Introduction
The Mermithidae is a family of nematodes parasitic in many kinds of insects, spiders, leeches, crustaceans and other invertebrates throughout the world (Poinar, Reference Poinar1975). In their life cycle, infective juveniles (pre-parasitic second-stage juveniles [J2]) hatch from eggs and actively seek and penetrate the host. The third-stage juvenile (J3) develops in the host, at which time the post-parasitic fourth-stage juvenile (J4) emerges, killing the host. The J4 develops into adults, which mate and lay eggs in the substrate. Juveniles hatched from these eggs will penetrate a new host and begin the cycle anew (Becnel & Johnson, Reference Becnel and Johnson1998).
There have been three reports of mermitids parasitizing Isopoda Latreille, 1817 to date: Poinar (1975) found juveniles in Armadillidium vulgare Latreille, 1804 and Porcellio scaber Latreille, 1804 in San Diego (United States) as well as the nematode Thaumamermis cosgrovei Poinar, 1981 in soil from the surrounding area; Doucet & Cagnolo (1998) found Agamermis decaudata parasitizing Castnia dedalus in Córdoba, Argentina; and Yoshino & Waki (Reference Yoshino and Waki2021) recently discovered infected Ligidium sp. (Isopoda: Liigidae) with an undetermined species collected from forest areas of Kanagawa Prefecture, Japan. In the present work, we found A. vulgare (Crustacea: Isopoda) individuals to be infected with Agamermis sp. in Magdalena city, Buenos Aires, Argentina. To the best of our knowledge, this is the fourth record of a mermithid infection in the order Isopoda.
Material and methods
Specimens of A. vulgare were collected by hand and from compost in Magdalena city, Buenos Aires, Argentina (35° 05′ 00″ S 57° 31′ 00″ W) during the April–September period of 2022 (autumn–winter). Isopods were identified by the key of Pérez-Schultheiss (Reference Pérez-Schultheiss2010). Nematodes were obtained by the dissection of A. vulgare specimens in Petri dishes with distilled water under a stereomicroscope and measured using a Leica DM 500 microscope. Photographs were taken with an Olympus DP-71 camera and a Leica DM 500 microscope. In order to confirm the nematodes identification, a molecular approach was performed. Genomic DNA was extracted using 50 μl of a 5% suspension of Chelex (Bio-Rad) in deionized water and 2 μl of proteinase K (20 mg/ml), followed by overnight incubation at 56°C, boiling at 95°C for 10 min and centrifugation at 14.000 g for 10 min. Thirty-five μl of the supernatant was transferred to a new Eppendorf tube and 1.5 μl was utilized as the template for polymerase chain reaction (PCR). The 18S rRNA partial sequences were amplified using the primers MermF 18S (5′-CAAGGACGAAAGTTAGAGGTTC-3′) and MermR 18S (5′-GGAAACCTTGTTACGACTTTTA-3′) according to Kobylinski et al. (Reference Kobylinski, Sylla, Black and Foy2012) with the GoTaq Master Mix (Promega Corporation, Madison, USA). The thermocycle conditions were as follows: 94°C for 5 min; 38 cycles of 94°C denaturation for 30 s; annealing 46°C for 40 s and extension 72°C for 80 s; and a single final extension period of 72°C for 3 min. PCR products were analysed by electrophoresis on 1% agarose gels and visualized by staining with ethidium bromide. The amplicons were sequenced in Macrogen Inc. (Korea) and edited with the Chromas software and the consensus sequence was obtained with Genedoc software (Kearse et al., Reference Kearse, Moir, Wilson, Stones-havas, Cheung and Sturrock2012). The obtained consensus sequences were compared with sequences in the BLAST tool available in the United States National Center for Biotechnology Information (NCBI) database (http://www.ncbi.nlm.nih.gov). The resulting sequences were submitted to the NCBI GenBank database (http://www.ncbi.nlm.nih.gov) under the accession number OP380610.
Global multiple alignment was made by using the ClustalO approach (https://www.ebi.ac.uk/Tools/msa/clustalo/). The 18S gene sequences from species belonging to the Mermithidae family were selected and the outgroup formed with sequences from Leptonchus granulosus (KR184128.1) and Funaria cacti (AY912037.1) as included. The model selection (K2P + I + G4) was made with the IQTree server (http://iqtree.cibiv.univie.ac.at/), and was chosen under the Bayesian information criterion. The Bayesian inference was reconstructed using two parallel analyses of Markov chain Monte Carlo with the MrBayes software (version 3.2.7a) (Ronquist et al., Reference Ronquist, Teslenko and van der Mark2012). The number of generations was established at 6 million, and the posterior probability (PP) distribution was determined. The sample frequency was made every 200 generations and the average standard deviation of split frequencies was observed to be less than 0.05. In order to analyse the parameters across all runs, the burn-in was set at 10%. Then, Tracer software (version 1.7.1) was used to corroborate that the parameters reach an effective sample size ≥ 200. Finally, a consensus tree was reconstructed after applying a burn-in of 10%. The final trees were visualized in FigTree software v 1.4.4. The robustness of the clades was assessed using PP ≥ 0.95 criterion.
Results and discussion
While conducting an assay with entomopathogenic nematodes, we serendipitously found a specimen of Agamermis sp. when dissecting a dead A. vulgare. In total, two Agamermis sp. (juveniles) were recovered from the cavity of 556 A. vulgare (prevalence of 0.36%). The mean intensity of infection was one (one parasite specimen per infected host). Of the 556 isopods dissected only eight were dead. Mermithids were found on two of these dead hosts.
Agamermis sp. was white in colour and had a long and cylindrical body that measures 4.6 cm in length and 0.027 cm width. Smooth cuticle with lateral lines that run on the body surface. Head of 76.5 μm in diameter, with six cephalic papillae of 63 μm long that surrounds the mouth opening. Amphids present. The nerve ring was situated at 292.3 μm from the anterior end and the body width at that level was 153 μm. Trophosome visible, extending three-quarters the length of the body. Tail length of 369 μm and anus width of 243 μm. Caudal appendage lacking (fig. 1A–C). These juveniles oriented themselves longitudinally in the host and could be seen with the naked eye. The absence of tail appendage and presence of a tail end ring provided robust evidence for identification of the genus (Kaiser, Reference Kaiser and Nickle1991).
Fig. 1. Agamermis spp. (juvenile): (A) head showing the mouth opening (Mo); (B) arrow showing the striae on the surface of the body; (C) tail; and (D) Bayesian tree of the Mermithidae family based on 18S rDNA data including Agamermis sp. CEP15 (OP380610). Numbers above branches represent Bayesian posterior probabilities. Scale bars: 65 μm
The obtained 18S gene sequence of Agamermis sp. named CEP-15 (accession number OP380610) was approximately 800 base pairs (bp) and, as it was expected, showed a high similarity with members of the Mermithidae family, being Agamermis sp. BH-2006 isolate ‘Riverside’ (accession number: DQ665653.1) the most similar (percentage identity: 100%) with a sequence of 735 bp and not the reported host. As it is seen in fig. 1D, the Bayesian inference showed a close relation between these two species (PP: 1.00). Also, they seem to be related to other species non-totally identified: a clade comprising Agamermis sp. FLS-2016 (KX173336.1) with length fragments of 755 bp, found in nymphs and adults of agricultural pests bugs in the south-eastern United States: Chinavia hilaris, Euschistus servus, Euschistus sp. (Hemiptera: Pentatomidae and Plataspidae, respectively) and Megacopta cribraria (Hemiptera: Plataspidae) (Stubbins et al., Reference Stubbins, Agudelo, ReayJones and Greene2016); Mermithid sp. JH-2004 (AY284743.1) (1728bp, host not reported); Mermithidae sp. KPMN_2FR (MZ603096.1) (820 bp) isolated from Simulium nigrogilvum Summers, 1911 (Diptera: Simuliidae) in central Thailand and Agamermis changshaensis (DQ628908.1) (759 bp); and Agamermis xianyangensis (EF617352.1) (817 bp), both with not reported hosts; The other clade consisted of Hexamermis agrotis (DQ530350.1) (757 bp) (host not reported) and Hexamermis popilliae (MF040823.1) (762 bp) parasitizing the Japanese beetle Popillia japonica Newman (Coleoptera: Scarabaeidae) in Italy (Mazza et al., Reference Mazza, Paoli and Strangi2017) where PP: 95.
Worldwide, six species of the genus Agamermis have been described: A. decaudata Cobb, Steiner & Christie, 1923; Agamermis unka kaburaki & Imamura, 1932; Agamermis cobbi Schuurmans, Stekhoven & Mawson, 1955; Agamermis sinuosa Kaiser, 1977; A. changshaensis Bao, Lou & Lou, 1992; and Agamermis catadecaudata Baker and Poinar, 1995. In Argentina, only A. decaudata was cited in representatives of two families of the order Orthoptera (i.e. Acrididae and Gryllidae) and in the isopod Castnia dedalus (de Doucet & Cagnolo, Reference De Doucet and Cagnolo1998; Camino & Achinelly, Reference Camino and Achinelly2011; Rusconi et al., Reference Rusconi, Camino and Achinelly2017). According to de Doucet & Cagnolo (Reference De Doucet and Cagnolo1998), the host specificity of A. decaudata is considered relatively narrow and related to physiological and ecological factors. Unfortunately, we could not reach species level due to the very low prevalence obtained and the fact that the nematodes died immediately after dissecting the pill bugs. In addition, this nematode was unable to complete its life cycle within the host since we only found it in dead isopods making the breeding impossible.
Acknowledgement
The authors thank D.C.V. María Laura Morote for editing the pictures.
Financial support
This study was partially supported by CONICET (PUE 2017-2021) and National University of La Plata (N804)..
Conflicts of interest
The authors declare that they have no conflicts of interest.
Ethical standards
The authors state that all procedures contributing to this work comply with the ethical standards of relevant national and institutional guidelines on the care and use of laboratory animals.
