7. Genomic regions identified in each P. westermani sample through BLASTn alignment to reference sequence NC002354.2.
Table A7. Genomic regions identified in each P. westermani sample through BLASTn alignment to reference sequence NC002354.2.
Sample IDCOX1 (Start–End)tRNA-Thr (Start–End)16S rRNA (Start–End)
CRB021–3435–100101–834
CRB031–3536–101102–839
CRB041–2627–9293–500
CRB091–3233–9899–825
CRB111–3031–9697–828
Note: These contigs are the same as listed in A1 (GenBank Accession ID provided therein).

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Figure 1. Maximum Likelihood (ML) phylogenetic tree based on a 463 bp region of the tRNA-Thr and 16S rRNA genes of P. westermani sequences from different hosts, constructed using the HKY substitution model. Sample sequences are highlighted and bootstrap support values (percentage) are indicated by node values.
Figure 1. Maximum Likelihood (ML) phylogenetic tree based on a 463 bp region of the tRNA-Thr and 16S rRNA genes of P. westermani sequences from different hosts, constructed using the HKY substitution model. Sample sequences are highlighted and bootstrap support values (percentage) are indicated by node values.
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Figure 2. Maximum likelihood (ML) phylogenetic tree based on 528 bp COX1 sequences from known freshwater crab intermediate hosts of P. westermani, constructed using the GTR + G + I model. Sample sequences are highlighted and bootstrap support values (percentage) are indicated by node values. The legend and line colors indicate the taxonomic groupings of the sequences.
Figure 2. Maximum likelihood (ML) phylogenetic tree based on 528 bp COX1 sequences from known freshwater crab intermediate hosts of P. westermani, constructed using the GTR + G + I model. Sample sequences are highlighted and bootstrap support values (percentage) are indicated by node values. The legend and line colors indicate the taxonomic groupings of the sequences.
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MDPI and ACS Style

Ricarte, D.R.; Cambronero, J.M.; Lorico, C.H.; Santos, H.J.; Arce, N.S., Jr.; de Cadiz, A.E. First Report of Isolapotamon sp. as a Potential Intermediate Host of Paragonimus westermani in Davao Oriental, Philippines. Parasitologia 2025, 5, 67. https://doi.org/10.3390/parasitologia5040067

AMA Style

Ricarte DR, Cambronero JM, Lorico CH, Santos HJ, Arce NS Jr., de Cadiz AE. First Report of Isolapotamon sp. as a Potential Intermediate Host of Paragonimus westermani in Davao Oriental, Philippines. Parasitologia. 2025; 5(4):67. https://doi.org/10.3390/parasitologia5040067

Chicago/Turabian Style

Ricarte, Diadem R., Joshua M. Cambronero, Carmela H. Lorico, Herbert J. Santos, Nestor S. Arce, Jr., and Aleyla E. de Cadiz. 2025. "First Report of Isolapotamon sp. as a Potential Intermediate Host of Paragonimus westermani in Davao Oriental, Philippines" Parasitologia 5, no. 4: 67. https://doi.org/10.3390/parasitologia5040067

APA Style

Ricarte, D. R., Cambronero, J. M., Lorico, C. H., Santos, H. J., Arce, N. S., Jr., & de Cadiz, A. E. (2025). First Report of Isolapotamon sp. as a Potential Intermediate Host of Paragonimus westermani in Davao Oriental, Philippines. Parasitologia, 5(4), 67. https://doi.org/10.3390/parasitologia5040067

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