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All issues Volume 32 (2025) Parasite, 32 (2025) 67 Full HTML
Open Access
Issue
Parasite
Volume 32, 2025
Article Number 67
Number of page(s) 15
DOI https://doi.org/10.1051/parasite/2025060
Published online 17 October 2025

© R. Gastineau et al., published by EDP Sciences, 2025

Licence Creative CommonsThis is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Introduction

In continental Europe, the invasion by more than a dozen species of terrestrial flatworms has been an increasing cause for concern in recent years [2, 12, 13, 18, 25, 33, 34, 55, 56, 59–62, 70, 76, 78, 90, 91, 94, 96]. Very recently (July 2025), three species of geoplanids were added to the list of invasive alien species of Union concern [32].

In the British Isles, the most widely established invasive flatworm species is the New Zealand flatworm, Arthurdendyus triangulatus (Dendy, 1894) Jones & Gerard, 1999, whose spread, impact and pest management options have been scrutinized since the 1970s [1, 8–11, 16, 21, 48, 75, 77]. Arthurdendyus triangulatus was added to the list of species of Union Concern in 2019 [31]. For this species, our team has recently described the complete and complex mitochondrial genome and its paralogous clusters of nuclear rRNAs [39].

For several years, genomic studies on Geoplanidae have been developed, revealing original features of these animals [36–40, 42, 43, 54, 55, 57, 58, 88, 89]. As a part of these investigations, we collected additional species in Northern Ireland. The first species was the indigenous Microplana scharffi (Graff, 1899), from the subfamily Microplaninae Pantin, 1953, for which we described the mitogenome and additional genomic features [40]. Specimens of Kontikia andersoni Jones, 1981 and Australoplana sanguinea (Moseley, 1877) were also collected. The aim of our study was initially to populate the databases with sequences of poorly investigated organisms. The scarcity of molecular studies was exemplified by K. andersoni, for which not a single sequence was available on GenBank prior to this study.

While processing the sequencing results, it appeared that both species showed molecular signals of the presence of two species of the genus Mitosporidium Haag, James, Pombert, Larsson, Schaer, Refardt & Ebert, 2015 [46, 47]. Ten years after its description, the genus Mitosporidium still only includes a single species, Mitosporidium daphniae Haag et al., 2015 [46, 47], an intracellular parasite of the guts of the planktonic crustacean Daphnia magna Straus, 1820 from northwestern Europe (Belgium, Germany, and the United Kingdom). Mitosporidium daphniae shares similarities with Microsporidia (e.g. their status as intracellular parasites, and the presence of a polar tube in spores), but it is clearly distinguished on the evolutionary level by having retained a restricted mitochondrial genome that contains a limited set of protein-coding genes (vs. no mitochondrion in Microsporidia). The circularity of this mitogenome remains to be verified.

The phylogenetic classification of Mitosporidium is not a matter of consensus, within the framework of the relationships between “true” Microsporidia and Rozellomycota and other basal Fungi [45]. Mitosporidium is either considered to be external to Microsporidia [30] or a member of “expended” Microsporidia [7], and sometimes designated as “short-branch microsporidian” [27]. In the most recent (2024) classification of Fungi, Mitosporidium is considered to be a member of the Phylum Rozellomycota, although its more precise placement is not provided [99]. This discussion is out of the scope of this paper; here we follow the interpretation of Mitosporidium as a member of the Microsporidia.

This serendipitous discovery of two species of Mitosporidium in invasive flatworms and its implications are described here, alongside all the information gathered in the course of this study concerning the genomics of the flatworm hosts and their preys. We also discuss whether these Mitosporidium species are actually parasites of the flatworms.

Materials and methods

Sampling

Flatworm collection took place in 2023. A specimen of K. andersoni was found by Mr Stewart Rosell (AFBI PhD student) in Castle Espie (latitude 54.53152, longitude -5.69632), a wetland reserve located in County Down, Northern Ireland, near a freshwater lagoon and below a decaying wooden board (Figs. 1–2). A specimen of Australoplana sanguinea was found in a private urban garden (latitude 54.588119, longitude -5.90957) in the main city of Belfast, also in County Down, below a concrete stone (Figs. 3–4) and adjacent to a compost box. Both specimens were killed by immersion in ethanol 96%, and kept stored within the ethanol before being sent to the National Museum of Natural History, France where they were registered in the collections under accession numbers MNHN JL467 for K. andersoni and MNHN JL472 for A. sanguinea.

thumbnail Figures 1–4

Geoplanids used in this study and their close environment. 1–2, Kontikia andersoni. 1, specimen MNHN JL467, unscaled; 2; origin of the specimen, below decaying wooden boards. 3–4, Australoplana sanguinea. 3, specimen MNHN JL472, unscaled; 4, origin of the specimen, below a concrete stone. Photographs by Stewart Rosell.

Genomics and bioinformatics

Due to its small size, the complete specimen of K. andersoni was used for sequencing, while just a posterior piece of 1 cm of A. sanguinea was cut and used. Samples were sent to the Beijing Genomics Institute to be sequenced on a DNBSEQ-G400 platform. Both samples returned a final quantity of ca. 140 M of 100 bp clean paired-end reads. Reads were analysed with Kraken 2 [102], version 2.1.3 against the NCBI core_nt database (k2_core_nt_20250609 release) with default parameters, using the THOT superdome flex server of the Laval University (Québec, Canada). Reads were assembled using SPAdes 4.0 [5] with k-mer 85, and the resulting contigs files were also analysed with Kraken 2. The contigs files were later datamined for the genes or genomes of interest by customised standalone blastn queries [15] using the mitogenome (OR835203) and the rRNA clusters (OR797296; OR797297) of A. triangulatus as references. Prey DNA was datamined as explained in previous publications [55]. Protein-coding genes of all mitochondrial genomes, whether of geoplanids or Mitosporidium spp., were annotated with the help of MITOS [28] with manual verifications done by blatsp queries of the open-reading frames displayed by ORFfinder (https://www.ncbi.nlm.nih.gov/orffinder/). Mitochondrial tRNA were found using ARWEN, v1.2 [67], while mitochondrial rRNA genes were found by manual alignment of the sequences with reference sequences from A. triangulatus using MEGAX [66], except for the nuclear rRNA genes whose boundaries were found with the help of Rfam [79]. LOGO representation of the amino-acid alignments were done on the LOGO website (https://weblogo.threeplusone.com/).

All data are available (see Data availability statement).

Phylogenies

For geoplanids, a recent multiprotein dataset [41] established with the amino-acid sequences of the 12 proteins was appended with the sequences of K. andersoni and A. sanguinea. Since, as explained later, ND4L could not be found for K. andersoni, it was replaced by a blank sequence in the dataset. For Mitosporidium spp., the mitochondrial protein phylogeny dataset from Haag et al. (2014) [46], which consists of ATP6, Cob, Cox1, Cox2 and Cox3, was appended with the corresponding microsporidian sequences detected in the flatworms. Corresponding sequences from Paramicrosporidium saccamoebae Corsaro et al., 2014 (GenBank: CM008827), a parasite of Amoeba, also considered a basal microsporidian [22], were also added. All proteins were independently aligned using MAFFT 7 [63] with the -auto option and the resulting alignment trimmed using trimAl [17] with the -automated1 and -keepseqs option. The best model of evolution was obtained on each alignment using ModelTest-NG [23] (default option). The alignments were then concatenated with Phyutility 2.7.1 [86]. All phylogenies were conducted using IQ-TREE 2.2.0 [72] with 1 000 ultrafast bootstrap replicates, and with a dataset partitioned with respect to the best model of evolution found for each alignment. All alignments and partition files are available as explained in the Data availability statement.

Attempts to directly observe flatworm tissues to detect microsporidia spores

A small piece of specimen MNHN JL472 of A. sanguinea kept in ethanol was rehydrated in water and squashed between a slide and a cover glass. The slide was observed (by JLJ) with a differential interference contrast microscope at various magnifications for seeking spores. Since the whole specimen of K. andersoni was destroyed for molecular analysis, no such study was attempted. Stained sections of four specimens of A. sanguinea in the personal collection of one of us (LW) were re-examined, looking for microsporidia: MUZD 555 from Victoria H&E (Heidenhain’s Iron Haematoxylin); LW 890 from Tasmania (H&E, MSB trichrome); LW 1761 from South Australia (H&E, MSB trichrome), and LW 1806 A and B from Studley Hill, Lancs UK, via Hugh Jones (Trichrome).

Results

Taxonomic distribution of the reads and contigs

The vast majority of reads could not be classified by Kraken 2, with 88.39% and 83.87% of unclassified reads for K. andersoni and A. sanguinea, respectively. Only 1.45% and 0.9% of reads were assigned to Platyhelminthes. However, it should be noted that no reference nuclear genome is currently available for geoplanids in GenBank, which could explain the low assignment rates. The proportions of reads assigned to annelids were 0.24% and 0.23%, to arthropods 2.11% and 3.39% and to Mitosporidium spp. 0.08% and 0.17%, for K. andersoni and A. sanguinea, respectively. The search for traces of other potential hosts or parasites was also inconclusive. Kraken assigned approximately 450 reads to Gregarinasina (considered 0.00% by Kraken) and only 0.01% of reads to Amoebozoa.

As was the case for the reads, the majority of the contigs could not be classified. For K. andersoni, only 35.30% were assigned, but this percentage reached 48.72% for A. sanguinea. Out of these contigs, 0.53% and 0.44% were assigned to Platyhelminthes, which is lower than the percentage of reads previously assigned to this group. The percentages of contigs assigned to annelids were 0.15%/0.19%, to Arthropoda 6.25%/10.19%, to Mitosporidium spp. 0.01%/0.08%, to Gregarinasina 0.00%/0.00% and to Amoebozoa 0.03%/0.05% (for K. andersoni/A. sanguinea, respectively). It should be noted that these results somewhat contradict those obtained following the datamining for prey DNA, as explained below, especially for the overrepresentation of contigs assigned to Arthropoda.

Geoplanids: paralogous rRNA genes

Like in all Geoplanids in which this was investigated, the two species studied here have two paralogous copies of the nuclear rRNA cluster of genes. Their nomenclature will follow those recently introduced [39]. For K. andersoni, partial (427 bp) sequences of both copies of the 18S gene could be obtained from the contigs file, and showed 95.32% identity with each other. The low coverage copy (LCC), or type I, had coverage of 133.58× (GenBank: PV468224) and the high coverage copy (HCC), or type II, had coverage of 305.58× (GenBank: PV468223). For A. sanguinea, a partial 1,026 bp sequence of type I was obtained with coverage of 41.84× (GenBank: PV468328). Only 714 bp could be retrieved for type II, with coverage of 604.57× (GenBank: PV468329). After trimming and alignment with Clustal omega, the fragments showed only 92.10% identity with each other.

Geoplanids: mitogenomes and phylogeny

For K. andersoni, the complete mitogenome could not be assembled; it was recovered as several contigs that could not be merged. However, it was possible to extract both rRNA genes plus eleven of the protein coding genes from these contigs (Table 1). ATP6 displayed an alternative TTG codon, and ND1 started with GTG. ND4, ND5 and cob were partial. ND4 was partial in 5′, while cob and ND5 were partial in 3′ ending. It was thus impossible to verify whether or not ND5 has a premature stop codon, as generally observed among species of Southern Hemisphere Rhynchodeminae [39]. Since it was also not possible to find ND4L, it is impossible to assess whether or not there is overlap between ND4L and ND4, as also reported [39]. The putative protein encoded by cox2 is 461 amino acids long.

Table 1

Genes from the mitochondrial genome of Kontikia andersoni.

For A. sanguinea, a 17 505 bp contig containing all genes was retrieved with 164.14× coverage. The genome could, however, not be circularized because of the lack of redundancy between its endings and the presence of repetitions, as it is known to occur among rhynchodemins [39]. For easier reading, it is represented as circular in Figure 5. The mitogenome codes for the 12 conserved protein coding genes, 20 tRNA and two rRNA (GenBank: PV491411). It was not possible to find tRNA-Thr, which is commonly lost or at least too divergent to be detected among geoplanids. Also, it was not possible to find one of the two tRNA-Ser usually found in rhynchodemins: more precisely, the tRNA-Ser that clusters with tRNA-Leu, tRNA-Tyr and tRNA-Gly, between the two rRNA genes. ARWEN suggested that a second tRNA-Ser might be present, but on the negative strand, and since all mitogenomes of geoplanids so far had their genes on the same strand, this result was not retained. The mitogenome shows several other features: ND5 shows a premature stop because of the presence of tRNA-Ser; there is a 32-bp overlap between ND4L and ND4; ND3 displays an alternative TTG start codon; and the putative Cox2 protein is 464 AA long.

thumbnail Figure 5

Australoplana sanguinea, mitogenome. The mitogenome is 17 505 bp in length and is represented as circular. The mitogenome codes for 12 protein coding genes, 20 tRNA and 2 rRNA.

In the multiprotein phylogeny, both taxa belong to a strongly supported clade (100% support) that also contains A. triangulatus, Marionfyfea adventor Jones & Sluys, 2016 and four species of Caenoplana Moseley, 1877 (Fig. 6).

thumbnail Figure 6

Maximum likelihood phylogenetic tree of geoplanids, based on concatenated amino acid sequences of 12 mitochondrial proteins. *For Kontikia andersoni, ND4L, which was not found, was replaced by a blank sequence in the dataset. Subfamilies are indicated on the right. Support indicated at the nodes.

Detection of prey DNA

Kontikia andersoni. A 9 884 contig with coverage of 200.79× was found. Best megablast query returned the completely covered 3 405 bp long partial 28S gene of Lumbricus sp. THS-2006 (DQ790041, [92]) with 99.79% identity. An attempt was made to again assemble the reads with a k-mer of 55. When datamining the resulting contigs file, it was possible to find a 1 043 bp contig with low coverage of 6.08× that returned 100% identity with partial 658 bp cox1 sequences labelled as Lumbricus festivus (Savigny, 1826) (FJ937302, [84] and JN419206, [29]).

Australoplana sanguinea. A 2 674 bp long fragment with high coverage of 405.95× was retrieved. Megablast query returned 18S hits for several species of earthworms, with the same percentage of identity of 99.94%. Names, authorities, accession numbers, lengths and references are indicated in Table S1 (Supplementary File 1). Also, a 15 822 bp contig with coverage of 60.42× was found, that matches with the mitogenome of a lumbricid. The contig has TAs at both ends, and contains all the conserved coding parts of a mitogenome. Best megablast result for the query of the cox1 gene was a partial 658 bp cox1 gene belonging to the lumbricid Dendrobaena octaedra (Savigny, 1826) (GenBank: JQ909014 [80]) with identity 96.48%, suggesting that a member of the genus Dendrobaena was a prey.

For none of our samples could any 18S from an Arthropoda, Amoebozoa or Gregarinasina be found, although for Arthropoda, Kraken’s results suggested otherwise.

Presence of Mitosporidium-like sequences

Four sequences, which we ascribe to Mitosporidium spp., were found among the contigs files for both samples, with pairs of corresponding sequences found within each file. These sequences correspond to two different species. The complete cluster of nuclear rRNA was retrieved from both samples. The cluster was 6 245 bp long for K. andersoni with 273.26× coverage (GenBank: PV480898) and 6 420 bp long for A. sanguinea with 73.01× coverage (GenBank: PV480899). In Table 2, the lengths of the different parts of the cluster and their conservation are compared for the two sequences discovered in this study. Megablast queries of the 18S part returned only two results with similarity >90%. One belongs to the reference sequence from Mitosporidium daphniae (GenBank: MF278562) from Haag et al. (2014) [46], the other is ascribed to an uncultured and unidentified Cryptomycota from a water sample coming from a bog located in the state of Michigan, USA (GenBank: MZ923257), described in Quandt et al. (2023) [81].

Table 2

Lengths of the different parts of the cluster of nuclear rRNA and their conservation in the two sequences discovered in this study.

The Mitosporidium-like mitogenomes will be referred to herein as Mtspo_JL467 (Fig. 7) and Mtspo_JL472 (Fig. 8), depending on the flatworm specimen they were found to be associated with. Their sizes were 14 870 bp (GenBank: PV491412) and 11 995 bp (GenBank: PV491413), with coverages of 26.33× and 34.89×, respectively. Their gene content was identical for the conserved protein-coding genes and rRNA genes, but differed regarding the non-conserved open-reading frames (ORFs) (Table 3) and the tRNA (Table 4). Except for the tRNA, the mitogenome of Mitosporidium sp. Mtspo_JL472 is colinear with that of M. daphniae, although it contains one less large non-conserved ORF. The mitogenome of Mitosporidium sp. Mtspo_JL472 shows a change in the position of the cob gene.

thumbnail Figure 7

Mitogenome of Mitosporidium sp. JL467 (Mtspo_JL467) from Kontikia andersoni. The mitogenome is 14 870 bp in length. The mitogenome could not be circularised and is represented as linear.

thumbnail Figure 8

Mitogenome of Mitosporidium sp JL472 (Mtspo_JL472) from Australoplana sanguinea. The mitogenome is 11 985 bp in length. The mitogenome could not be circularised and is represented as linear.

Table 3

Protein-coding genes, rRNA genes and non-conserved ORFs content of the mitochondrial genomes of Mitosporidium daphniae, Mitosporidium sp. Mtspo_JL467 and Mitosporidium sp. Mtspo_JL472.

Table 4

tRNA content of the mitochondrial genomes of Mitosporidium daphniae, Mitosporidium sp. Mtspo_JL467 and Mitosporidium sp. Mtspo_JL472.

Among the non-annotated ORFs, orf130 from Mtspo_JL467 and orf138 from Mtspo_JL472 were found to be well conserved with orf138 from M. daphniae (GenBank: QWQ66181). An alignment of these three putative proteins is provided as a LOGO figure (Fig. 9). In the central part, there is a nearly entirely conserved 20 amino-acid sequence that is highlighted on the figure. However, InterProScan could not find any noticeable conserved domain, and blastp queries just returned orf138 from M. daphniae as single result. This ORF is located between ATP9 and cox3 in the three Mitosporidium mitogenomes available. For orf116 from Mtspo_JL467, blastp queries suggest that it might be a truncated LAGLIDADG endonuclease. Blastp queries on orf298 from Mtspo_JL467 and orf287 from Mtspo_JL472 returned orf292 and orf295 from M. daphniae as best results. All seem to have retained at least partial LAGLIDADG domains, as can be seen in Figure 10.

thumbnail Figure 9

Alignment, presented as a LOGO figure, of the three putative proteins orf130 from Mitosporidium Mtspo_JL467, orf138 from Mitosporidium Mtspo_JL472 and orf138 from Mitosporidium daphniae. There is a nearly entirely conserved 20-amino-acid sequence in the central part (highlighted in yellow in the figure).

thumbnail Figure 10

Alignment, presented as a LOGO figure, of the three putative proteins orf298 from Mitosporidium Mtspo_JL467, orf287 from Mitosporidium Mtspo_JL472 and orf295 from Mitosporidium daphniae. A partial LAGLIDADG domain was retained in the three species (highlighted in yellow in the figure).

There were noticeable differences in the content of tRNA between the three Mitosporidium mitogenomes. It should, however, be noted that M. daphniae was annotated using a different set of software, which could lead to discrepancies. All mitogenomes so far seem to lack tRNA-Asp, tRNA-Thr and tRNA-Cys.

The multiprotein phylogeny (Fig. 11) returned results similar to Haag et al. (2014) [46]. The three Mitosporidium species were in a strongly supported clade, sister to Rozella allomycetis (Doweld) Letcher, which displays a very long branch, and Paramicrosporidium saccamoebae was sister-group to the clade containing Mitosporidium and Rozella. Mitosporidium daphniae and Mitosporidium sp. Mtspo_JL472 were the closest related, which can be put into perspective with the complete conservation of the order of the protein-coding genes and rRNA genes among these two taxa.

thumbnail Figure 11

Maximum likelihood phylogenetic tree of the three Mitosporidium species and their relatives, based on concatenated amino acid sequences. Based on the dataset by Haag et al., 2014 [46], added with the sequence of Paramicrosporidium saccamoebae and the sequences obtained in the present study for Mitosporidium Mtspo_JL467 and Mitosporidium Mtspo_JL472. The three Mitosporidium species are in a strongly supported clade, sister to Rozella allomycetis, which displays a very long branch, and P. saccamoebae is sister-group to the clade containing Mitosporidium and Rozella. Support indicated at the nodes.

Search for microsporidia in flatworm tissues

We have not obtained direct evidence of microsporidia infection of flatworm tissue. No observation was made on K. andersoni. For A. sanguinea, our search for microsporidia on tissues kept in ethanol of the infected specimen MNHN JL472 was negative. Our search on stained serial sections of other specimens revealed evidence of gregarine infestation with the exception of the specimen from Tasmania. None exhibited anything in their intestinal diverticula that had the morphology of microsporidia, nor anything approaching Graff’s peculiar sporozoan [98].

(In)formal description of two new species of Mitosporidium

Microsporidia are now known to belong to the Kingdom Fungi, but since they were considered unicellular protozoans in the past, the International Code of Zoological Nomenclature (ICZN) is still used for them. Therefore the rules of the current ICZN (1999) [50] apply to the description of new species. Two of these rules are the need for a description of the species (Article 13.1) and deposition of type-specimens (Article 16.4). We cannot satisfy the first of these rules, since our attempt to visualise spores in the A. sanguinea MNHN JL472 specimen failed, and the K. andersoni MNHN JL467 specimen was destroyed; for the second rule, we could consider that the remaining part of the body of specimen MNHN JL472 containing microsporidia is the type-bearing specimen, but this would be impossible for MNHN JL467, which was destroyed. Therefore, we present below what is currently known for each species, without ascribing a formal binomial name.

Both species are ascribed to the genus Mitosporidium Haag et al., 2015 (Microsporidia) on the basis of high similarity of several DNA sequences (Tables 2–4).

Kingdom Fungi

Subkingdom Rozellomyceta

Genus Mitosporidium Haag, James, Pombert, Larsson, Schaer, Refardt & Ebert, 2015

(note that according to the ICZN, the date of the taxon is 2015, not 2014, since the 2014 paper did not comply with the Code) [46, 47].

Mitosporidium sp. JL467

Locality: Castle Espie, Northern Ireland

Host: Kontikia andersoni Jones, 1981, Platyhelminthes, Tricladida, Geoplanidae

Possible hosts: species of Lumbricus, earthworm preys of the flatworm (see discussion)

Morphology: unknown

DNA characterisation: see Tables 2–4.

Mitosporidium sp. JL472

Locality: Belfast, Northern Ireland

Host: Austroplana sanguinea (Moseley, 1877), Platyhelminthes, Tricladida, Geoplanidae

Possible hosts: species of Lumbricus (Supplemental Table S1) or species of Dendrobaena, earthworm preys of the flatworm (see Discussion)

Morphology: unknown

DNA characterisation: see Tables 2–4.

Discussion

Insights into two invasive terrestrial flatworm species

Kontikia andersoni supposedly originates from New Zealand. In Europe, it has been recorded in Cornwall, the Isles of Scilly, the Isle of Man, Scotland, Northern Ireland, and the Republic of Ireland [53, 100]. Various reports suggest that K. andersoni feeds on a wide range of prey that includes arthropods (especially Collembola), molluscs (slugs) and annelids (earthworms). The species has recently been introduced to the sub-Antarctic Macquarie Island [44, 101] where it is quickly spreading [49].

Australoplana sanguinea originates from South–East Australia, and is present in Tasmania, New Zealand and the Chatham Islands. Since its first record in the Isles of Scilly in 1980, it has colonized most of the British Isles including Ireland, Wales and Scotland and even the Channel Islands [3, 51, 52, 71, 85]. It seems particularly abundant in Cornwall and North–West England and is mostly known to feed on earthworms. For both species, no reports are known from continental Europe.

The original goal of this study was to document the genetic sequences of K. andersoni and A. sanguinea, for later use in molecular taxonomy and phylogeny. In this regard, and although the sequencing of K. andersoni was far from optimal, this task has been fulfilled. Genetic databases are now enriched with sequences of both species. It was possible in both cases to obtain partial sequences of both versions of the paralogous 18S genes [19, 20, 39]. The multiprotein phylogeny clearly separates K. andersoni from Parakontikia ventrolineata (Dendy, 1892) Winsor, 1991 and Australopacifica atrata (Steel, 1897), two species that have previously been classified in the genus Kontikia Froehlich, 1955. This result strongly suggest that molecular phylogeny might challenge the current classification of several Kontikia species.

Identification of prey

It is not possible to separate the organs when performing a molecular analysis on a fragment of the body of a flatworm, even more so when a whole specimen is used; for this reason, the sequences obtained include the flatworm itself, the contents of the digestive tract and therefore the prey, and (if present) the parasites, whether those of the flatworm or those of the prey.

For Kontikia andersoni, our molecular results based on both 28S and cox1 sequences suggest that the prey included species of Lumbricus, probably L. festivus. For Austroplana sanguinea, results based on a mitogenome suggest that the prey was a species of Dendrobaena, and results based on partial 18S suggested that the prey included earthworms that could not be identified at the species level, which is expected for 18S sequences [6]. This leaves us with the certainty that the prey of the two flatworms were earthworms.

No evidence of arthropod DNA (neither 18S nor mitochondrial DNA) was found in the contigs file by data mining, although Kraken assigned between 2% and 3.4% of the reads to Arthropoda, and an even higher percentage of the contigs. Some of the largest contigs assigned to Arthropoda by Kraken were extracted and submitted to a Megablast query on the NCBI server. They did not return any results. Based on this, and on the discrepancies mentioned, we consider this to be primarily an artefact of the taxonomic assignment, which is probably impaired by the absence of a reference nuclear genome from a Geoplanidae in the database. In previous studies, our gut DNA datamining protocol [55] has successfully detected traces of multiple prey types within a single specimen. We must therefore conclude that no detectable amount of insect DNA was present in our samples, and that the Kraken assignments should be interpreted with caution. This point is particularly noteworthy, given that the only previously described species of Mitosporidium is a parasite of an arthropod.

Considerations on Mitosporidium species

Based on sequence similarity across multiple loci, we have no doubt that the closest relative of our two species is Mitosporidium daphniae, and we therefore assign them to the genus Mitosporidium.

There were noticeable differences between Mtspo_JL467 and all related sequences, for both its nuclear rRNA or mitochondrial genome and despite the fact that the sample originates from the same area as Mtspo_JL472. This suggests that the radiation of Mitosporidium species includes major variations.

We would also like to note the conservation of the ca. 130–140 AA long ORF. Although it is impossible to assess whether or not this is a functional gene, in case more mitogenomes of Mitosporidium spp. became available, it would be worth checking the presence and conservation of this ORF.

Hypotheses and speculations about the hosts of the two new species of Mitosporidium

On the basis of the available evidence, it is not currently possible to assess the exact host-parasite relationship between the two geoplanids investigated and the presence of Mitosporidium spp.

We briefly present here our reasoned doubts and speculations concerning host identity.

Hypothesis 1:

The Mitosporidium spp. are parasites of flatworms. This is the simplest explanation, but we have not obtained direct evidence of microsporidia infection of flatworm tissue. This should be attempted by means of light and electron microscopy of various worm tissues. It is known that parasitic flatworms (Neodermata) can be parasitised by microsporidia [4, 68, 73, 87, 95]. However, no microsporidia appear to have been reported for terrestrial flatworms (Tricladida, Geoplanidae).

Hypothesis 2:

The Mitosporidium spp. are parasites of earthworms, which are common prey for both flatworm species. Our results on the DNA of the prey indeed show that both flatworms consume earthworm species. However, only very few microsporidia parasitic in earthworms are known from an early light and electron microscopy study [14, 24], with none described with modern molecular methods.

If species of Mitosporidium are indeed parasites of earthworms and exhibit strict host specificity, then under Hypothesis 2, our findings could only be explained if two flatworm species, co-occurring in the same geographical area, each preyed upon a distinct earthworm species, and each of those earthworms was itself infected by a distinct Mitosporidium species. Such a scenario appears statistically far less plausible than Hypothesis 1, which posits that each Mitosporidium species is a parasite of a single flatworm species. In this latter case, our results would represent two independent flatworm–Mitosporidium host–parasite associations.

Both flatworm species studied here are native to the Australia–New Zealand region. This suggests the existence of radiation of Mitosporidium in the Geoplanidae of this region, which should be verified.

Alternative hypotheses may be proposed. The only species of Mitosporidium formerly described, M. daphniae, is a parasite of a freshwater crustacean. Neither geoplanids nor their earthworm preys have aquatic stages nor contact with freshwater animals; the hypothesis of a freshwater host is therefore rejected. The Mitosporidium detected here could be hyperparasites, i.e. parasites of parasites infecting flatworms or earthworms, such as amoebae. Indeed, some “basal” microsporidian species, such as Paramicrosporidium saccamoebae, parasitise amoebae [22]. However, given the absence of amoebal DNA in our samples, we dismiss this hypothesis.

The great similarities between the GenBank partial 18S sequence MZ923257 [81] with M. daphniae and Mtspo_JL472 are puzzling. This sequence was derived from an aquatic sample [81]. If this sequence belongs to a species of Mitosporidium, What was its host?

Conclusion

In this study, we characterised multiple sequences from two invasive flatworm species in Northern Ireland and, unexpectedly, detected the genetic signatures of two distinct microsporidian species – one in each flatworm host: Mitosporidium sp._JL472 in Australoplana sanguinea and Mitosporidium sp._JL467 in Kontikia andersoni. The two microsporidian species are genetically distinct yet both clearly belong to the genus Mitosporidium. Due to the limitations imposed by the ICZN, we are currently unable to formally describe these species or assign them Latin binomials.

As our study was based on a “cocktail” of DNA – including DNA of flatworms, earthworms and microsporidia – some uncertainty remains regarding the actual host of the microsporidia. We acknowledge that our conclusions rely on a single specimen per flatworm species, and that a definitive determination of host specificity will require additional sampling. Nevertheless, we consider the hypothesis that each Mitosporidium species is specific to its respective land flatworm host to be the more plausible explanation.

Following this study, the genus Mitosporidium comprises three species: one parasitic on a freshwater crustacean [46] and two parasitic on land flatworms. This significantly broadens the known host range of the genus.

A possible reason for the success of an invasive species when introduced into a new territory is the absence of predators and parasites, which allows the invasive species to reproduce exponentially; this is known as the Enemy Release Hypothesis [64]. Therefore, the discovery of parasites in invasive species is of particular interest. The case of the invasive yellow-legged (Asian) hornet (Vespa velutina nigrithorax Lepeletier, 1836) is emblematic [74], with the description of several parasites or pathogens [35, 65, 97], although none seem to reduce the spread of the species. Mitosporidium daphniae has been reported to exert a negative, albeit marginal, effect on the lifetime fecundity of its crustacean host [45, 83]. At present, nothing is known about the potential pathogenic effects of the newly identified Mitosporidium species on their respective hosts, and this question warrants further investigation.

Acknowledgments

We would like to acknowledge Mr Stewart Rosell, Mr David Craig and Ms Jo-Anne McKeown for the collection of flatworms and photographs. Prof. Lise Dupont from Université Paris Est Créteil, France, kindly provided advice about earthworm parasites and barcoding. Prof. Karen Luisa Haag from Universidad Federal de Río Grande del Sur, Brazil, kindly discussed with us some aspects of the biology of Mitosporidium prior to the submission of the paper. Dr. Aurore Dubuffet from Université Clermont Auvergne, France, provided information about microsporidia in earthworms. We thank the reviewers for their excellent comments and suggestions.

Funding

This work was co-funded by the Ministry of Science under the “Regional Excellence Initiative” Program for 2024–2027 (RID/SP/0045/2024/01).

Conflicts of interest

The Editor-in-Chief of Parasite is one of the authors of this manuscript. COPE (Committee on Publication Ethics, http://publicationethics.org), to which Parasite adheres, advises special treatment in these cases. In this case, the peer-review process was handled by an Associate Editor, Jérôme Depaquit; the manuscript was reviewed by three reviewers.

Use of AI

Artificial intelligence (AI) tools were not employed in the design or execution of the experiments, nor in the primary preparation of this manuscript. ChatGPT (OpenAI, San Francisco, CA, USA) was used on a limited basis to test the drafting of a few paragraphs; all text was subsequently reviewed, edited and validated by the authors.

Data availability statement

All data have been submitted to GenBank and have received accession numbers, as indicated in the text. In addition, the sequencing reads have been deposited in the SRA and are available under BioProject PRJNA1247450. Kraken reports, prey DNA, alignments and partition files are available on Zenodo: https://doi.org/10.5281/zenodo.17131052.

Author contribution statement

RG initiated the study, performed all molecular analyses and wrote most of the results; AKM collected specimens and provided comments on earthworms; LW examined slides, investigated old literature and commented on parasitology of geoplanids; JLJ examined specimens, coordinated the study and added a parasitological perspective, especially in the discussion. Figures were made by RG and JLJ. All authors read and commented several versions of the manuscript, including the final one.

Supplementary Files

Supplementary Table 1: Australoplana sanguinea Megablast query for a 2 674 bp long fragment with high coverage of 405.95×. The query returned 18S hits for several species of earthworms, with the same percentage of identity of 99.94%. Names, authorities, accession numbers, lengths, reference and presence in Ireland are indicated. References include [26, 69, 82, 93]. Access here

References

  1. Alford D. 1998. Potential problems posed by non-indigenous terrestrial flatworms in the United Kingdom. Pedobiologia, 42(5–6), 574–578. [Google Scholar]
  2. Álvarez-Presas M, Mateos E, Tudo A, Jones H, Riutort M. 2014. Diversity of introduced terrestrial flatworms in the Iberian Peninsula: a cautionary tale. PeerJ, 2, e430. [Google Scholar]
  3. Anderson R. 1986. The land planarians of Ireland (Tricladida: Terricola) a summary of distribution records, Irish Naturalists’ Journal, 22(4), 141–146. [Google Scholar]
  4. Azevedo C, Canning EU. 1987. Ultrastructure of a microsporidian hyperparasite, Unikaryon legeri (Microsporida), of trematode larvae. Journal of Parasitology, 73(1), 214–223. [Google Scholar]
  5. Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, Kulikov AS, Lesin VM, Nikolenko SI, Pham S, Prjibelski AD. 2012. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. Journal of Computational Biology, 19(5), 455–477. [CrossRef] [PubMed] [Google Scholar]
  6. Barraux A, Noël S, Roy V, Dupont L. 2024. Challenges of molecular barcode-based identification of earthworm specimens for biodiversity assessment. Frontiers in Ecology and Evolution, 12, 1358984. [Google Scholar]
  7. Bass D, Czech L, Williams BA, Berney C, Dunthorn M, Mahé F, Torruella G, Stentiford GD, Williams TA. 2018. Clarifying the relationships between Microsporidia and Cryptomycota. Journal of Eukaryotic Microbiology, 65(6), 773–782. [CrossRef] [PubMed] [Google Scholar]
  8. Blackshaw RP, Stewart VI. 1992. Artioposthia triangulata (Dendy, 1894), a predatory terrestrial planarian and its potential impact on lumbricid earthworms. Agricultural Zoology Reviews, 5, 201–219. [Google Scholar]
  9. Boag B, Evans K, Yeates G, Johns P, Neilson R. 1995. Assessment of the global potential distribution of the predatory land planarian Artioposthia triangulata (Dendy) (Tricladida: Terricola) from ecoclimatic data. New Zealand Journal of Zoology, 22(3), 311–318. [Google Scholar]
  10. Boag B, Palmer LF, Neilson R, Chambers SJ. 1994. Distribution and prevalence of the predatory planarian Artioposthia triangulata (Dendy) (Tricladida: Terricola) in Scotland. Annals of Applied Biology, 124(1), 165–171. [Google Scholar]
  11. Boag B, Yeates GW. 2001. The potential impact of the New Zealand flatworm, a predator of earthworms, in Western Europe. Ecological Applications, 11(5), 1276–1286. [Google Scholar]
  12. Bouguerche C, Roth J, Boström S, Justine J-L. 2025. Lövplattmask Obama nungara – en invasiv landplanarie funnen i Sverige. Fauna og flora, 120(2), 36–45. [Google Scholar]
  13. Bouguerche C, Roth J, Justine J-L. 2025. Flatworms in the land of the midnight sun: first record of the invasive species Obama nungara (Platyhelminthes, Geoplanidae) in Sweden, its northernmost location in continental Europe. bioRxiv, 2025.03. 24.644988. [Google Scholar]
  14. Burke JM. 1970. A microsporidian in the epidermis of Eisenia foetida (Oligochaeta). Journal of Invertebrate Pathology, 16(1), 145–147. [Google Scholar]
  15. Camacho C, Coulouris G, Avagyan V, Ma N, Papadopoulos J, Bealer K, Madden TL. 2009. BLAST+: architecture and applications. BMC Bioinformatics, 10, 1–9. [CrossRef] [PubMed] [Google Scholar]
  16. Cannon RJC, Baker RHA, Taylor MC, Moore JP. 1999. A review of the status of the New Zealand flatworm in the UK. Annals of Applied Biology, 135(3), 597–614. [Google Scholar]
  17. Capella-Gutiérrez S, Silla-Martínez JM, Gabaldón T. 2009. trimAl: a tool for automated alignment trimming in large-scale phylogenetic analyses. Bioinformatics, 25(15), 1972–1973. [CrossRef] [PubMed] [Google Scholar]
  18. Carbayo F, Álvarez-Presas M, Jones HD, Riutort M. 2016. The true identity of Obama (Platyhelminthes: Geoplanidae) flatworm spreading across Europe. Zoological Journal of the Linnean Society, 177, 5–28. [Google Scholar]
  19. Carranza S, Baguñà J, Riutort M. 1999. Origin and evolution of paralogous rRNA gene clusters within the flatworm family Dugesiidae (Platyhelminthes, Tricladida). Journal of Molecular Evolution, 49(2), 250–259. [Google Scholar]
  20. Carranza S, Giribet G, Ribera C, Baguñà J, Riutort M. 1996. Evidence that two types of 18S rDNA coexist in the genome of Dugesia (Schmidtea) mediterranea (Platyhelminthes, Turbellaria, Tricladida). Molecular Biology and Evolution, 13, 824–832. [Google Scholar]
  21. Christensen OM, Mather JG. 1998. The “New Zealand flatworm”, Artioposthia triangulata, in Europe: the Faroese situation. Pedobiologia, 42, 532–540. [Google Scholar]
  22. Corsaro D, Walochnik J, Venditti D, Steinmann J, Müller K-D, Michel R. 2014. Microsporidia-like parasites of amoebae belong to the early fungal lineage Rozellomycota. Parasitology Research, 113(5), 1909–1918. [Google Scholar]
  23. Darriba D, Posada D, Kozlov AM, Stamatakis A, Morel B, Flouri T. 2019. ModelTest-NG: A new and scalable tool for the selection of DNA and protein evolutionary models. Molecular Biology and Evolution, 37(1), 291–294. [Google Scholar]
  24. De Puytorac P, Tourret M. 1963. Étude de kystes d’origine parasitaire (microsporidies ou grégarines) sur la paroi interne du corps des vers Megascolecidae. Annales de Parasitologie Humaine et Comparée, 38(6), 861–874. [Google Scholar]
  25. de Waart SA, Vanhove MPM, Justine J-L, Kmentová N. 2025. Going Dutch: European distribution of non-native land flatworm species belonging to Geoplaninae and Bipaliinae with focus on the Netherlands. NeoBiota, 99, 285–321. [Google Scholar]
  26. Díaz Cosín DJ, Novo M, Fernández R, Marchán DF, Gutiérrez M.2014. A new earthworm species within a controversial genus: Eiseniona gerardoi sp. n. (Annelida, Lumbricidae)-description based on morphological and molecular data, ZooKeys, 1(399), 71. [Google Scholar]
  27. Doliwa A, Grabner D, Sures B, Dunthorn M. 2023. Comparing Microsporidia-targeting primers for environmental DNA sequencing. Parasite, 30, 52. [Google Scholar]
  28. Donath A, Jühling F, Al-Arab M, Bernhart SH, Reinhardt F, Stadler PF, Middendorf M, Bernt M. 2019. Improved annotation of protein-coding genes boundaries in metazoan mitochondrial genomes. Nucleic Acids Research, 47(20), 10543–10552. [Google Scholar]
  29. Donnelly RK, Harper GL, Morgan AJ, Pinto-Juma GA, Bruford MW. 2014. Mitochondrial DNA and morphological variation in the sentinel earthworm species Lumbricus rubellus. European Journal of Soil Biology, 64, 23–29. [Google Scholar]
  30. Dubuffet A, Chauvet M, Moné A, Debroas D, Lepère C. 2021. A phylogenetic framework to investigate the microsporidian communities through metabarcoding and its application to lake ecosystems. Environmental Microbiology, 23(8), 4344–4359. [CrossRef] [PubMed] [Google Scholar]
  31. European Union. 2019. Commission implementing regulation (EU) 2019/1262 of 25 July 2019 amending Implementing Regulation (EU) 2016/1141 to update the list of invasive alien species of Union concern. Official Journal of the European Union, 26.7.2019, 199/1. [Google Scholar]
  32. European Union. 2025. Commission Implementing Regulation (EU) 2025/1422 of 17 July 2025 amending Implementing Regulation (EU) 2016/1141 to update the list of invasive alien species of Union concern. Official Journal of the European Union, (18.7.2025). [Google Scholar]
  33. Fourcade Y. 2021. Fine-tuning niche models matters in invasion ecology. A lesson from the land planarian Obama nungara. Ecological Modelling, 457, 109686. [Google Scholar]
  34. Fourcade Y, Winsor L, Justine J-L. 2022. Hammerhead worms everywhere? Modelling the invasion of bipaliin flatworms in a changing climate. Diversity and Distributions, 28, 844–858. [Google Scholar]
  35. Gabín-García LB, Bartolomé C, Guerra-Tort C, Rojas-Nossa SV, Llovo J, Maside X. 2021. Identification of pathogens in the invasive hornet Vespa velutina and in native Hymenoptera (Apidae, Vespidae) from SW-Europe. Scientific Reports, 11(1), 11233. [Google Scholar]
  36. Gastineau R, Justine J-L. 2020. Complete mitogenome of the invasive land flatworm Parakontikia ventrolineata, the second Geoplanidae (Platyhelminthes) to display an unusually long cox2 gene. Mitochondrial DNA part B, 5(3), 2115–2116. [Google Scholar]
  37. Gastineau R, Justine J-L, Lemieux C, Turmel M, Witkowski A. 2019. Complete mitogenome of the giant invasive hammerhead flatworm Bipalium kewense. Mitochondrial DNA Part B, 4(1), 1343–1344. [Google Scholar]
  38. Gastineau R, Lemieux C, Turmel M, Justine J-L. 2020. Complete mitogenome of the invasive land flatworm Platydemus manokwari. Mitochondrial DNA Part B, 5(2), 1689–1690. [Google Scholar]
  39. Gastineau R, Lemieux C, Turmel M, Otis C, Boyle B, Coulis M, Gouraud C, Boag B, Murchie AK, Winsor L, Justine J-L. 2024. The invasive land flatworm Arthurdendyus triangulatus has repeated sequences in the mitogenome, extra-long cox2 gene and paralogous nuclear rRNA clusters. Scientific Reports, 14(1), 7840. [Google Scholar]
  40. Gastineau R, Murchie AK, Gey D, Winsor L, Justine J-L. 2024. The terrestrial flatworm Microplana scharffi (Geoplanidae, Microplaninae): mitochondrial genome, phylogenetic proximity to the Bipaliinae and genes related to regeneration. Zootaxa, 5523(2), 211–221. [Google Scholar]
  41. Gastineau R, Otis C, Boyle B, Winsor L, Justine J-L. 2025. The mitogenome of the Northern Hemisphere native terrestrial flatworm Rhynchodemus sylvaticus (Leidy, 1851) (Platyhelminthes, Geoplanidae). Mitochondrial DNA Part B, 10(7), 558–562. [Google Scholar]
  42. Gastineau R, Ravelomanana A, Ramzy RR, Koken M, Soavolamanoro SE, Otis C, Boyle B, Gey D, Winsor L, Justine J-L. 2025. First confirmed records with molecular data of the terrestrial flatworm Bipalium kewense Moseley, 1878 (Tricladida, Geoplanidae) in Madagascar, Egypt, and South Africa. Check List, 21(3), 557–564. [Google Scholar]
  43. Gastineau R, Winsor L, Justine J-L. 2022. The complete mitogenome of the potentially invasive flatworm Australopacifica atrata (Platyhelminthes, Geoplanidae) displays unusual features common to other Rhynchodeminae. ZooKeys, 1110, 121–133. [Google Scholar]
  44. Greenslade P, Stevens MI, Edwards R. 2007. Invasion of two exotic terrestrial flatworms to subantarctic Macquarie Island. Polar Biology, 30, 961–967. [Google Scholar]
  45. Grossart H-P, Wurzbacher C, James TY, Kagami M. 2016. Discovery of dark matter fungi in aquatic ecosystems demands a reappraisal of the phylogeny and ecology of zoosporic fungi. Fungal Ecology, 19, 28–38. [Google Scholar]
  46. Haag KL, James TY, Pombert J-F, Larsson R, Schaer TM, Refardt D, Ebert D. 2014. Evolution of a morphological novelty occurred before genome compaction in a lineage of extreme parasites. Proceedings of the National Academy of Sciences of the United States of America, 111(43), 15480–15485. [Google Scholar]
  47. Haag KL, James TY, Pombert J-F, Larsson R, Schaer TM, Refardt D, Ebert D. 2015. Correction for “Evolution of a morphological novelty occurred before genome compaction in a lineage of extreme parasites”. Proceedings of the National Academy of Sciences of the United States of America, 112(10), e1162. [Google Scholar]
  48. Haria A, McGrath S, Moore J, Bell J, Blackshaw R. 1998. Impact of the New Zealand flatworm (Artioposthia triangulata) on soil structure and hydrology in the UK. Science of the Total Environment, 215(3), 259–265. [Google Scholar]
  49. Houghton M, Terauds A, Shaw J. 2022. Rapid range expansion of an invasive flatworm, Kontikia andersoni, on sub-Antarctic Macquarie Island. Biological Invasions, 24(12), 3769–3783. [Google Scholar]
  50. International Commission on Zoological Nomenclature. 1999. International Code of Zoological Nomenclature: The International Trust for Zoological Nomenclature. 306 pp. [Google Scholar]
  51. Jones H. Undated. Australoplana sanguinea (Dendy, 1891) Infosheet available from https://www.alderneywildlife.org/sites/default/files/2023–04/Australian%20flatworm%20info%20sheet%20-%20Dr%20Hugh%20Jones.pdf. [Google Scholar]
  52. Jones H, Boag B. 1996. The distribution of New Zealand and Australian terrestrial flatworms (Platyhelminthes: Turbellaria: Tricladida: Terricola) in the British Isles – the Scottish survey and MEGALAB WORMS. Journal of Natural History, 30(7), 955–975. [Google Scholar]
  53. Jones HD. 2005. Identification: British land flatworms. British Wildlife, 16(3), 189–194. [Google Scholar]
  54. Justine J-L, Gastineau R, Gey D, Robinson DG, Bertone MA, Winsor L. 2024. A new species of alien flatworm in the Southern United States. PeerJ, 12, e17904. [Google Scholar]
  55. Justine J-L, Gastineau R, Gros P, Ruzzier E, Charles L, Winsor L. 2022. Hammerhead flatworms (Platyhelminthes, Geoplanidae, Bipaliinae): mitochondrial genomes and description of two new species from France, Italy, and Mayotte. PeerJ, 10, e12725. [Google Scholar]
  56. Justine J-L, Gastineau R, Winsor L. 2024. Land flatworms (Geoplanidae) in France and French overseas territories: ten years of research. Zoologia (Curitiba), 41, e24004. [Google Scholar]
  57. Justine J-L, Gey D, Thévenot J, Gastineau R, Jones HD. 2020. The land flatworm Amaga expatria (Geoplanidae) in Guadeloupe and Martinique: new reports and molecular characterization including complete mitogenome. PeerJ, 8, e10098. [Google Scholar]
  58. Justine J-L, Marie AD, Gastineau R, Fourcade Y, Winsor L. 2022. The invasive land flatworm Obama nungara in La Réunion, a French island in the Indian Ocean, the first report of the species for Africa. Zootaxa, 5154(4), 469–476. [Google Scholar]
  59. Justine J-L, Thévenot J, Winsor L. 2014. Les sept plathelminthes invasifs introduits en France. Phytoma, (674), 28–32. [Google Scholar]
  60. Justine J-L, Winsor L, Gey D, Gros P, Thévenot J. 2014. The invasive New Guinea flatworm Platydemus manokwari in France, the first record for Europe: time for action is now. PeerJ, 2, e297. [Google Scholar]
  61. Justine J-L, Winsor L, Gey D, Gros P, Thévenot J. 2018. Giant worms chez moi! Hammerhead flatworms (Platyhelminthes, Geoplanidae, Bipalium spp., Diversibipalium spp.) in metropolitan France and overseas French territories. PeerJ, 6, e4672. [Google Scholar]
  62. Justine J-L, Winsor L, Gey D, Gros P, Thévenot J. 2020. Obama chez moi! The invasion of metropolitan France by the land planarian Obama nungara (Platyhelminthes, Geoplanidae). PeerJ, 8, e8385. [Google Scholar]
  63. Katoh K, Standley DM. 2013. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Molecular Biology and Evolution, 30(4), 772–780. [CrossRef] [Google Scholar]
  64. Keane RM, Crawley MJ. 2002. Exotic plant invasions and the enemy release hypothesis. Trends in Ecology & Evolution, 17(4), 164–170. [Google Scholar]
  65. Kim I-K, Kim C-J, Choi J-H, Kang HJ, Choi MB. 2025. Stylopization by Xenos spp. (Xenidae, Strepsiptera) in invasive alien hornet, Vespa velutina, in South Korea. Parasite, 32, 10. [Google Scholar]
  66. Kumar S, Stecher G, Li M, Knyaz C, Tamura K. 2018. MEGA X: molecular evolutionary genetics analysis across computing platforms. Molecular Biology and Evolution, 35(6), 1547. [CrossRef] [PubMed] [Google Scholar]
  67. Laslett D, Canbäck B. 2008. ARWEN: a program to detect tRNA genes in metazoan mitochondrial nucleotide sequences. Bioinformatics, 24(2), 172–175. [CrossRef] [PubMed] [Google Scholar]
  68. Lovy J, Friend S. 2017. Phylogeny and morphology of Ovipleistophora diplostomuri n. sp. (Microsporidia) with a unique dual-host tropism for bluegill sunfish and the digenean parasite Posthodiplostomum minimum (Strigeatida). Parasitology, 144(14), 1898–1911. [Google Scholar]
  69. Martinsson S, Klinth M, Erséus C. 2021. Distribution and genetic diversity of two earthworms, Helodrilus oculatus and Satchellius mammalis (Clitellata: Lumbricidae) in Scandinavia. Fauna Norvegica, 41, 1–14. [Google Scholar]
  70. Mazza G, Menchetti M, Sluys R, Solà E, Riutort M, Tricarico E, Justine J-L, Cavigioli L, Mori E. 2016. First report of the land planarian Diversibipalium multilineatum (Makino & Shirasawa, 1983) (Platyhelminthes, Tricladida, Continenticola) in Europe. Zootaxa, 4067(5), 577–580. [Google Scholar]
  71. McCarthy T. 1993. The exotic land planarian Geoplana sanguinea (Moseley) naturalized in the Galway area (Turbellaria, Tricladida, Terricola). Irish Naturalists’ Journal, 24(6), 257–258. [Google Scholar]
  72. Minh BQ, Schmidt HA, Chernomor O, Schrempf D, Woodhams MD, Von Haeseler A, Lanfear R. 2020. IQ-TREE 2: new models and efficient methods for phylogenetic inference in the genomic era. Molecular Biology and Evolution, 37(5), 1530–1534. [CrossRef] [PubMed] [Google Scholar]
  73. Miquel J, Kacem H, Baz-González E, Foronda P, Marchand B. 2022. Ultrastructural and molecular study of the microsporidian Toguebayea baccigeri n. gen., n. sp., a hyperparasite of the digenean trematode Bacciger israelensis (Faustulidae), a parasite of Boops boops (Teleostei, Sparidae). Parasite, 29, 2. [CrossRef] [EDP Sciences] [PubMed] [Google Scholar]
  74. Monceau K, Bonnard O, Thiéry D. 2014. Vespa velutina: a new invasive predator of honeybees in Europe. Journal of Pest Science, 87(1), 1–16. [Google Scholar]
  75. Moore JP, Dynes C, Murchie AK. 1998. Status and public perception of the “New Zealand flatworm”, Artioposthia triangulata (Dendy), in Northern Ireland. Pedobiologia, 42, 563–571. [Google Scholar]
  76. Mori E, Giulia M, Panella M, Montagna M, Winsor L, Justine J-L, Menchetti M, Schifani E, Melone B, Mazza G. 2022. Opening Pandora’s box: the invasion of alien flatworms in Italy. Biological Invasions, 24, 205–216. [Google Scholar]
  77. Murchie AK, Gordon AW. 2013. The impact of the “New Zealand flatworm”, Arthurdendyus triangulatus, on earthworm populations in the field. Biological Invasions, 15(3), 569–586. [Google Scholar]
  78. Murchie AK, Justine J-L. 2021. The threat posed by invasive alien flatworms to EU agriculture and the potential for phytosanitary measures to prevent importation. Technical note prepared by IUCN for the European Commission. https://hal.science/hal-03547527. [Google Scholar]
  79. Ontiveros-Palacios N, Cooke E, Nawrocki EP, Triebel S, Marz M, Rivas E, Griffiths-Jones S, Petrov AI, Bateman A, Sweeney B. 2025. Rfam 15: RNA families database in 2025. Nucleic Acids Research, 53(D1), D258–D267. [Google Scholar]
  80. Porco D, Decaëns T, Deharveng L, James SW, Skarżyński D, Erséus C, Butt KR, Richard B, Hebert PD. 2013. Biological invasions in soil: DNA barcoding as a monitoring tool in a multiple taxa survey targeting European earthworms and springtails in North America. Biological invasions, 15(4), 899–910. [Google Scholar]
  81. Quandt CA, Marino JA, Simmons DR, Davis WJ, Hassett BT, Picard KT, James TY. 2023 Evaluating the diversity of the enigmatic fungal phylum Cryptomycota across habitats using 18S rRNA metabarcoding. Fungal Ecology, 64:101248. [Google Scholar]
  82. Raś D, Csuzdi C, Urbisz A, Gajda Ł, Małota K, Świątek P. 2025. Ovaries of Lumbricidae earthworms (Annelida, Crassiclitellata), from morphology to ultrastructure. European Zoological Journal, 92(1), 97–122. [Google Scholar]
  83. Refardt D, Ebert D. 2012. The impact of infection on host competition and its relationship to parasite persistence in a Daphnia microparasite system. Evolutionary Ecology, 26, 95–107. [Google Scholar]
  84. Richard B, Decaëns T, Rougerie R, James S, Porco D, Hebert P. 2010. Re‐integrating earthworm juveniles into soil biodiversity studies: species identification through DNA barcoding. Molecular Ecology Resources, 10(4), 606–614. [Google Scholar]
  85. Santoro G, Jones HD. 2001. Comparison of the earthworm population of a garden infested with the Australian land flatworm (Australoplana sanguinea alba) with that of a non-infested garden. Pedobiologia, 45(4), 313–328. [Google Scholar]
  86. Smith SA, Dunn CW. 2008. Phyutility: a phyloinformatics tool for trees, alignments and molecular data. Bioinformatics, 24(5), 715–716. [CrossRef] [PubMed] [Google Scholar]
  87. Sokolova YY, Overstreet RM, Heard RW, Isakova NP. 2021. Two new species of Unikaryon (Microsporidia) hyperparasitic in microphallid metacercariae (Digenea) from Florida intertidal crabs. Journal of Invertebrate Pathology, 182, 107582. [CrossRef] [PubMed] [Google Scholar]
  88. Solà E, Álvarez-Presas M, Frías-López C, Littlewood DTJ, Rozas J, Riutort M. 2015. Evolutionary analysis of mitogenomes from parasitic and free-living flatworms. PLoS One, 10(3), e0120081. [Google Scholar]
  89. Soo OYM, Gastineau R, Verdon G, Winsor L, Justine J-L. 2023. Rediscovery of Bipalium admarginatum de Beauchamp, 1933 (Platyhelminthes, Tricladida, Geoplanidae) in Malaysia, with molecular characterisation including the mitogenome. Zootaxa, 5277(3), 585–599. [Google Scholar]
  90. Soors J, Mees J, Sevrin D, Van den Neucker T. 2022. Marionfyfea adventor Jones & Sluys (2016), a non-native land planarian new for Belgium (Platyhelminthes: Tricladida: Geoplanidae). Belgian Journal of Zoology, 152, 113–116. [Google Scholar]
  91. Soors J, Van den Neucker T, Halfmaerten D, Neyrinck S, De Baere M. 2019. On the presence of the invasive planarian Obama nungara (Carbayo, Álvarez-Presas, Jones & Riutort, 2016) (Platyhelminthes: Geoplanidae) in an urban area in Belgium. Belgian Journal of Zoology, 149(1), 43–47. [Google Scholar]
  92. Struck TH, Schult N, Kusen T, Hickman E, Bleidorn C, McHugh D, Halanych KM. 2007. Annelid phylogeny and the status of Sipuncula and Echiura. BMC Evolutionary Biology, 7, 57. [Google Scholar]
  93. Sun J, James SW, Jiang J, Yao B, Zhang L, Liu M, Qiu J, Hu F. 2017. Phylogenetic evaluation of Amynthas earthworms from South China reveals the initial ancestral state of spermathecae. Molecular Phylogenetics and Evolution, 115, 106–114. [Google Scholar]
  94. Thunnissen NW, de Waart SA, Collas FP, Jongejans E, Hendriks AJ, van der Velde G, Leuven RS. 2022. Risk screening and management of alien terrestrial planarians in The Netherlands. Management of Biological Invasions, 13(1), 81–100. [Google Scholar]
  95. Toguebaye BS, Quilichini Y, Diagne PM, Marchand B. 2014. Ultrastructure and development of Nosema podocotyloidis n. sp. (Microsporidia), a hyperparasite of Podocotyloides magnatestis (Trematoda), a parasite of Parapristipoma octolineatum (Teleostei). Parasite, 21, 44. [CrossRef] [EDP Sciences] [PubMed] [Google Scholar]
  96. Vila-Farré M, Sluys R, Mateos E, Jones HD, Romero R. 2011. Land planarians (Platyhelminthes: Tricladida: Geoplanidae) from the Iberian Peninsula: new records and description of two new species, with a discussion on ecology. Journal of Natural History, 45(15–16), 869–891. [Google Scholar]
  97. Villemant C, Zuccon D, Rome Q, Muller F, Poinar GO, Justine JL. 2015. Can parasites halt the invader? Mermithid nematodes parasitizing the yellow-legged Asian hornet in France. PeerJ, 3, e947. [CrossRef] [PubMed] [Google Scholar]
  98. von Graff L. 1899. Monographie der Turbellarien. II. Tricladida Terricola (Landplanarien). Atlas von Achtundfünfzig Tafeln zur Monographie der Turbellarien II. Tricladida Terricola (Landplanarien). Pls. I–LVIII., Leipzig: Wilhelm Engelmann. I–XII + 574 pp. [Google Scholar]
  99. Wijayawardene NN, Hyde KD, Mikhailov KV, Péter G, Aptroot A, Pires-Zottarelli CLA, Goto BT, Tokarev YS, Haelewaters D, Karunarathna SC, de Kirk PMA. Santiago ALCM, Saxena RK, Schoutteten N, Wimalasena MK, Aleoshin VV, Al-Hatmi AMS, Ariyawansa KGSU, Assunção AR, Bamunuarachchige TC, Baral H-O, Bhat DJ, Błaszkowski J, Boekhout T, Boonyuen N, Brysch-Herzberg M, Cao B, Cazabonne J, Chen X-M, Coleine C, Dai D-Q, Daniel H-M, da Silva SBG, de Souza FA, Dolatabadi S, Dubey MK, Dutta AK, Ediriweera A, Egidi E, Elshahed MS, Fan X, Felix JRB, Galappaththi MCA, Groenewald M, Han L-S, Huang B, Hurdeal VG, Ignatieva AN, Jerônimo GH, de Jesus AL, Kondratyuk S, Kumla J, Kukwa M, Li Q, Lima JLR, Liu X-Y, Lu W, Lumbsch HT, Madrid H, Magurno F, Marson G, McKenzie EHC, Menkis A, Mešić A, Nascimento ECR, Nassonova ES, Nie Y, Oliveira NVL, Ossowska EA, Pawłowska J, Peintner U, Pozdnyakov IR, Premarathne BM, Priyashantha AKH, Quandt CA, Queiroz MB, Rajeshkumar KC, Raza M, Roy N, Samarakoon MC, Santos AA, Santos LA, Schumm F, Selbmann L, Selçuk F, Simmons DR, Simakova AV, Smith MT, Sruthi OP, Suwannarach N, Tanaka K, Tibpromma S, Tomás EO, Ulukapı M, Van Vooren N, Wanasinghe DN, Weber E, Wu Q, Yang EF, Yoshioka R, Youssef NH, Zandijk A, Zhang G-Q, Zhang J-Y, Zhao H, Zhao R, Zverkov OA, Thines M, Karpov SA. 2024. Classes and phyla of the kingdom Fungi. Fungal Diversity, 128(1), 1–165. [Google Scholar]
  100. Wilkinson G. 2016. The non-native flatworm, Kontikia andersoni Jones 1981 (Seriata, Geoplanidae) in Scotland. Glasgow Naturalist, 26(3), 81–82. [Google Scholar]
  101. Winsor L, Stevens M. 2005. Terrestrial flatworms (Platyhelminthes: tricladida: terricola) from sub-antarctic Macquarie Island. Kanunnah, 1, 17–32. [Google Scholar]
  102. Wood DE, Lu J, Langmead B. 2019. Improved metagenomic analysis with Kraken 2. Genome Biology, 20(1), 257. [Google Scholar]

Cite this article as: Gastineau R, Murchie AK, Winsor L & Justine J.-L. 2025. An Irish cocktail of flatworm, earthworm and parasite DNAs: genomics of invasive land flatworms (Geoplanidae) reveal infestations by two new Mitosporidium species (Microsporidia). Parasite 32, 67. https://doi.org/10.1051/parasite/2025060.

All Tables

Table 1

Genes from the mitochondrial genome of Kontikia andersoni.

In the text
Table 2

Lengths of the different parts of the cluster of nuclear rRNA and their conservation in the two sequences discovered in this study.

In the text
Table 3

Protein-coding genes, rRNA genes and non-conserved ORFs content of the mitochondrial genomes of Mitosporidium daphniae, Mitosporidium sp. Mtspo_JL467 and Mitosporidium sp. Mtspo_JL472.

In the text
Table 4

tRNA content of the mitochondrial genomes of Mitosporidium daphniae, Mitosporidium sp. Mtspo_JL467 and Mitosporidium sp. Mtspo_JL472.

In the text

All Figures

thumbnail Figures 1–4

Geoplanids used in this study and their close environment. 1–2, Kontikia andersoni. 1, specimen MNHN JL467, unscaled; 2; origin of the specimen, below decaying wooden boards. 3–4, Australoplana sanguinea. 3, specimen MNHN JL472, unscaled; 4, origin of the specimen, below a concrete stone. Photographs by Stewart Rosell.
In the text
thumbnail Figure 5

Australoplana sanguinea, mitogenome. The mitogenome is 17 505 bp in length and is represented as circular. The mitogenome codes for 12 protein coding genes, 20 tRNA and 2 rRNA.
In the text
thumbnail Figure 6

Maximum likelihood phylogenetic tree of geoplanids, based on concatenated amino acid sequences of 12 mitochondrial proteins. *For Kontikia andersoni, ND4L, which was not found, was replaced by a blank sequence in the dataset. Subfamilies are indicated on the right. Support indicated at the nodes.
In the text
thumbnail Figure 7

Mitogenome of Mitosporidium sp. JL467 (Mtspo_JL467) from Kontikia andersoni. The mitogenome is 14 870 bp in length. The mitogenome could not be circularised and is represented as linear.
In the text
thumbnail Figure 8

Mitogenome of Mitosporidium sp JL472 (Mtspo_JL472) from Australoplana sanguinea. The mitogenome is 11 985 bp in length. The mitogenome could not be circularised and is represented as linear.
In the text
thumbnail Figure 9

Alignment, presented as a LOGO figure, of the three putative proteins orf130 from Mitosporidium Mtspo_JL467, orf138 from Mitosporidium Mtspo_JL472 and orf138 from Mitosporidium daphniae. There is a nearly entirely conserved 20-amino-acid sequence in the central part (highlighted in yellow in the figure).
In the text
thumbnail Figure 10

Alignment, presented as a LOGO figure, of the three putative proteins orf298 from Mitosporidium Mtspo_JL467, orf287 from Mitosporidium Mtspo_JL472 and orf295 from Mitosporidium daphniae. A partial LAGLIDADG domain was retained in the three species (highlighted in yellow in the figure).
In the text
thumbnail Figure 11

Maximum likelihood phylogenetic tree of the three Mitosporidium species and their relatives, based on concatenated amino acid sequences. Based on the dataset by Haag et al., 2014 [46], added with the sequence of Paramicrosporidium saccamoebae and the sequences obtained in the present study for Mitosporidium Mtspo_JL467 and Mitosporidium Mtspo_JL472. The three Mitosporidium species are in a strongly supported clade, sister to Rozella allomycetis, which displays a very long branch, and P. saccamoebae is sister-group to the clade containing Mitosporidium and Rozella. Support indicated at the nodes.
In the text

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