CC BY
7
Protistology 17 (4): 244-258 (2023) | doi:10.21685/1680-0826-2023-17-4-5 Pl'OtiStOlO&y
Original article
Mesnilia travisiae gen. nov., sp. nov. (Microsporidia: Metchnikovellida), a parasite of archigregarines Selenidium sp. from the polychaete Travisia forbesii: morphology, molecular phylogeny and phylogenomics
Ekaterina V. Frolova12*, Mikhail P. Raiko13, Natalya I. Bondarenko12, Gita G. Paskerova2, Timur G. Simdyanov4, Alexey V. Smirnov2, and Elena S. Nassonova1*
institute of Cytology, Russian Academy of Sciences, 194064 St. Petersburg, Russian Federation
2 Department of Invertebrate Zoology, St. Petersburg University, 199034 St. Petersburg, Russian Federation
3 Centre for Algorithmic Biotechnology. Institute for Translational Biomedicine, St. Petersburg University, 199004 St. Petersburg, Russian Federation
4 Department of Invertebrate Zoology, Faculty of Biology, Lomonosov Moscow State University, 119991 Moscow, Russian Federation
| Submitted October 27, 2023 | Accepted December 13, 2023 |
Summary
Spore sacs and free spores of a metchnikovellid were found in archigregarines Selenidium sp. isolated from polychaetes Travisia forbesii. The studied worms were collected in the subtidal areas of the Kandalaksha Gulf of the White Sea and of Zelenetskaya Bay of the Barents Sea. Spore sacs of these hyperparasites had an elongated shape with a slight flexion. They had one polar plug and contained 12—14 rounded spores. Both spore sacs and free spores were in direct contact with the cytoplasm of the host cell. Contrary to a canonical idea about development of metchnikovellids, sac-bound sporogony was often observed in this parasite without traces of ongoing free sporogony. A combination of morphological features and host range distinguishes the studied isolates from any known genus and species of metchnikovellids. Phylogenetic analysis based on the SSU rRNA gene and BUSCO phylogenomics, showed that studied isolates form a new lineage of metchnikovellids. We proposed a new genus Mesnilia gen. nov. and described a new species, Mesnilia travisiae sp. nov. (Microsporidia: Metchnikovellida) to accommodate these organisms. Phylogenetic analysis showed that there is a mixed metchnikovellid infection in the population of polychaetes T. forbesii from Zelenetskaya Bay. We found molecular evidence for presence of the second metchnikovellid species in this host, which has yet to be characterised at the morphological level. In phylogenetic and phylogenomic trees, this 'cryptic' parasite grouped with another new metchnikovellid discovered in the populations of Pygospio elegans collected in Zelenetskaya Bay. New isolates described in this
https://doi.org/10.21685/1680-0826-2023-17-4-5
© 2023 The Author(s)
Protistology © 2023 Protozoological Society Affiliated with RAS
Corresponding author: Elena S. Nassonova. Institute of Cytology RAS, Tikhoretsky Ave. 4, 194064 St. Petersburg, Russia; [email protected] Ekaterina V. Frolova. Institute of Cytology RAS, Tikhoretsky Ave. 4, 194064 St. Petersburg, Russia; [email protected], st050266@ student.spbu.ru
paper form two new lineages in the phylogenomic tree of metchnikovellids. This study confirmed widespread occurrence of mixed metchnikovellid infections in infrapopulations of gregarines from polychaetes.
Key words: Microsporidia, Metchnikovellida, gregarines, Apicomplexa, polychaetes, hyperparasitism, White Sea, Barents Sea, SSU rDNA phylogeny, BUSCO phylogenomics
Introduction
Metchnikovellida is a group of hyperparasitic microsporidia. They parasitise gregarines found in intestines of marine invertebrates, mainly polychaetes (Vivier, 1975; Sprague, 1977; Larsson, 2014). Recent in-depth investigations involving phylogenetic and phylogenomic analyses, have conclusively placed metchnikovellids as a basal branch within a broader clade that also encompasses canonical microsporidia (Mikhailov et al., 2017; Galindo et al., 2018; Nassonova et al., 2021; Bojko et al., 2022). Metchnikovellids differ from canonical microsporidia in many morphological and developmental traits including the structure of invasion apparatus (Larsson, 2014). Within the life cycle of metchnikovellids, two distinct types of sporogony can be identified: (1) 'free' sporogony resulting in formation of multiple spores located either directly in the host cytoplasm or within vacuoles, and (2) 'sac-bound' sporogony resulting in production ofthick-walled spore sacs encapsulating several spores (Vivier and Schrével, 1973; Sokolova et al., 2014). The number of spores in the sac is a species-specific feature. In some it is strictly defined (Frolova et al., 2021), while in other species it can vary within a certain range (Paskerova et al., 2016). The shape of spore sacs, their size and presence of thicker polar parts (so-called 'polar thickenings' or 'polar plugs') are used as a key feature in the system of metchnikovellids developed over a century ago (Caullery and Mesnil, 1914; Caullery and Mesnil, 1919).
Since the first description (Caullery and Mesnil, 1897) about 30 species of metchnikovellids have been documented (Vivier, 1975; Larsson, 2014; Sokolova et al., 2014; Paskerova et al., 2016; Frolova et al., 2022). Caullery and Mesnil (1914, 1919) established three genera, Metchnikovella, Amphiamblys and Amphiacantha, originally grouped into the family Metchnikovellidae. These genera differ drastically in shape, size and the proportions between the length and width of the spore sacs.
Metchnikovella has oval, cylindrical or fusiform spore sacs with rounded ends. The sacs often have thicker polar plugs at one or at both ends. The length of spore sac exceeds the width less than ten times. Amphiamblys is characterised by long rod-shaped spore sacs with rounded ends. The length exceeds the width 10 times or more. Amphiacantha has fusiform spore sacs with sharp ends that usually extend with thread-like prolongations. Dogiel (1922) found an unusual metchnikovellid species in archigregarines Selenidium sp. from Travisia forbesii in the Barents Sea. This species has bottle-shaped spore sacs with one rounded end and one tapering end with a polar plug. He established a new genus and species for this organism, which he called Caulleryetta mesnili (Dogiel, 1922). This genus was not justified later in the revision of the family Metchnikovellidae by Vivier (1975) and in the further works of some authors (Sprague, 1977; Canning and Vâvra, 2000; Schrével and Desportes, 2013). However, Sprague et al. (1992) included the genus Caulleryetta in the 'checklist of available generic names' of mic-rosporidia and noted that it should be considered valid until proven otherwise. This genus was also listed by Issi and Voronin (2007), Becnel et al. (2014) and Cali et al. (2017).
The genus Metchnikovella Caullery et Mesnil, 1897 historically housed the majority of known metchnikovellids. Compared to the relatively uniform genera Amphiacantha and Amphiamblys, the genus Metchnikovella, as it was defined by Caullery et Mesnil (1914, 1919) and Vivier (1975), exhibits a remarkable diversity in the morphology of spore sacs. The shape of spore sacs varies in an exceptional way: from oval to cylindrical and fusiform. Depending on the species, they contain from 8 to 32 oval or rounded spores arranged in one — three rows. As it was mentioned above, the spore sacs of most Metchnikovella species possess one or two polar plugs. However, some species show no pronounced polar plugs. The type species, M. spionis, has remarkably elongated polar plugs at both ends of the spore sac. The spore sacs and free spores of
Metchnikovella spp. can be surrounded by vacuoles or located in direct contact with the host cytoplasm. Unfortunately, due to the low resolution of light microscopes in former times, older descriptions often lack information whether the hyperparasites develop within the vacuoles.
Therefore, despite having a limited set of distinguishing characters, species classified as Metch-nikovella spp. displayed a greater variety of features compared to other metchnikovellid genera. Due to high polymorphism of the shape of spore sacs of the type species M. spionis, Caullery and Mesnil even suggested restricting the genus Metchnikovella to this only species, and noted that all other species within the genus were placed there provisionally (Caullery and Mesnil, 1919). Keeping in mind an exceptional morphological polymorphism of Metchnikovella, Larsson (2014) proposed to transfer the species that form oval spore sacs with a single polar plug and spherical spores to the genus Caulleryetta Dogiel, 1922.
Phylogenetic reconstructions further support the assumption that Metchnikovella is an artificial assemblage, as it does not form a monophyletic clade (Nassonova et al., 2021; Frolova et al., 2021; Frolova et al., 2022). However, lack of modern data for the type species Metchnikovella spionis Caullery et Mesnil, 1897, and Caulleryetta mesnili Dogiel, 1922, as well as the limited number ofmetchnikovellid taxa in phylogenetic trees, hinders the reconstruction of robust phylogeny of metchnikovellids.
Despite the years of intensive monitoring of the parasite fauna of Travisia forbesii in the Barents Sea, we have not succeeded in reisolating Caulleryetta mesnili. Instead, we isolated another metchnikovellid with a unique set of morphological features. This hyperparasite was also observed in archigregarines from T. forbesii collected in the White Sea. We present here its morphological description and provide molecular data for this organism, named Mesnilia travisiae gen. nov., sp. nov. Furthermore, our study provided molecular evidence for the existence of another yet hidden metchnikovellid from the same host species. This hyperparasite remains morphologically unidentified. In SSU rDNA trees and in phylogenomic reconstructions, this parasite groups with a new metchnikovellid found within the scope of our recent screenings of Selenidium pygospionis inhabiting populations of Pygospio elegans from Zelenetskaya Bay (Frolova et al., 2023). Here, we established the new genus and the new species of metchnikovellids from the Travisia forbesii, obtained sequences offour
new isolates ofmetchnikovellids from T. forbesii and P. elegans and demonstrated two new lineages in the phylogenetic tree of metchnikovellids.
Material and methods
Polychaetes Travisia forbesii Johnston, 1840 were collected from two locations: the subtidal zone near the White Sea Biological Station of M.V. Lo-monosov Moscow State University (WSBS) in Ve-likaja Salma, the Kandalaksha Bay ofthe White Sea (66°33'12.0"N 33°06'17.0"E) in August 2017 and 2020, and near the Biological Station of Murmansk Marine Biological Institute ofthe Russian Academy of Sciences (MMBI) in Zelenetskaya Bay of the Barents Sea (69°06'43.3"N 36°05'56.1"E) in August 2020—2023. Polychaetes Pygospio elegans were collected in the littoral zone in the Zelenetskaya Bay in August 2021.
For specimens collected near WSBS in 2017, dissections and gut symbiont examinations were conducted in the WSBS laboratory using an MBS 9 stereomicroscope (LOMO, Russia). Archigregarines displaying signs of infection, were placed on a cover glass and examined using a Leica DM2500 microscope equipped with differential interference contrast (DIC) and documented with a Leica DFC 420C digital camera. Polychaetes collected from the Barents Sea, were transported alive to the Department of Invertebrate Zoology, St. Petersburg University, and kept in containers at +6 °C with seawater. The seawater was refreshed every second day. Examination of potentially infected archigregarines was carried out using a Leica M205C dissection microscope equipped with Rottermann contrast. Infected archigregarines were examined using a Leica DM2500 microscope equipped with DIC, and documented with a Leica DFC295 digital camera. If the presence of metchnikovellids in archigregarines was confirmed with light microscopy, a large amount of Millipore-filtered (0.45 ^m) seawater was added under the coverslip, which resulted in detaching the cells from the object slide. The archigregarines containing free spores and spore sacs were individually collected from the slide using a hair-thin tapered-tip Pasteur pipette, washed in a fresh portion of Millipore-filtered seawater, and placed in 200 ^l PCR tubes with 1—2 ^l ofwater. Each tube was checked for the presence ofa gregarine using a Leica M205C dissection microscope.
DNA extraction from infected archigregarines was performed using Arcturus® PicoPure® DNA
Extraction Kit (Thermo Fisher Scientific, Waltham, MA, USA). Subsequently, DNA was amplified by Multiple Displacement Amplification (MDA) using a Repli-g Single Cell Amplification Kit (Qiagen, Hilden, Germany), according to the manufacturer's instructions. For verification of MDA reactions, the SSU rRNA gene was amplified by PCR using a 1:10 diluted MDA product as a template with microsporidia-specific primers: 18F, 530R (Weiss and Vossbrinck, 1999) and 1353TnR (Nassonova et al., 2021). PCR program parameters were the following: initial denaturation (5 min at 95 °C) followed by 35 cycles of 30 s at 95 °C, 50 s at 50 °C and 90 s at 72 °C, followed by 7 min at 72 °C for final elongation. Amplicons were purified using a Cleanup Mini Purification Kit (Eurogen, Moscow, Russia) or with ExoSAP-ITTM PCR Product Cleanup Reagent (Thermo Fisher Scientific, Waltham, MA, USA). The Sanger sequencing reactions were carried out using the AppliedBiosys-temsTM BigDye™ Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific, Waltham, MA, USA) and sequenced using Applied BiosystemsTM 3500*L Genetic Analyzer (Thermo Fisher Scientific, Waltham, MA, USA). The final length of the assembled contigs was around 1300—1400 bp.
Verified MDA products were used to prepare the libraries with TruSeq Nano DNA Library Preparation Kit (Illumina, San Diego, CA, USA) and sequenced using Illumina HiSeq 2500 system at the Core Facility Centre "BioBank" of the St. Petersburg University Research Park (https:// researchpark.spbu.ru/en/biobank-eng) according to the manufacturer's protocols. Quality control check of raw sequence data was performed using FastQC (http://www.bioinformatics.babraham. ac.uk/projects/fastqc/). Reads were trimmed using Trimmomatic (http://www.usadellab.org/cms/? page=trimmomatic). The assembler SPAdes v.3.15 in a single-cell mode was used for de novo genome assembly (Nurk et al., 2013). To separate hyperparasite contigs from the host material and bacterial contamination in the obtained metagenome assemblies the binning was performed using MaxBin v.2.2.7. To determine the completeness of the identified target genomes, the BUSCO v.5.2.2 package (Manni et al., 2021) with the fungi_odb10 (with "parasitic check") and microsporidia_odb10 datasets was used.
Phylogenomic analysis was carried out using a set of single-copy orthologs in the Fungi BUSCO database (fungi_odb10) and BUSCO Phyloge-nomics utility script (McGowan, 2019), with de-
fault parameters in the SUPERMATRIX mode. The analysis included a selection of genomes and transcriptomes ofmicrosporidia, rozellids, aphelids, fungi available in the GenBank database. A set ofgenomes of Holozoa (choanoflagellates, ichthyo-sporeans, and filasterians) was used as an outgroup. For each identified ortholog, a multiple alignment was constructed using the MUSCLE algorithm (Edgar, 2004). The resulting alignments were filtered and trimmed with the trimAl tool (Capella-Gutierrez et al., 2009). Alignments of individual orthologs were combined into a united concatenated alignment (53 taxa, 73 orthologs, 20783 amino acid positions) that was used for phylogenetic reconstruction using (a) maximum likelihood method (IQ-TREE v. 1.6.12, single partition, model LG+F+I+G4, ultrafast bootstrap) (Nguyen et al., 2015; Hoang et al., 2018) and (b) Bayesian analysis (MrBayes v. 3.2.7a, GTR model, gamma-distributed rate variation across sites and a proportion of invariable sites) (Ronquist et al., 2012).
For SSU rDNA phylogenetic analysis we constructed an alignment, containing all available sequences of metchnikovellids and a selection of 'core microsporidia'. A set of 'short-branch microsporidia' (sensu Bass et al., 2018) was used as an outgroup. Sequences were aligned using MAFFT v. 7.490 (Katoh and Standley, 2013) with the 'favour accuracy' mode as implemented in the CIPRES portal (Miller et al., 2010). A mask was created by the G-blocks algorithm (as implemented in SeaView v. 4.6.1 — Gouy et al., 2010) and was further manually expanded to include the maximal possible number of nucleotide positions.
The maximum likelihood (ML) phylogenetic analysis was conducted using IQ-TREE launched at the CIPRES portal, with all parameters estimated by the program. The best-fit model, TIM3+F+I+G4, was chosen according to the Bayesian information criterion. The tree was tested using non-parametric bootstrapping with 1000 pseudoreplicates. Bayesian analysis was performed with MrBayes v. 3.2.6 at the CIPRES portal, employing the GTR model with у correction for intersite rate variation (eight categories) and the covarion model. Trees were run as two separate chains (using default heating parameters) for 5 million generations, at which point they had ceased converging (the final average standard deviation of the split frequencies was <0.01), and the first 25% of generations were discarded for burn-in.
The SSU rRNA gene sequences obtained in this study were deposited with the GenBank under
Table 1. Occurrence of archigregarines and metchnikovellid hyperparasites in polychaetes Travisia forbesii from the White Sea (WSBS) and the Barents Sea (MMBI) in 2017-2023.
Sampling site, year Dissected worms Selenidium-infected worms Worms with metchnikovellid-infected Selenidium sp.
N N %* N %** %***
WSBS, 2017 10 7 70 2 20 29
WSBS, 2020 4 3 75 0 0 0
MMBI, 2020 14 7 50 2 14 29
MMBI, 2021 35 13 37 3 9 23
MMBI, 2022 59 32 54 1 2 3
MMBI, 2023 102 56 55 11 11 20
Note: WSBS - the White Sea Biological Station of M.V. Lomonosov Moscow State University; MMBI - the Biological Station of Murmansk Marine Biological Institute of the Russian Academy of Sciences; N - the number of worms; * the ratio of Selenidium-infected worms to the total number of dissected worms; ** the ratio of worms with metchnikovellid-infected Selenidium sp. to the total number of dissected worms; *** the ratio of worms with metchnikovellid-infected Selenidium sp. to the total number of worms with archigregarines.
the accession numbers: PP057783-PP057786. The dataset used in the phylogenomic analyses were deposited in Figshare at https://figshare. com/s/912285b8 6769afe24e0f (doi: 10.6084/m9. figshare.24777507).
Results
Occurrence, prevalence and morphology of met-
CHNIKOVELLIDS FROM Travisia forbesii
The archigregarines Selenidium sp. were frequently found in the gut of T. forbesii. The rate of infection varied from 37 to 55% in the polychaetes collected in the Barents Sea and from 70 to 75% in the worms sampled in the White Sea (Table 1). Each worm hosted from one to about 30 archigregarines, which were either attached to the intestine epithelium or resided freely in the gut lumen. The frequency of occurrence of metchnikovellids in T. forbesii was relatively low. It varied from 2 to 14% in the samples from the Barents Sea and in a broader range (0—20%) in the specimens from the White Sea. The ratio of metchnikovellid-infected Selenidium sp. to uninfected ones varied significantly (from 0 to 29%) across different sites and years.
Infected archigregarines usually exhibited vacu-olated cytoplasm containing numerous inclusions corresponding to various stages of metchnikovellid infection (Fig. 1, A). However, in some cases, the cytoplasm of infected cell appeared almost homogeneous (Fig. 1, B). Gentle pressure of an archigregarine cell with a coverslip revealed numerous spore sacs, positioned longitudinally in
the host cytoplasm, as well as proliferative stages and free spores (Fig.1, C—D). The mature spore sacs had an elongated shape with a slight flexion and one prominent polar plug (Fig. 1, E). The sacs contained 12—14 rounded spores. Notably, differences in the size of spore sacs were observed between isolates collected in the White and Barents Seas, as detailed in Table 2. Furthermore, worms collected from the White Sea exhibited higher intensity of infection (Fig. 2), with archigregarines containing from 12 to 35 spore sacs per individual (n = 14). In contrast, infected archigregarines from the Barents Sea contained from 1 to 11 spore sacs per host cell (n = 19).
In immature spore sacs, no spores were visible inside, and the sac wall appeared thin (Fig. 1, A—B). The spores within mature sacs were rounded in shape and measured 1.3 — 2.5 * 0.9 — 1.6 ^m (avg (average) = 1.95 * 1.2, n = 25). Free spores were observed alongside spore sacs of varying maturity, typically located in the terminal regions ofthe archigregarine cell (Fig. 1, D). Free spores were oval and measured 1.5 - 2.8 * 0.8 - 1.7 ^m (avg = 2.1 * 1.4, n = 21).
New metchnikovellid isolate infecting selenidium
pygospionis FROM pygospio elegans
Four metchnikovellid species were described in the population of P. elegans in the White and Barents Seas (reviewed in Frolova et al. 2023). During our recent screenings of parasitic fauna in the populations of P. elegans, in addition to the four other species previously described, we unexpectedly found a new metchnikovellid, provisionally designated as MD2_b01_MMBI2021. It had oval (rarely
Ш
Fig. 1. Metchnikovellid Mesnilia travisiae TB2_b02_MMBI2020, a parasite of the archigregarine Selenidium sp. isolated from the polychaete Travisia forbesii collected in the Barents Sea, DIC. A, B - Early stages of infection and immature spore sacs; C - mature spore sacs and stages of free sporogony - Plasmodium and sporoblasts; merged from two pictures taken at different focus depths; D — mature spore sac and free spores at the anterior end of the gregarine; E - an isolated mature spore sac of M. travisiae; note a polar plug at one pole of each spore sac. Abbreviations: im - immature spore sacs, pp - polar plug, hn - host nucleus, sbs - sporoblasts, pl - plasmodium, ss - spore sac, fs - free spores. Arrowheads point at early stages of sporogony. Scale bars: 10 ^m.
irregularly oval, pear-shaped), sometimes slightly bent or curved spore sacs with one polar plug (Fig. 3). The spore sacs measured 8.9 — 11.1 x 4.2 — 4.7 ^m in maximal dimension (avg = 9.9 x 4.4 ^m, n = 8). They contained 8-12 spores per sac. Sac-bound spores were oval and measured 1.7 — 2.8 x 1.3 — 1.5 ^m (avg = 2.2 x 1.4 ^m, n = 14). Free spores were also oval and measured 2.3 — 3.2 x 1.3 — 1.9 ^m (avg = 2.9 x 1.6 ^m, n = 12). Both free spores and spore sacs seemed to be in direct contact with the host cytoplasm. Morphologically, it resembled
M. dogieli, but at the molecular level, these two metchnikovellids were very distant; the identity of SSU rRNA gene sequences was 65.5%.
SSU rDNA PHYLOGENY
The obtained phylogenetic reconstructions of Metchnikovellida based on the SSU rRNA gene is congruent with the results of phylogenetic analyses published previously (Mikhailov et al., 2017; Galindo et al., 2018; Frolova et al., 2021; Nassonova
Fig. 2. Metchnikovellid Mesnilia travisiae MT_WSBS2017, a parasite of the archigregarine Selenidium sp. isolated from the polychaete Travisia forbesii collected in the White Sea, DIC. Mature (A) and immature (B) spore sacs of M. travisiae in the cytoplasm of the archigregarines. Abbreviations: ss - spore sac, pp - polar plug. Scale bars: 30 ^m.
et al., 2021; Frolova et al., 2022; Mikhailov et al., 2022): metchnikovellids are a sister to 'core' microsporidia. In the SSU rDNA phylogenetic tree (Fig. 4), metchnikovellids from archigregarines and eugregarines form a robustly supported clade which, together with the sequence of morphologically unidentified parasite from the blastogregarine Siedleckia cf. nematoides (GHVV01457926, here and further the accession numbers of sequences in GenBank are provided) (Mikhailov et al., 2022), groups into a moderately supported superclade.
Three clades of metchnikovellids were always recovered. One was fully supported by all methods
and comprised two sequences of Amphiacantha spp. (KX214676, KX214677), environmental clone p1_44 (KX214678) and the sequence of Metchni-kovella spiralis (MW344837), as was previously shown by Frolova et al. (2021, 2022). In most reconstructions, the sequences of M. dobrovolskiji (OP225322) and M. incurvata (0XFS01000707) branched close to this clade, although with low support (Fig. 4). These species always branched sequentially in Bayesian analyses, but formed a clade in most ML analyses. The statistical support for both these topologies was negligible. The second clade of metchnikovellids united two sequences of
Table 2. Size variation in spore sacs of Mesnilia travisiae parasitising archigregarines Selenidium sp. from Travisia forbesii collected in the White Sea (isolate MT_WSBS2017) and in the Barents Sea
(isolate TB2_b02_M M BI2020).
Isolates Length (pm) Width (|jm) Average size ± SE (jm); n
MT_WSBS2017 9.0 - 17.6 4.4 - 5.9 13.1 ± 0,28 x 5.2 ± 0,05; 52
TB2_b02_MMBI2020 7.7 - 16.3 2.4 - 4.6 12.4 ± 0,28 x 3.3 ± 0,11; 36
Note: n - a number of measurements; SE - standard error of the mean.
w
f
/Г^ , * if* •
Г $i
Fig. 3. Metchnikovellid isolate MD2_MMBI2021 ex Selenidium pygospionis from the polychaetes Pygospio elegans collected from the littoral zone of Zelenentskaya Bay of the Barents Sea, DIC. Abbreviations: ss — spore sac, fs — free spores. Scale bar: 20 ^m.
Amphiamblys spp. (KX214672, KX214674) and the sequence of Metchnikovella dogieli (MT969020). This group had moderate support (BS = 73; PP = 0.95). The sequences of new isolates formed a third clade, which was moderately supported (BS = 76; PP = 1.0) and occurred to be a sister clade to the rest of metchnikovellids, except the basal lineage corresponding to an 'uncultured' parasite from Siedleckia cf. nematoides.
The SSU rRNA gene sequences of the metchnikovellid isolates from the White Sea (MT_ WSBS2017) and Barents Sea (TB2_b02_MMBI 2020) were almost identical (99.2% identity). Phy-logenetic analysis showed that there was a mixed metchnikovellid infection in the infrapopulations of archigregarines T. forbesii polychaetes from Zelenetskaya Bay population. We found molecular evidence for the presence of a second metchnikovellid species (the sequence labelled TB2_b04_ MMBI2020, Fig. 4), which remained uncharac-terised yet at the morphological level. The sequence identity between this 'hidden' species and the isolates described above (MT_WSBS2017 and TB2_b02_MMBI2020) was 69.4%. This 'hidden' species branched together with a yet undescribed metchnikovellid parasitising Selenidium pygospionis from Pygospio elegans isolated in Zelenetskaya Bay
in 2021 (isolate MD2_b01_MMBI2021, Fig. 3).
BUSCO PHYLOGENOMICS
To increase the resolution and robustness of phylogenetic reconstructions, we used the BUSCO-based phylogenomic analysis. The general topology of the phylogenomic tree corresponded to earlier published ones (Mikhailov et al., 2017; Galindo et al., 2018; Nassonova et al. 2021). Compared to the SSU rDNA tree, the clades of core microsporidia and metchnikovellids were fully supported (Fig. 5). Within the metchnikovellid clade, the grouping of Amphiamblys sp. and Metchnikovella dogieli was robustly supported. M. incurvata also branched together with them, like in earlier SSU rDNA trees with limited sampling of metchnikovellids (Frolova et al., 2021; Nassonova et al., 2021). The support for this branching was always high. New isolates studied in the present paper formed two clades. Both morphologically identified isolates MT_WSBS2017 and TB2_b02_MMBI2020 from Travisia forbesii grouped together. The yet hidden isolate TB2_b04_ MMBI2020 from T. forbesii branched separately from other isolates from the same polychaete and grouped together with yet undescribed metchnikovellid MD2_b01_MMBI2021 parasitising S. pygospionis from P. elegans. Both these clades were fully supported.
Discussion
The metchnikovellid from Travisia forbesii de-picted here, has elongated spore sacs with one polar plug. Within the frames of the current taxonomy of Metchnikovellida, it clearly differs from the genera Amphiamblys and Amphiacantha. Based on the shape and size of spore sacs, it could be classified as a member of the morphologically heterogenous genus Metchnikovella Caullery et Mesnil,1914. Because of the presence of the polar plug only at one end of the spore sac, we can also consider it a member of the genus Caulleryetta sensu Larsson (2014). However, the latter genus is problematic and requires special consideration before classifying any new members in it.
The genus Caulleryetta was established by Dogiel in 1922 for a metchnikovellid, isolated from the archigregarine Selenidium sp. inhabiting the polychaete Travisia forbesii. This species, which he named C. mesnili, had pyriform spore sacs with a short thin neck ending in a plug, typically containing 8—12 spores. Dogiel (1922, p. 574) compared them
Fig. 4. SSU rDNA phylogeny of Microsporidia and related lineages including the sequences of Mesnilia travisiae and other isolates retrieved in this study (in bold). The tree was calculated using 1323 nucleotide positions. IQ-TREE (TIM3+F+I+G4 model; ultrafast bootstrap) / MrBayes (GTR model + gamma correction, 8 rate categories + covarion). Black dots indicate full support by all methods (bootstrap support [BS] > 99%, posterior probability [PP] > 0.99). Open circles correspond to BS > 95% and PP > 0.95.
with "an egg, elongated from the narrow end, or a small bottle". He provided description of young and mature spores sacs ('cysts') and several line drawings, but did not include a formal taxonomic diagnosis of the new genus and species. So, the genus Caulleryetta at that time was not formally established. Vivier (1975, p. 353) considered this genus as invalid and introduced a new combination "Metchnikovella mesnili (Dogiel, 1922)". Sprague (1977) also did not list Caulleryetta among metchnikovellid genera. However, in 1992 he provided English translation of selected sections of Dogiel's description (originally written in French) and composed a taxonomic summary for this genus, thus accepting its validity (Sprague, 1992, p. 310). Issi and Voronin (2007, p. 1017) provided a formal diagnosis of the genus Caulleryetta Dogiel, 1922 as "microsporidia with elongated oval sporophorous vesicles, one end of
which is rounded, and the other tapers like the neck of a bottle. Usually forms 5-10 sporophorous vesicles, located in a row before and after the nucleus of the host cell" (translated from Russian; the spore sacs characteristic of metchnikovellids are considered as one of the variants of sporophorous vesicles of microsporidia, therefore Issi and Voronin following Canning and Vavra (2000), called spore sacs as sporophorous vesicles).
No type material or slides of Caulleryetta mesnili was established by Dogiel. A careful search in the archives and collections at the Department of Invertebrate Zoology ofSaint Petersburg University, where Dogiel worked, did not recover any additional data on this organism. So, the species Caulleryetta mesnili Dogiel, 1922 remains poorly studied and needs re-isolation, preferably from the type habitat (Strait Ekaterininskaya Gavan', Kola Bay, Barents
- Encephalitozoon cuniculi GB-M1 GCF_000091225.1
- Vairimorpha ceranae PA081199 GCF_000988165.1
-Pancytospora epiphaga JUm1396 GCA_024243955.1
-Enteropsectra breve JUm2551 GCA_024243835.1
_i-Enterospora canceri GB1 GCA_002087915.1
I-Enterocytozoon hepatopenaei TH1 GCA_002081675.1
-Hepatospora eriocheir GB1 GCA_002087885.1
i-Anncaliia algerae PRA339 GCA_000385875.2
'-Tubulinosema ratisbonensis Franzen GCA_004000155.1
—Antonospora locustae CLX GCA_007674295.1
_i- Vavraia culicis subsp. floridensis GCF_000192795.1
' '-Trachipleistophora hominis GCA_000316135.1
-Pseudoloma neurophilia MK1 GCA_001432165.1
-Cucumispora dikerogammari Dv6 GCA_014805705.1
-Dictyocoela muelleri Ou54 GCA_016256075.1
tí
- Astathelohania contejeani T1 GCA_014805555.1
-Hamiltosporidium magnivora Be-OM-2 GCA_004325065.1
-Edhazardia aedis USNM 41457 GCA_000230595.3
-Nematocida displodere JUm2807 GCA_001642395.1
-Nematocida homosporus JUm1504 GCA 024244095.1
- Nematocida parisii ERTml GCF_000250985.1
Amphiamblys sp. WSBS2006 GCA_001875675.1 Metchnikovella dogieli WSBS2016_MD
Metchnikovella incurvata LNA5 GCA 003600395.1 Mesnilia travisiae TB2_b02_MMBI2020 Mesnilia travisiae MT_WSBS2017
- metchnikovellid ex Selenidium sp. ex Travisia forbesii TB2_b04 MMBI2020 metchnikovellid ex Selenidium pygospionis MD2_b01_MMBI2021
-Mitosporidium daphniae UGP3 GCF_000760515.2
-Paramicrosporidium saccamoebae KSL3 GCA 002794465.1
— Rozella allomycis CSF55 GCA_000442015.1 Spizellomyces punctatus DAOM BR117 GCF_000182565.1 Batrachochytrium dendrobatidis JAM81 GCF 000203795.1 Catenaria anguillulae PL171 GCA 002102555.1 — Conidiobolus coronatus NRRL28638 GCA_001566745.1 Coemansia reversa NRRL1564 GCA 002705745.1 Mortierelia alpina ATCC 32222 GCA_000240685.2 Lichtheimia ramosa KPH11 GCA_008728235.1 Rhizopus arrhizus 97-1192 GCA_000697195.1
Laccaria bicolor S238N-H82 GCF_000143565.1
Puccinia graminis f. sp. tritici CRL 75-36-700-3 GCF_000149925.1 - Schizosaccharomyces pombe 972h- GCF_000002945.1
- Saccharomyces cerevisiae S288C GCF 000146045.2
CORE MICROSPORIDIA
Metchnikovellida
FUNGI
Parapheiidium tribonemae EP00158 Amoeboapheiidium occidental FD01 GCA_023515795.1 -Parvularia atlantis EP00160
-Fonticula alba ATCC 38817 GCF 000388065.1
| Aphelida Nuclearlida
-1 91/1.0
ichthyophonus hoferi GCA_002751075.1
Sphaeroforma arctica JP610 GCF 001186125.1
Saipingoeca rosetta ATCC 50818 GCF 000188695 Monosiga brevicoilis MX1 GCF_000002865.3 Capsaspora owczarzaki ATCC 30864 GCF 000151315.2 — Coraiiochytrium iimacisporum Hawaii GCA 002811645.1
HOLOZOA (outgroup)
Fig. 5. Phylogenomic tree of Holomycota showing the position of a new species of metchnikovellids and other isolates retrieved in this study (in bold). The tree was reconstructed using a concatenated alignment ("supermatrix") prepared with a dataset of BUSCO single-copy protein domains (73 orthologs, 20783 amino acid positions) for 47 representatives of the Holomycota clade and 6 other Amorphea species used as an outgroup. The phylogeny was reconstructed using the maximum likelihood method (IQ-TREE v. 1.6.12, single partition, model LG+I+G4, ultrafast bootstrap) and Bayesian analysis (MrBayes 3.2.7a, GTR model, gamma-distributed rate variation across sites and a proportion of invariable sites). The support values are as follows: bootstrap values (BS, IQ-TREE), posterior probability (PP, MrBayes). Clades sharing full support gained with both methods (> 99% BS, > 0.99 PP) are indicated by black dots. An open circle corresponds to BS > 95% and PP > 0.95.
Sea) and type host (polychaete Travisia forbesii).
Larsson (2014, p. 622-623) re-defined the borders of the genus Caulleryetta. He believed the number ofplugs to be the primary character, and proposed the following diagnosis for the genus Caulleryetta: "Spore sacs oval, one end with a polar plug. Both sporogonies produce spores of the same shape. Spores almost spherical, slightly pointed over the polar sac" (Larsson, 2014, p. 622). He transferred to this genus all metchnikovellids with the single polar plug. As a result, six more species, formerly classified
as members of the genus Metchnikovella, became members of the genus Caulleryetta (see Larsson, 2014, p. 623). According to his classification, only species forming spore sacs with two polar plugs remained within the genus Metchnikovella. He re-defined the latter genus as follows: "Spore sacs cylindrical or fusiform, more or less curved, with rounded ends containing polar plugs. Length not exceeding 10 times the width. Spores are oval. Both sporogonies produce spores of approximately the same shape" (Larsson, 2014, p. 621).
Fig. 6. Cut-off from the tree shown in Fig. 4, demonstrating the wide distribution and broad variation in shape and size of hyperparasites with Caulleryetta-like morphology (sensu Larsson, 2014) among the metchnikovellid lineages. Drawings of the spore sacs are to scale.
Molecular phylogeny did not support the revision proposed by Larsson (2014). It was shown that species with oval sacs having one polar plug are scattered throughout the phylogenetic tree of metch-nikovellids and do not form a monophyletic clade (Frolova et al., 2021; Frolova et al., 2022) (Fig. 6). The species that, according to Larsson's definition, should be included in this genus, belong to different phylogenetic lineages of metchnikovellids. Thus, the available data suggest that the genus Caulleryetta sensu Larsson (2014) is an artificial group. It appears to be a paraphyletic assemblage. Moreover, the type species of this genus — C. mesnili Dogiel, 1922 differs in morphology of spore sacs and spores from all other metchnikovellids forming spore sacs with one polar plug (six species transferred by Larsson (2014) to the genus Caulleryetta: C. berliozi, C. brasili, C. hovas-sei, C. nereidis, C. oviformis, C. wohlfarthi, three species sequenced and described recently by us under the generic name Metchnikovella: M. dogieli (Paskerova et al., 2016), M. spiralis (Frolova et al., 2021) and M. dobrovolskiji (Frolova et al., 2022) and three isolates characterized in the present paper). In this situation, the most parsimonious solution seems to return to the initial definition of Caulleryetta as a monotypic genus (Dogiel, 1922; Spra-gue, 1992; Issi and Voronin, 2007) and return other species transferred by Larsson back to the genus Metchnikovella. In fact, it returns us to the classifications by Sprague (1992) and Issi and Voronin (2007).
The isolates that we described in the present paper are from the same host and super-host as C. mesnili Dogiel, 1922. However, in contrast to this species, they have elongated and slightly bent spore sacs with 12-14 spores, which appear to be slightly oval, not rounded. These spores and spore sacs resemble to some extent those of M. dogieli (Paskerova et al., 2016) and to a lesser extent those of M. incurvata (Sokolova et al., 2013), but certainly not those of C. mesnili.
All lifecycle stages of studied metchnikovellids from T. forbesii as well as M. incurvata and M. dogieli develop in direct contact with the host cytoplasm. However, unlike the metchnikovellids from T. forbesii, M. incurvata has fusiform, slightly incurved (boomerang-shaped) spore sacs, they are larger (22-27 ^m versus 7.7-17.6 ^m), possess two polar plugs and more spores per sac (up to 16 versus 12-14). Compared to M. dogieli, which also has slightly bent spore sacs (however, oval rather than elongated) and only one polar plug, the shape and size of the spore sacs of the studied isolates were more uniform. Regardless of their size or degree of maturity, the sacs of the studied isolates remain elongated with a slight flexion. This is probably caused by different structure of the sac wall and its rigidity between the compared hyperparasites. The spore sacs of metchnikovellid from T. forbesii are generally smaller and the size polymorphism is significantly less than in the case of M. dogieli. Among
sequenced metchnikovellids, M. dobrovolskijiis also characterized by spore sacs with 12 spores and one polar plug, however these sacs are irregularly oval, sometimes pear-shaped, both free spores and spore sacs are enclosed in vacuoles (Frolova et al., 2022); that sharply contradict with the morphological features of isolates described from T. forbesii.
Another unusual trait of the metchnikovellids from T. forbesii is that the stages of sac-bound sporo-gony may occur in the absence of free sporogony. It contradicts the current ideas about the life cycle of metchnikovellids. Typically, free sporogony precedes sac-bound one, resulting in the occurrence of gregarines with free spores or with both free spores and spore sacs (Frolova et al., 2023). In the studied metchnikovellids, the archigregarines, only with spore sacs were often seen. Early formation of spore sacs could be considered as an adaptation enabling fast production of thick-walled spore sacs, resistant to environmental conditions. It may be a situational response to unfavourable environmental conditions.
The unique morphological features of the isolates from T. forbesii are complemented by their independent position in the phylogenetic and phylo-genomic trees.
There are the following variants for taxonomic placement of described here metchnikovellid from T. forbesii:
(1) to place it in the genus Caulleryetta using expanded definition by Larsson (2014). This choice appears to be weak, as we just discussed above the need to limit Caulleryetta back to Dogiel's description and diagnosis of Issi and Voronin. The studied metch-nikovellid evidently does not fit this stringent definition;
(2) to describe it as one more Metchnikovella, thus increasing the heterogeneity of this genus and postponing the taxonomic problems for the future, until (maybe) more data on similar organisms will become available;
(3) to create a new genus for it, basing on its isolated phylogenetic position and clear differences from Caulleryetta sensu Dogiel. No one of species currently placed in the genus Metchnikovella is phylogenetically close to the isolates from T. forbesii. In this case we avoid adding more paraphyly to the assemblage called Metchnikovella. This solution seems to be preferable. Therefore, based on the morphological features and phylogenetic position of the studied isolates, we suggest the establishment of a new genus — Mesnilia, in honour of Félix Étienne Pierre Mesnil (1868—1938), a French zoologist, biologist, botanist, mycologist and algologist, one ofthe
founders of research on the metchnikovellids. The type species for the new genus is named Mesnilia travisiae gen. nov., sp. nov. after the super-host, Travisia forbesii.
Our study highlights the existence of a 'hidden' metchnikovellid species even in well-studied hosts, as evidenced by molecular detection of uncultured metchnikovellid TB2_b04_MMBI2020 from the super-host T. forbesii and by discovery of isolate MD2_b01_MMBI2021 from Pygospio elegans. These findings are further evidence of widespread co-occurring infections of metchnikovellid in infra-populations of gregarines from polychaetes (Soko-lova et al., 2014; Frolova et al., 2023). The concept of the host specificity (also known as common assumption 'one host — one parasite') obviously does not work for metchnikovellids. As of now, no less than five species of metchnikovellids are known from the super-host P. elegans, of them, no fewer than three species (including MD2_ b01_MMBI2021) parasitise the archigregarine Selenidium pygospionis and two species infecting the eugregarine Polyrabdinapygospionis (Frolova et al. 2023). Our study once again stressed this complication in isolating, studying and identifying hy-perparasitic microsporidia.
We were specially searching for Caulleryetta mesnili in the locations at the Barents Sea during 2020—2023, but never isolated it. Theoretically, we cannot exclude that yet hidden and morphologically undescribed isolate TB2_b04_MMBI2020 from T. forbesii corresponds to C. mesnili. The isolate MD2_b01_MMBI2021, infecting Selenidium py-gospionis in the polychaete Pygospio elegans, groups together with the above mentioned isolate TB2_ b04_MMBI2020 in the phylogenomic tree (Fig. 5). The former isolate is not well-characterised yet at the morphological level, but from the available field images (Fig. 3) we cannot conclude that it is similar in morphology to Dogiel's Caulleryetta. It has oval, sometimes slightly bent or curved spore sacs with one polar plug, and only rarely irregularly oval, pear-shaped spore sacs were seen. This reduces, but does not completely exclude the chances that the still morphologically unstudied isolate TB2_b04_ MMBI2020 from T. forbesii is C. mesnili.
The isolates described in this study (two isolates of new species Mesnilia travisiae, one yet hidden species from T. forbesii and a new isolate infecting Selenidium pygospionis from Pygospio elegans), form two new lineages in the tree of metch-nikovellids. Despite the increment in the number of obtained sequences, the phylogenetic tree of
metchnikovellids based on the SSU rRNA gene sequences remains unstable. The phylogenomic analysis shows better resolution and results in highly supported tree; however, it still includes limited set of metchnikovellid taxa.
It is becoming evident that Metchnikovellida is a widely distributed and species-rich group of microsporidia. This stresses the need for further study of metchnikovellid diversity in order to improve phylogenetic analyses by adding more species and to study complex multilevel parasitic systems involving hyperparasites.
Taxonomic summary
Phylum Microsporidia Balbiani, 1882
Class Rudimicrosporea Sprague, 1977
Order Metchnikovellida Vivier, 1975
Genus Mesnilia gen. nov. Diagnosis. Spore sacs are elongated, with one polar plug. Both sporogonies are in direct contact with host cytoplasm. Spore sacs non-accompanied by free spores may be observed in host cytoplasm.
Mesnilia travisiae sp. nov.
Diagnosis. Free spores are oval (1.5—2.8 * 0.8 — 1.7 ^m). Spore sacs are elongated with a slight flexion (7.7—17.6 * 2.4—5.9 ^m), with rounded ends and a prominent polar plug at one end. Sac-bound spores counted 12—14 per sac. Sac-bound spores are rounded (1.3-2.5 * 0.9-1.6 ^m).
Differences from closely related species. The species differs from other metchnikovellids by the combination of characters: the size and shape of the spore sacs, the number of spores per sac, the number of polar plugs, the super-host and host range. It exhibits significant differences in the SSU rRNA gene sequence and in protein-coding gene sequences.
Type locality. Zelenetskaya Bay of the Barents Sea (69°06'43.3"N 36°05'56.1"E). Subtidal zone.
Type habitat. Marine.
Type host and super-host. Archigregarine Selenidium sp. (Apicomplexa: Selenidiidae) from the polychaete Travisiaforbesii (Annelida: Travisiidae).
Location in the host. Gregarine cytoplasm.
Type material. Images ofthe live archigregarines are stored in the image collection of the Department of Invertebrate Zoology, St Petersburg University. Frozen purified genomic DNA of the infected archigregarines as well as the individual infected
gregarine cells fixed in 96% ethanol are stored at the same department.
Etymology. This genus was named in honour of Félix Étienne Pierre Mesnil (1868—1938), one ofthe founders of the studies on metchnikovellids, French zoologist, biologist, botanist, mycologist and algo-logist. The species was named after the super-host, Travisiaforbesii.
Gene sequences. SSU rRNA gene sequences of M. travisiae have been deposited in the GenBank under the accession numbers OR887354-OR887355
Acknowledgements
This study was supported by the Russian Science Foundation — project № 23-74-00071. This study utilised equipment of the Core Facility Centres 'Biobank', 'Development of Molecular and Cell Technologies' and 'Culturing of microorganisms' ofthe Research Park ofSaint Petersburg University. Authors thank the staff of the White Sea Biological Station of M.V. Lomonosov Moscow State University and the Biological Station "Dalnie Zelentsy" of the Murmansk Marine Biological Institute of Kola Science Centre of the Russian Academy of Sciences for providing facilities for field sampling and material processing, as well as for their kind and friendly approach. The authors would like to thank the fellow colleagues, Oksana Kamyshatskaya and Yelisei Mezentsev, for their help in digging the polychaetes in a wide range of weather conditions (including unfavourable ones).
References
Bass D., Czech L., Williams B.A.P., Berney C. et al. 2018. Clarifying the relationships between Microsporidia and Cryptomycota. J. Eukaryot. Microbiol. 65: 773-782. https://doi.org/10.1111/ jeu.12519
Becnel J.J., Takvorian P.M. and Cali A. 2014. Checklist of available generic names for Microsporidia with type species and type hosts. In: Mic-rosporidia: pathogens of opportunity (Eds: Weiss L.M. and Becnel J.J.). John Wiley and Sons, Inc., Ames, Iowa, pp 671-686. https://doi.org/10.1002/ 9781118395264.app1
Bojko J., Reinke A.W., Stentiford G.D., Williams B. et al. 2022. Microsporidia: a new taxonomic, evolutionary, and ecological synthesis. Trend. Para-
sitol. 38: 642-659. https://doi.org/10.1016/j.pt. 2022.05.007
Cali A., Becnel J.J. and Takvorian P.M. 2017. Microsporidia. In: Handbook of the protists. 2nd ed. (Eds: Archibald J.M. et al.). Springer, Cham, Switzerland, pp. 1559-1618. https://doi.org/10. 1007/978-3-319-28149-0_27
Canning E. and Vávra J. 2000. Phylum Microsporida Balbiani, 1882. In: An illustrated guide to the protozoa: organisms traditionally referred to as protozoa, or newly discovered groups. Vol. 1, 2nd ed. (Eds: Lee J.J. et al.). Allen Press, Lawrence, Kan, pp. 39-126.
Capella-Gutiérrez S., Silla-Martínez J.M. and Gabaldyn T. 2009. trimAl: a tool for automated alignment trimming in large-scale phylogenetic analyses. Bioinformatics. 25 (15): 1972-1973. https://doi.org/10.1093/bioinformatics/btp348
Caullery M. and Mesnil F. 1897. Sur un type nouveau (Metchnikovella n.g.) d'organismes parasites des Grégarines. C. R. Séances Soc. Biol. 4 (49): 960-962.
Caullery M. and Mesnil F. 1914. Sur les Metchnikovellidae et autres protistes parasites des Grégarines d'Annélides. C. R. Séances Soc. Biol. 2 (77): 527-532.
Caullery M. and Mesnil F. 1919. Metchniko-vellidae et autres protistes parasites des Grégarines d'Annélides. Ann. Inst. Pasteur. 33 (4): 209-240.
Dogiel V.A. 1922. Sur un nouveau genre de Metchnikovellidae. Ann. Inst. Pasteur. 36: 574-577.
Edgar R.C. 2004. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 32: 1792-1797. https://doi.org/ 1093/nar/gkh340
Frolova E.V., Paskerova G.G., Smirnov A.V. and Nassonova E.S. 2021. Molecular phylogeny and new light microscopic data of Metchnikovella spiralis (Microsporidia: Metchnikovellidae), a hyperparasite of eugregarine Polyrhabdina sp. from the polychaete Pygospio elegans. Parasitology. 148 (7): 779-786. https://doi.org/10.1017/S0031182021000603
Frolova E.V., Paskerova G.G., Smirnov A.V., Nassonova E.S. 2022. Metchnikovella dobrovolskiji sp. nov. (Microsporidia: Metchnikovellida), a parasite of archigregarines Selenidium pygospionis from the polychaete Pygospio elegans. Protistology. 16 (3): 226-235. https://doi.org/10.21685/1680-0826-2022-16-3-7
Frolova E.V., Paskerova G.G., Smirnov A.V. and Nassonova E.S. 2023. Diversity, distribution, and development ofhyperparasitic microsporidia in
gregarines within one super-host. Microorganisms. 11 (1): 152. https://doi.org/10.3390/microorga-nisms11010152
Galindo L. J, Torruella G., Moreira D., Timpano H. et al. 2018. Evolutionary genomics of Metchnikovella incurvata (Metchnikovellidae): an early branching microsporidium. Genome Biol. Evol. 10: 2736-2748. https://doi.org/10.1093/gbe/evy205
Gouy M., Guindon S. and Gascuel O. 2010. SeaView version 4: a multiplatform graphical user interface for sequence alignment and phylogenetic tree building. Mol. Biol. Evol. 27: 221-224. https:// doi.org/10.1093/molbev/msp259
Hoang D.T., Chernomor O., von Haeseler A., Minh B.Q. and Vinh L.S. 2018. UFBoot2: Improving the ultrafast bootstrap approximation. Mol. Biol. Evol. 35: 518-522. https://doi.org/10.1093/ molbev/msx281
Issi I.V. and Voronin V.N. 2007. Phylum Microsporidia Balbiani 1882. In: Protista: handbook on zoology. Pt 2. (Eds: Frolov A.O. and Krylov M.V.). Nauka, St. Petersburg, pp. 994-1045 (in Russian with English summary).
Katoh K., Standley D.M. 2013. MAFFT multiple sequence alignment software version 7: Improvements in performance and usability. Mol. Biol. Evol. 30: 772-780. https://doi.org/10.1093/ molbev/mst010
Larsson J.I.R. 2014. The primitive Microsporidia. In: Microsporidia: Pathogens ofopportunity. 1st ed. (Eds: Weiss L.M. and Becnel J.J.). John Wiley and Sons, Inc., Ames, Iowa, pp. 605-634. https:// doi.org/10.1002/9781118395264.ch24
Manni M., Berkeley M. R., Seppey M., Sim-âo F. A. et al. 2021. BUSCO update: novel and streamlined workflows along with broader and deeper phylogenetic coverage for scoring of euka-ryotic, prokaryotic, and viral genomes. Mol. Biol. Evol. 38 (10): 4647-4654. https://doi.org/ 10.1093/ molbev/msab199
McGowan J. 2019. BUSCO phylogenomics utility script. https://github.com/jamiemcg/BUS-CO_phylogenomics
Mikhailov K.V., Simdyanov T.G. and Aleo-shin V.V. 2017. Genomic survey of a hyperpara-sitic microsporidian Amphiamblys sp. (Metchnikovellidae). Genome Biol. Evol. 9 (3): 454-467. https://doi.org/10.1093/gbe/evw235
Mikhailov K.V., Nassonova E.S., Shishkin Y.A., Paskerova G.G. et al. 2022. Ribosomal RNA of the metchnikovellids in gregarine trans-criptomes and rDNA of the microsporidia sensu
lato in environmental metagenomes. Biol. Bull. Rev. 12 (3): 213-239. https://doi.org/10.1134/ S2079086422030069
Miller M. A., Pfeiffer W. and Schwartz T. 2010. Creating the CIPRES Science Gateway for inference of large phylogenetic trees. In: 2010 Gateway Computing Environments Workshop (GCE). Presented at the 2010 Gateway Computing Environments Workshop (GCE), IEEE, New Orleans, LA, USA, pp. 1-8. https://doi.org/10.1109/ GCE.2010.5676129
Nassonova E.S., Bondarenko N.I., Paskerova G.G., Kovâcikovâ M. et al. 2021. Evolutionary relationships of Metchnikovella dogieli Paskerova et al., 2016 (Microsporidia: Metchnikovellidae) revealed by multigene phylogenetic analysis. Para-sitol. Res. 120 (2): 525-534. https://doi.org/10. 1007/s00436-020-06976-x
Nguyen L.-T., Schmidt H.A., von Haeseler A. and Minh B.Q. 2015. IQ-TREE: A fast and effective stochastic algorithm for estimating maximum likelihood phylogenies. Mol. Biol. Evol. 32: 268-274. https://doi.org/10.1093/molbev/msu300
Nurk S., Bankevich A., Antipov D., Gurevich A.A. et al. 2013. Assembling single-cell genomes and mini-metagenomes from chimeric MDA products. J. Comput. Biol. 20 (10): 714-737. https://doi.org/ 10.1089/cmb.2013.0084
Paskerova G.G., Frolova E.V., Kovâcikovâ M., Panfilkina T.S. et al. 2016. Metchnikovella dogieli sp. n. (Microsporidia: Metchnikovellida), a parasite of archigregarines Selenidium sp. from polychaetes Pygospio elegans. Protistology. 10 (4): 148-157. https://doi.org/10.21685/1680-0826-2016-10-4-4 Ronquist F., Teslenko M., van der Mark P., Ayres D. L. et al. 2012. MrBayes 3.2: efficient Baye-sian phylogenetic inference and model choice across a large model space. Syst. Biol. 61: 539-542. https:// doi.org/10.1093/sysbio/sys029
Schrével J. and Desportes I. 2013. Biology of gregarines and their host-parasite interactions. Ch.
2. In: Treatise on zoology-anatomy, taxonomy, biology. The gregarines (Eds: Desportes I. and Schrével J.). Brill, Leiden, Boston, pp 25-195. https://doi.org/10.1163/9789004256057_004
Sokolova Y.Y., Paskerova G.G., Rotari Y.M., Nassonova E.S. and Smirnov A.V. 2013. Fine structure of Metchnikovella incurvata Caullery and Mesnil, 1914 (Microsporidia), a hyperparasite of gregarines Polyrhabdina sp. from the polychaete Pygospio elegans. Parasitology. 140: 855-867. https://doi.org/10.1017/S0031182013000036
Sokolova Y.Y., Paskerova G.G., Rotari Y.M., Nassonova E. S. and Smirnov A. V. 2014. Description of Metchnikovella spiralis sp. n. (Microsporidia: Metchnikovellidae), with notes on the ultrastructure ofmetchnikovellids. Parasitology. 141: 1108-1122. https://doi.org/10.1017/S0031182014000420
Sprague V. 1977. Classification and phylogeny. In: Comparative Pathobiology, Vol. 2. Systematics of the Microsporidia (Eds.: Bulla L. A. and Cheng T. C.), Plenum Press, New York, USA, pp. 1-30.
Sprague V., Becnel J.J. and Hazard E.I. 1992. Taxonomy of phylum Microspora. Crit. Rev. Microbiol. 18 (5-6): 285-395. https://doi.org/10.3109/ 10408419209113519
Vivier E. 1975. The microsporidia of the protozoa. Protistologica. 9 (3): 345-361.
Vivier E. and Schrével J. 1973. Étude en mic-roscopie photonique et électronique de différents stades du cycle de Metchnikovella hovassei et observations sur la position systématique des Metch-nikovellidae. Protistologica. 9: 95-118.
Weiss L.M. and Vossbrinck C.R. 1999 Molecular biology, molecular phylogeny, and molecular diagnostic approaches to the Microsporidia. In: The Microsporidia and microsporidiosis (Eds: Wittner M. and Weiss L.). Amer. Soc. Microbiol., Washington D.C., pp 129-171. https://doi.org/10.1128/ 9781555818227.ch4
