{"id":1774,"date":"2025-08-16T13:43:27","date_gmt":"2025-08-16T04:43:27","guid":{"rendered":"https:\/\/lmi.jp\/articles\/?p=1774"},"modified":"2025-11-26T14:28:02","modified_gmt":"2025-11-26T05:28:02","slug":"morphological-and-genetic-analysis-of-diploscapter-coronatus-insights-into-identification-challenges-and-species-relationships","status":"publish","type":"post","link":"https:\/\/lmi.jp\/articles\/2025\/08\/16\/morphological-and-genetic-analysis-of-diploscapter-coronatus-insights-into-identification-challenges-and-species-relationships\/","title":{"rendered":"Morphological and Genetic Analysis of Diploscapter coronatus: Insights into Identification Challenges and Species Relationships"},"content":{"rendered":"\n


\u00a0Norihito Morimoto<\/strong><\/a>, PhD*1,2<\/sup>, Yoshie Nishida<\/strong><\/a>, PhD*2<\/sup>, Masataka Korenaga,<\/strong><\/a> PhD*1,3<\/sup><\/p>\n\n\n\n

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\u2003 Cite<\/span><\/span><\/i><\/i><\/span><\/summary>
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Morimoto N, Nishida Y, Korenaga M. Morphological and Genetic Analysis of Diploscapter coronatus: Insights into Identification Challenges and Species Relationships. Lab Med Int 2025; 4(2): 69-74. doi: 10.51041\/lmi.4.2_69<\/p>\n<\/div><\/details>\n<\/div>\n\n\n\n

Original
Lab Med Int 2025; 4(2): 69-74<\/p>\n\n\n\n

 
Correspondence: Departments of Medical Laboratory Science, Kochi Gakuen University, 292-26 Asahitenjin-cho, Kochi 780-0955, Japan. E-mail: nmorimoto”@”kochi-gu.ac.jp.
Received November 21, 2024; accepted January 9, 2025
*1 <\/sup>Department of Medical Laboratory Science, Kochi Gakuen University
*2 <\/sup>Department of Clinical Laboratory, Kochi Medical School Hospital, Kochi University
*3 <\/sup>Department of Parasitology, Kochi University Medical School, Kochi University<\/strong><\/span><\/p>\n\n\n\n

Download PDF<\/span><\/a><\/div>\n\n\n\n

ABSTRACT<\/strong><\/h2>\n\n\n\n

Objective:<\/strong> This study aims to address the challenges of identifying Diploscapter coronatus<\/em> by morphological and genetic analysis. The nematode was previously identified morphologically in fecal samples from a patient with Henoch-Sch\u00f6nlein purpura. We used genetic methods to complement and refine species identification.
Materials and Methods:<\/strong> Nematodes isolated from the patient\u2019s feces were fixed in formalin, and morphological observations were made under a light microscope. PCR amplification of the SSU rRNA and Hsp90 genes was performed on DNA extracted from the nematodes during the same period. The resulting DNA was cloned, sequenced, and subjected to phylogenetic analysis using the neighbor-joining method.
Results:<\/strong> Morphological observations confirmed that the nematode shared key features with D. coronatus<\/em>. The SSU rRNA analysis showed 99% similarity with 11 Diploscapter<\/em> species, including D. coronatus<\/em>. However, Hsp90 gene analysis placed the nematode in the D. lycostoma<\/em> cluster, revealing inconsistencies between morphological and genetic data.
Conclusion:<\/strong> The present study highlights the complexity of species identification within the genus Diploscapter <\/em>that results from the limited genetic information and overlapping morphological characters. The results underscore the need for more extensive genetic data and comprehensive phylogenetic analyses to resolve these identification challenges and better understand species relationships within the genus.<\/p>\n\n\n\n


\u3014Lab Med Int 2025; 4(2): 69-74\u3015<\/p>\n\n\n\n

Key Words<\/strong><\/h3>\n\n\n\n


Diploscapter coronatus<\/em>, Diploscapter<\/em> spp., genetic analysis, small subunit ribosomal RNA \uff08SSU rRNA\uff09, heat shock protein 90 \uff08Hsp90\uff09<\/p>\n\n\n\n

I. Introduction<\/strong><\/h2>\n\n\n\n

 Diploscapter coronatus<\/em> was first described by Cobb in 1893 as a free-living nematode typically found in soil surrounding decaying plants and roots1\uff09<\/strong><\/sup>. Its detection in human samples is very rare, although its presence has been noted in past cases of anoxia and alkaline urine2\uff09, 3\uff09<\/strong><\/sup>. In addition, recent reports have identified nematodes suspected of being this species in clinical samples such as urine and feces4\uff09, 5\uff09<\/strong><\/sup>. Several species, including D. coronatus<\/em>, have been documented within the genus Diploscapter<\/em>. In 2002, we detected this nematode in fecal samples from a patient with Henoch-Sch\u00f6nlein purpura, identified by its morphological features6\uff09<\/strong><\/sup>. Recently, both morphological and genetic methods have been used for species identification in nematode studies7\uff09<\/strong><\/sup>. In this study, we report the analysis of a sample of D. coronatus<\/em>, previously identified by morphological observations and now complemented by genetic identification methods.<\/p>\n\n\n\n

II. Materials and Methods<\/strong><\/h2>\n\n\n\n

1. Samples<\/strong>
Nematodes found in patient feces in 2002 were cultured in nematode growth medium \uff08NGM\uff09, then fixed in 10% buffered formalin and stored at room temperature. Morphological observations were carried out on these formalin-fixed nematodes. During the same period, genomic DNA was extracted from D. coronatus<\/em> nematodes derived from the patient and cultured in NGM using a QIAamp DNA Micro Kit \uff08Qiagen, Hilden, Germany\uff09 and stored frozen at \u221280\u2103. This study was approved by the Ethics Committee of Kochi University Medical School, Japan \uff08No.30-87\uff09.<\/p>\n\n\n\n

2. Morphology<\/strong>
Morphological observations and measurements of the formalin-fixed nematodes were made using a light microscope and considered in conjunction with the observations we have previously reported6\uff09<\/strong><\/sup>. Using De Man\u2019s formula8<\/strong>\uff09<\/sup>, specific ratios and indices were calculated based on measurements of different parts of the nematode\u2019s body to describe its morphological characteristics.<\/p>\n\n\n\n

3. Polymerase Chain Reaction <\/strong>\uff08PCR<\/strong>\uff09 and Cloning<\/strong>
The primers used for the small subunit ribosomal RNA \uff08SSU rRNA\uff09 gene were obtained from Holterman et al9\uff09<\/strong><\/sup>. \uff082006\uff09, and those for the 90 kDa heat shock protein \uff08Hsp90\uff09 gene were obtained from Zeng Qi Zhao7\uff09<\/strong><\/sup> \uff082013\uff09, respectively. The 20 \u00b5L PCR reactions contained 10 \u00b5L KAPA2G Fast PCR Kit \uff08Kapa Biosystems, USA\uff09, 1 \u00b5L \uff080.05 \u00b5M\uff09 of each forward and reverse primer, and 2 \u00b5L of DNA template. The thermal cycling program was as follows: denaturation at 95\u2103 for 3 min, followed by 30 cycles of denaturation at 94\u2103 for 30 s, annealing at 55\u2103 for 30 s, and extension at 72\u2103 for 45 s. A final extension was performed at 72\u2103 for 10 min. The amplified DNA was purified using a QIAGEN Gel Extraction Kit \uff08QIAGEN, Valencia, CA, USA\uff09. The cDNA was ligated into a T-vector using the DynaExpress TA PCR Cloning Kit \uff08BioDynamics Laboratory, Tokyo, Japan\uff09 and then transformed into competent Escherichia coli<\/em> DH5\u03b1 cells \uff08FUJIFILM Wako, Tokyo, Japan\uff09 using the heat shock method. Finally, the recombinant plasmid was extracted from the transformed E. coli<\/em>, using EZ-10 Spin Column Plasmid DNA Miniprep Kit \uff08BIO BASIC, Ontario, Canada\uff09.<\/p>\n\n\n\n

4. Sequencing and Phylogenetic Analysis<\/strong>
Purified plasmids were sequenced using the Big Dye\u2122 Terminator Cycle Sequencing Ready Reaction Mix v3.1 Kit. Cycle sequencing products were purified using the FastGene Dye Terminator Removal Kit \uff08NIPPON Genetics, Tokyo, Japan\uff09 and analyzed on an ABI PRISM 310 Genetic Analyzer \uff08Applied Biosystems, Foster City, CA, USA\uff09. The genetic sequences obtained were subjected to similarity searches using BLAST10\uff09<\/strong><\/sup> from the National Center for Biotechnology Information \uff08NCBI\uff09. DNA sequences were aligned using Clustal OMEGA11\uff09<\/strong><\/sup> and a phylogenetic tree was constructed using the Neighbor-Joining method with MEGA 1112\uff09<\/strong><\/sup>. The root of the phylogenetic tree was constructed using the nucleotide sequence of Rhabditis myriophila<\/em> \uff08U81588\uff09 for SSU, and Acrobeloides amurensis<\/em> \uff08DQ340377\uff09 and Cephalobus cubaensis<\/em> \uff08DQ340378\uff09 for HSP90.<\/p>\n\n\n\n

\"\"<\/figure>\n\n\n\n

Figure1<\/strong> Microscopic morphology of adult nematodes.
Whole body of both adult female\uff08 A\uff09 and male exhibiting the presence of spicules\uff08 \u25b3\uff09 and caudal alae\uff08 \u25b2\uff09 in the tail\uff08 B\uff09. Enlarged view of the head of an adult female\uff08 C\uff09. Scale bar indicating 100 \u03bcm.<\/p>\n\n\n\n

III. Results<\/strong><\/h2>\n\n\n\n

1. Morphological Observations<\/strong>
The microscopic images of the male and female nematodes in this study are shown in Figure 1<\/strong>. The length of the adult worms ranged from 344 to 522 \u00b5m, with a width of 22 to 39 \u00b5m \uff08Table 1<\/strong>\uff09. They had two characteristic pairs of lips on the head, with the dorsal and ventral lips being hook-shaped and the second pair branching anteriorly, consistent with the characteristics of the genus Diploscapter<\/em> \uff08Figure 1B<\/strong>\uff09. To compare this nematode with the genus Diploscapter<\/em>, coefficients calculated from the De Man measurements are presented in Table 1<\/strong>. Morphologically, both the size and the De Man ratios were similar to those of D. coronatus<\/em>.<\/p>\n\n\n\n

2. Genetic Findings<\/strong>
A homology search using BLAST showed that the SSU rRNA region of this nematode exhibited 99% similarity to 11 Diploscapter<\/em> species. The similarity to the two included D. coronatus<\/em> sequences was 99.3%. In the Hsp90 region, similarity ranged from 89.8% to 99.4% for D. lycostoma<\/em> and from 86.8% to 89.7% for D. coronatus<\/em> \uff08Table 2<\/strong>\uff09. Phylogenetic analysis classified the Diploscapter <\/em>species into clusters based on the four SSU regions, with the nematode in this study placed in a cluster that included D. coronatus<\/em> \uff08Figure 2<\/strong>\uff09. In contrast, using the Hsp90 data, the Diploscapter<\/em> species were classified into clusters corresponding to D. coronatus<\/em>, D. formicidae<\/em> and D. lycostoma<\/em> with the nematode in this study placed in the D. lycostoma<\/em> cluster \uff08Figure 3<\/strong>\uff09.<\/p>\n\n\n\n

Table 1<\/strong> Comparative measurements of adluts Diploscapter spp.*<\/p>\n\n\n\n

\"\"<\/figure>\n\n\n\n

*The De Man ratios \uff08a to c\u2019+V\uff09 Favoured by plant and soil nematologists are as follows: a=body length\/maximun width; b=body
length\/length of esophagus; c=body length\/tail length; V= Percentage of body length from the head to the vulva.
**Length body\uff08 \u03bcm\uff09.<\/p>\n\n\n\n

Table 2<\/strong> Homology search in the SSU rRNA region using BLAST<\/p>\n\n\n\n

\"\"<\/figure>\n\n\n\n

Homology search using BLAST was performed with the nucleotide sequences obtained from sequencing the plasmid
DNA, which contained the PCR product of the SSU region of this nematode.<\/p>\n\n\n\n

IV. Discussion<\/strong><\/h2>\n\n\n\n

Morphologically, it has been estimated that there are approximately 15 species within the genus Diploscapter<\/em>13\uff09<\/strong><\/sup>. However, to date, comprehensive genetic analyses and electron microscopic evaluations have not been carried out for most species. The nematode analyzed in this study matched the morphological characteristics of D. coronatus<\/em> observed in previous studies14\uff09-17\uff09<\/strong><\/sup>. In addition, the b-ratio \uff08body length\/esophagus length\uff09 of D. coronatus<\/em> differed from that of D. lycostoma<\/em> in the De Man ratios referenced for nematodes17\uff09<\/strong><\/sup>. From these findings, the nematode in this case demonstrated a b-ratio of 4.4-5.6, which morphologically identified it as D. coronatus<\/em>.<\/p>\n\n\n\n

The information available in NCBI on the genus Diploscapter<\/em> is particularly limited, and only three species are fully documented: D. coronatus<\/em>, D. lycostoma<\/em> and the newly described D. formicidae<\/em>. The nematode in this study showed high similarity to D. coronatus<\/em> in the SSU rRNA gene, while in the Hsp90 region, it exhibited higher similarity to D. lycostoma<\/em>. We currently lack sufficient genetic data on the SSU rRNA region in D. lycostoma<\/em>. In addition, the genetic information available for the Hsp90 region lacks corresponding, detailed morphological descriptions. These factors likely contribute to the inconsistencies observed using genetic identification methods. In practice, when performing a BLAST search for the Hsp90 gene sequence of D. lycostoma<\/em> \uff08EF467042\uff09, the similarity to other D. lycostoma<\/em> sequences ranges from 91.4% to 93.6%, and to D. coronatus<\/em> sequences ranges from 85.8% to 85.9%. Identification by genetic methods is therefore challenging.<\/p>\n\n\n\n

Interestingly, G. Markin et al<\/em>18\uff09<\/strong><\/sup> reported that D. coronatu<\/em>s \uff08Cobb 18931\uff09<\/strong><\/sup>\uff09 is synonymous with D. lycostoma<\/em> \uff08Volk 1950\uff0919\uff09<\/strong><\/sup>. The paucity of samples available for phylogenetic analysis may suggest that the resulting phylogenetic tree does not accurately reflect the true evolutionary relationships between the classified groups.<\/p>\n\n\n\n

To date, while D. coronatus<\/em> has been detected in human samples4\uff09, 5\uff09<\/strong><\/sup>, there have been no reports of other species within the genus Diploscapter<\/em> being detected in humans. <\/p>\n\n\n\n

In addition, the detection of Diploscapter formicidae<\/em>7\uff09<\/strong><\/sup> and Diploscapter lycostoma<\/em>19\uff09, 20\uff09<\/strong><\/sup> in association with ants suggests a possible correlation between species and host specificity. In general, when a rare parasite is detected in humans, species identification is often performed based on the similarity of gene sequences from a particular genetic region, and genetic identification for various organisms has advanced in clinical examination. However, for the genus Diploscapter<\/em>, the current genetic information available is insufficient, leading to uncertainties in species identification. In fact, Arai et al21\uff09<\/strong><\/sup> reported that although they attempted to genetically identify Diploscapter<\/em> from human samples, a definitive identification of D. coronatus<\/em> could not be achieved because of limited nucleotide sequence information.<\/p>\n\n\n\n

Interestingly, although males are thought to be absent in the genus Diploscapter<\/em>, we have reported the presence of males in this genus6\uff09<\/strong><\/sup>. Furthermore, the presence of males in D. coronatus<\/em> has also been reported by other researchers4\uff09, 22\uff09<\/strong><\/sup>. If males could be found in other species of the genus Diploscapter<\/em>, it might be possible to identify species based on characteristics such as male reproductive organs.<\/p>\n\n\n\n

\"\"<\/figure>\n\n\n\n

Figure 2<\/strong> Neighbor-joining phylogenetic trees inferred from SSU rRNA gene\uff08 A\uff09 and HSP90 gene\uff08 B\uff09.
The phylogenetic trees include sequences from 9 species for SSU rRNA DNA and from 11 species for the
HSP90 gene within the genus Diploscapter, along with sequences from other related taxa. Bootstrap values are
shown at the nodes. The scale bar indicates the number of nucleotide substitutions per site. Sequences from
Rhabditis myriophila and other outgroup species were used to root the tree.<\/p>\n\n\n\n

Table 3 <\/strong>Homology search in the HSP90 region using BLAST<\/p>\n\n\n\n

\"\"<\/figure>\n\n\n\n

Homology search using BLAST was performed with the nucleotide sequences obtained from sequencing the plasmid
DNA, which contained the PCR product of the HSP90 region of this nematode.<\/p>\n\n\n\n

V. Conclusion<\/strong><\/h2>\n\n\n\n

The identification of species within the genus Diploscapter<\/em> requires more extensive morphological and molecular data, with particular emphasis on comparing genetic information among different species to advance phylogenetic analyses. In addition, the ability to specifically detect species will likely provide important insights into host specificity.<\/p>\n\n\n\n

VI. Acknowledgements including funding<\/strong><\/h2>\n\n\n\n

We thank Bevin McGeever, PhD, from Edanz \uff08https:\/\/jp.edanz.com\/ac\uff09 for editing a draft of this manuscript.<\/p>\n\n\n\n

Authorship contributions<\/strong>
All authors were involved in the preparation of the manuscript and reviewed the final manuscript.<\/p>\n\n\n\n

Disclosure of Conflicts of Interest<\/strong>
We declare that we have no conflicts of interest. <\/p>\n\n\n\n

References<\/strong><\/h2>\n\n\n\n
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