First Report of Mermithidae (Enoplea: Mermithida) Parasitizing Adult Stable Flies in Japan

During our study period on surveillance and control measures for flies in farms (Shimizu et al., 2023), adult stable flies were collected from three farms (F1, F2, and F3) across different geographic locations in the Gifu Prefecture, Japan (Table 1 and Figure 1A). Flies were captured once annually from 2021 to 2023 at farms F1 and F2, and once at farm F3 in 2022. Flies inside and outside the livestock barns were captured using butterfly nets. After collection, the flies were transferred to our laboratory and killed by placing them at −80°C. At the time of the use of flies, three nematodes were unexpectedly isolated individually from three flies. The isolated nematodes were fixed in 70% ethanol. Nematodes 1 (Gifu), 2 (Yourou), and 3 (Nakatsugawa) were isolated from stable flies captured from farms F1, F2, and F3, respectively (Table 1).

Figure 1.

For morphological observation, the specimens were cleared in a glycerol-ethanol solution (5% glycerol in 70% ethanol) by the evaporation of ethanol and were mounted on glass slides with 50% glycerol solution. The specimens were viewed under Nikon Optiphot (Nikon, Tokyo, Japan) and Olympus BX50 (Olympus, Tokyo, Japan) microscopes equipped with a commercial camera Lucida.

A molecular approach was used to identify the nematode species. Total DNA was extracted from the middle part of the nematode body using a DNeasy Blood and Tissue Kit (QIAGEN, Venlo, Netherlands). Several primer sets were used to amplify 18S and 28S rDNA (Supplementary Table S2). PCR was performed with GoTaq Hot Start Green Master Mix (Promega, Madison, WI, USA) using a Veriti thermal cycler (Applied Biosystems, Foster City, CA, USA). The PCR conditions are listed in Supplementary Table S2. The PCR products were purified using NucleoSpin Gel and PCR Clean-up (Macherey-Nagel, Duren, Germany), and the nucleotide sequences were determined via direct sequencing using a BigDye Terminator Cycle Sequencing Kit v3.1 (Applied Biosystems). DNA sequences were confirmed and edited using Genetyx-Win version 13 software (Genetyx, Tokyo, Japan), and the species were identified based on the results of the Basic Local Alignment Search Tool (BLAST) analysis. The obtained sequences were deposited in the DDBJ/EMBL/GenBank database under the following accession numbers: LC788412 to LC788414 for 18S rDNA and LC788415 to LC788417 for 28S rDNA.

The DNA sequences of the mermithids available in the NCBI database are highly variable in length. Therefore, to include reference sequences from known species in the phylogenetic analysis, we aligned 327 to 986 bp for the 18S and 296 to 822 bp for the 28S rDNA sequences. Furthermore, to include reference sequences with known hosts in the phylogenetic analysis, we aligned 327 to 1011 bp of the 18S rDNA sequences. Phylogenetic trees were constructed using the maximum-likelihood method with the Kimura two-parameter model, and the reliability of the branches was evaluated using 1,000 replicates. In total, 76 sequences of previously reported mermithid nematodes from Japan and other countries were used for the phylogenetic analysis (Supplementary Table S3).

The morphological characteristics of the mermithids are shown in Figures 1B—1F. The nematodes were wire-like in shape and had a milky-white cuticle sheath. The specimen from farm F1 was ca. 12.5 cm in length and ca. 0.25 mm in width. Among the nematodes, the sample from farm F1 (Figure 1A) could be studied morphologically, as it had relatively complete head and tail extremities (Figures 1C–1F). The head had a slender esophagus, and the tail had an appendage on the posterior end. Thus, this nematode was a juvenile belonging to the family Mermithidae. Based on the general appearance and host range, the other two degenerated individuals from farms F2 and F3 could belong to the same family and stage (Poinar, 1975). This is further supported by the sequence analysis described in the following sections.

The lengths of the three 18S rDNA sequences were as follows: Gifu, 1,662 bp; Yourou, 1,733 bp; and Nakatsugawa, 1,719 bp. BLAST analysis showed that nematodes from Gifu and Youro shared 99.94% (1603/1604) and 99.82% (1633/1636) identity, respectively, with Hexamermis sp. (LC661691) isolated from Glaucias subpunctatus (Hemiptera) in Mie, Japan (Watanabe et al., 2020). In addition, the Nakatsugawa specimen shared 99.21% (630/635) identity with Amphimermis enzoni (MT021436) isolated from Ischnura fluviatilis and Rhionaeschna bonariensis (Odonata) in Argentina (Rusconi et al, 2020) and 99.10% (774/781) with Amphimermis sp. (EF617354) isolated in China. The lengths of the three 28S rDNA sequences were as follows: Gifu, 1,103 bp; Yourou, 1,103 bp; and Nakatsugawa, 1,082 bp. BLAST analysis showed that Gifu and Yourou exhibited 90.39% (715/791) and 90.52% (716/791) identity, respectively, with Hexamermis agrotis (KC784667) isolated in Turkey. In addition, Nakatsugawa shared 96.18% sequence identity with Amphimermis sp. (EF617372) isolated in China.

Phylogenetic trees based on the alignment of 18S and 28S rDNA sequences classified the mermithids from Gifu and Yourou into clusters containing Ovomermis sinensis reported in China (MN3679544 to MN367957, and DQ520879) and Poland (KU177046), and Hexamermis sp. (LC661690 and LC661691) reported in Japan, whereas the specimen from Nakatsugawa fell into a cluster containing Amphimermis sp. reported in Argentina (MT021436) and China (EF617354, EF617355, and EF617372) (Figure 2).

Figure 2.

A phylogenetic tree, constructed using 18S rDNA sequences and referencing known host information, revealed that specimens from Gifu and Yourou formed a distinct cluster encompassing hosts such as Isopoda, Hemiptera, Hymenoptera, and Lepidoptera (Figure 3). In contrast, the Nakatsugawa specimen was positioned within a cluster that included Hemiptera and Odonata hosts. Additionally, these mermithids could be categorized into the same clusters as other mermithids recorded from Japan, rather than in clusters within which the host species were categorized (Figure 3). Phylogenetic analysis of 13 unidentified species of mermithids that parasitize insects in Japan (registered in GenBank) categorized the three mermithids we sampled into clusters with O. sinensis, Hexamermis sp., or Amphimermis sp., with one exception (MW649131 isolated from Erisoma auratum in Hokkaido; Figure 4, Supplementary Table S4).

Figure 3.

Figure 4.