Mermithid nematodes (Enoplea: Mermithidae) parasitize a wide range of invertebrates (Nickle, 1972; Platzer, 2007; Poinar, 2015), and previous studies have reported several cases of mermithid parasitism in Japan (Supplementary Table S1). Mermithids have gained attention as biological alternatives to chemical insecticides for the control of agricultural pests (Nickle, 1981; Hidaka and Andow, 2017) and disease vectors (Molloy, 1981; Platzer, 1981; Sanad et al., 2017) because of their ability to cause host mortality. However, biological knowledge of most mermithids, including their host preferences, distribution, and parasitism rates, remains limited. Additionally, the species-level classification of mermithids is challenging because of the scarcity of published morphological information and DNA sequence data for Mermithidae.
Stable flies (Diptera: Muscidae:
In this study, we observed nematodes resembling mermithids inside adult stable flies at three farms in different regions of the Gifu Prefecture, Japan. To the best of our knowledge, there has been no report on nematodes in adult stable flies in Japan. To characterize them morphologically and molecularly, microscopic observations and sequence analyses of the 18S and 28S rDNA genes were carried out. Subsequently, the phylogenetic relationships of the species to the available Mermithidae sequences were analyzed based on the rDNA sequences.
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).
Detection of parasitic nematodes from
Dairy cattle farm | |||||
F1 | Gifu | 11 Oct 2021 | 80 | 1 | 1a |
15 Sep 2022 | 140 | 0 | 0 | ||
20 Sep 2023 | 112 | 0 | 0 | ||
Subtotal | 332 | 1 | 1 | ||
F2 | Yourou | 8 Oct 2021 | 17 | 1 | 1b |
18 Oct 2022 | 38 | 0 | 0 | ||
11 Oct 2023 | 27 | 0 | 0 | ||
Subtotal | 82 | 1 | 1 | ||
Sheep farm | |||||
F3 | Nakatsugawa | 29 Jul 2022 | 72 | 1 | 1c |
Total | 486 | 3 | 3 |
Nematode 1 (Gifu).
Nematode 2 (Yourou).
Nematode 3 (Nakatsugawa).
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).
Three nematodes were found individually in three of the 486 stable flies from three farms in Gifu, Japan (Table 1). At each of the farms (F1, F2, and F3), only a single adult stable fly of the 332, 82, and 72 flies, respectively, sampled was parasitized by nematodes, indicating a low prevalence of the nematode. Previous studies have reported negative effects on hosts infected with mermithids, including deformation (Muñoz-Muñoz et al., 2016; Mazza et al., 2017). However, differences in the general appearance were not observed between the three infected flies and the other flies.
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
Phylogenetic trees based on the alignment of 18S and 28S rDNA sequences classified the mermithids from Gifu and Yourou into clusters containing
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
This is the first report of mermithids isolated from adult stable flies in Japan. This study identified mermithid parasitization in merely three out of 486 flies. Notably, the occurrence of mermithid parasitism in adult stable flies has been previously reported only once, originating from cattle farms and suburbs in the USA (Smith et al., 1987). These results suggest that mermithid parasitism is extremely rare in adult stable flies.
Morphological identification of mermithids must be performed using adults, which constitute the free stage of these parasites, as the genitalic structures and other organs necessary for identification are fully formed specifically in this stage (Poinar, 1979). However, the Mermithidae nematodes obtained in this study were in the larval form; therefore, accurate identification could not be performed. Species identification based on morphological observation remains unclear, whereas sequence analysis is a well-developed powerful tool for genetic identification (St-Onge et al., 2008; Yoshino et al., 2021). Molecular phylogenetic analysis revealed that the mermithids parasitizing adult stable flies formed two different clusters with
In terrestrial mermithids, including
Mermithids that parasitize adult stable flies tended to be classified into the same clusters as mermithids previously recorded in Japan, and most unidentified species of mermithids that parasitize insects in Japan were classified into clusters with