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Micron 77 (2015) 25–31

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Micron

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igh-pressure freezing and freeze-substitution fixation reveal theltrastructure of immature and mature spermatozoa of thelant-parasitic nematode Trichodorus similis (Nematoda;riplonchida; Trichodoridae)

ehnam Lak a, Vladimir V. Yushin b,c, Dieter Slos a, Myriam Claeys a, Wilfrida Decraemer a,d

Wim Bert a,∗

Nematology Research Unit, Department of Biology, Ghent University, K.L. Ledeganckstraat 35, 9000 Ghent, BelgiumA.V. Zhirmunsky Institute of Marine Biology, FEB RAS, Vladivostok 690041, RussiaFar East Federal University, Vladivostok, RussiaRoyal Belgian Institute of Natural Sciences, Department of Invertebrates, Vautierstraat 29, 1000 Brussels, Belgium

r t i c l e i n f o

rticle history:eceived 10 March 2015eceived in revised form 18 May 2015ccepted 19 May 2015

eywords:ryo-fixationibrous bodiesilopodiaembranous organellesuclear envelopeseudopod

a b s t r a c t

The spermatozoa from testis and spermatheca of the plant-parasitic nematode Trichodorus similisSeinhorst, 1963 (Nematoda; Triplonchida; Trichodoridae) were studied with transmission electronmicroscopy (TEM), being the first study on spermatogenesis of a representative of the order Triplonchidaand important to unravel nematode sperm evolution. Comprehensive results could only be obtainedusing high-pressure freezing (HPF) and freeze-substitution instead of chemical fixation, demonstratingthe importance of cryo-fixation for nematode ultrastructural research. The spermatozoa from the testis(immature spermatozoa) are unpolarized cells covered by numerous filopodia. They contain a centrally-located nucleus without a nuclear envelope, surrounded by mitochondria. Specific fibrous bodies (FB)as long parallel bundles of filaments occupy the peripheral cytoplasm. No structures resembling mem-branous organelles (MO), as found in the sperm of many other nematodes, were observed in immaturespermatozoa of T. similis. The spermatozoa from the uterus (mature or activated spermatozoa) are bipolarcells with an anterior pseudopod and posterior main cell body (MCB), which include a nucleus, mitochon-dria and MO appearing as large vesicles with finger-like invaginations of the outer cell membrane, or as

large vesicles connected to the inner cell membrane. The peripheral MO open to the exterior via pores. Inthe mature sperm, neither FBs nor filopodia were observed. An important feature of T. similis spermato-zoa is the late formation of MO; they first appear in mature spermatozoa. This pattern of MO formation isknown for several other orders of the nematode class Enoplea: Enoplida, Mermithida, Dioctophymatida,Trichinellida but has never been observed in the class Chromadorea.

© 2015 Elsevier Ltd. All rights reserved.

. Introduction

Data on sperm structure and spermatogenesis of Metazoare known to be informative to elucidate phylogenetic relations

Baccetti, 1985; Jamieson et al., 1995). Spermatozoon morphologynd development have also been used for taxonomic and phylo-enetic analyses of nematodes (Baccetti et al., 1983; Justine and

∗ Corresponding author. Tel.: +32 92645223.E-mail address: wim.bert@UGent.be (W. Bert).

ttp://dx.doi.org/10.1016/j.micron.2015.05.012968-4328/© 2015 Elsevier Ltd. All rights reserved.

Jamieson, 1999; Justine, 2002; Yushin and Malakhov, 2004). Nema-tode spermatozoa represent an aberrant type of male gamete,in that they are characterized by the absence of an axonemeand an acrosome and their possession of unique organelles suchas membranous organelles (MO) and fibrous bodies (FB) (Foor,1983; Bird and Bird, 1991; Justine and Jamieson, 1999; Justine,2002; Yushin and Malakhov, 2004, 2014). Most of the avail-

able information on the structure and development of nematodesperm was obtained from representatives of the extensive orderRhabditida (sensu De Ley and Blaxter, 2002). Most rhabditidspossess a relatively uniform sperm defined as the rhabditid pat-

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ern, which is usually considered as a reference for all nematodesYushin et al., 2011; Yushin and Malakhov, 2014). This typef nematode spermatozoon represents an amoeboid bipolar cellith an anterior pseudopod and a posterior main cell body with

condensed nucleus that lacks a nuclear envelope, mitochondria,O and FB (Baccetti et al., 1983; Justine and Jamieson, 1999; Justine,

002). MOs are unique aberrant organelles, characteristic of devel-ping and mature sperm of many nematodes studied, which appears large (0.5–1.0 �m diameter) vesicles with dense content andnternal finger-like projections of the outer cell membrane (Justine,002). The MOs are derived from Golgi bodies and appear as aart of MO–FB complexes. The prism-shaped paracrystalline FBsre composed of densely packed parallel filaments consisting ofhe unique cytoskeleton protein msp (major sperm protein) (Bakert al., 1998; Justine and Jamieson, 1999; Justine, 2002; Smith, 2006).he FB–MO complexes dissociate during the late stages of sper-atogenesis into separate FB and MO. After sperm activation inside

he female gonoducts, MOs join to the plasmalemma of the spermain cell body and release their content into the uterus lumen,

nd the empty MOs resemble membranous sacs being continuousith the sperm plasmalemma. Sperm activation is also accompa-

ied by the transformation of FBs into the msp-based cytoskeletonf a newly formed pseudopod (Justine and Jamieson, 1999; Justine,002; Smith, 2006). The pseudopod and amoeboid movement areypical characteristics of nematode sperm.

The subclass Enoplia, the putative most early diverging cladeithin the nematodes, shows an unusual diversity of structure and

atterns of male gamete formation (Baccetti et al., 1983; Justinend Jamieson, 1999; Yushin and Malakhov, 2014). Within this sub-lass, spermatogenesis of the order Enoplida has been relativelyell studied (Noury-Sraïri, 1994; Yushin and Malakhov, 1994, 1998,

999; Yushin et al., 2002; Yushin, 2003; Afanasiev-Grigoriev andushin, 2009). The nucleus of enoplid (order Enoplida) sperma-ozoon has a distinct nuclear envelope, while spermatozoa fromther nematodes outside the Enoplida always lack such an envelopeJustine, 2002; Yushin and Malakhov, 2014). Possessing a nuclearnvelope is considered a primitive feature (Baccetti et al., 1983;ushin and Malakhov, 1994, 2014) and in this aspect Enoplidaesembles other animals. These morphological data were taken intoccount, together with insufficient supported molecular phyloge-etic data, to suggest that Enoplia is indeed the most early diverginglade within the phylum Nematoda.

However, no information is available on sperm ultrastructure ofhe other order within the Enoplia, the order Triplonchida. Studiesn sperm morphology of this group are limited to light microscopicbservations. Even the most important triplonchids, the plant-arasitic Trichodoridae, have been very well studied by Decraemer1988, 1995) but the sperm has never been studied at ultrastruc-ural level. However, a first study of sperm ultrastructure of aepresentative of Triplonchida is crucial to complete the picture onnoplia spermatogenesis, indispensable for unraveling the intrica-ies of nematode sperm evolution and phylogenetic relationshipsithin the phylum Nematoda.

Importantly, our preliminary observations showed that tra-itional TEM fixation methods are insufficient to study theltrastructure of trichodorids, which is most likely the main rea-on explaining the absence of ultrastructural information for spermithin the order Triplonchida. An alternative to chemical fixa-

ion is cryo-fixation, i.e., physical preservation. Cryo-fixation haseveral distinct advantages over chemical fixation: a much fasterate of fixation, a simultaneous stabilization of all cellular compo-ents, superior preservation of fine structure (e.g., sharp membrane

elineation and clear visibility of cytoskeletal elements) and an

mproved preservation of antigenicity (Claeys et al., 2004). Thesedvantages are especially relevant for tissues that are difficulto preserve by conventional chemical fixation at ambient tem-

7 (2015) 25–31

peratures, such as nematode tissues. Especially, high-pressurefreezing and freeze-substitution fixation has been proposed asa very promising fixation technique for nematodes (Mcdonaldand Müller-Reichert, 2002; Claeys et al., 2004; Weimer, 2006;Mcdonald et al., 2007, Müller-Reichert et al., 2008; Fischer et al.,2014).

As a result of the new approach, in this paper we present:(1) the first transmission electron microscopy observations onsperm structure in males and females of the trichodorid nematodeTrichodorus similis, which allow to elucidate new aspects of nema-tode sperm morphology and development; (2) cryo-fixation as aprominent fixation method for nematode ultrastructural researchcompared to classical TEM methods.

2. Materials and methods

2.1. Sampling and extraction

Soil samples were taken with an auger from a horse pasture inBerendrecht, Antwerp, Belgium. After extraction with a modifiedBaermann tray, trichodorids were carefully picked out and femalesand males placed in separate embryo dishes with distilled waterusing a stereomicroscope (Leica, Austria). All specimens used forthis study were identified as T. similis Seinhorst, 1963.

2.2. Chemical fixation

In preliminary experiments, three species of the genus Tri-chodorus (T. variopapillatus Hooper, 1972, T. sparsus Szczygiel, 1968and T. similis) were processed by chemical methods. T. variopapil-latus and T. sparsus were fixed according to Yushin (2010) but with1% instead of 2% osmium tetroxide. T. similis was fixed according toSlos et al. (2015), but post-fixation took place in 2% osmium tetrox-ide overnight instead of for only 2 h, and dehydration was tried outwith and without following propylene oxide series.

2.3. High-pressure freezing (HPF) and freeze-substitution

Males and females were processed separately. Copper mem-brane carriers with a depth of 0.2 mm (Leica, Microsystems,Vienna, Austria) were treated and covered with egg lecithin(20 mg/ml). Each carrier was filled with 20% Bovine Serum Albu-min (BSA). 1–2 nematodes were transferred to the membranecarrier and immersed in BSA. The specimens were extremelyrapidly frozen (vitrification) with liquid nitrogen at a pressure of2000 bar (EMPact; Leica, Microsystems, Vienna, Austria). Freeze-substitution was carried out in the automated Leica AFS (Leica,Microsystems, Vienna, Austria). The carriers were transferred fromEMPact to AFS under liquid nitrogen and each carrier was placed inan eppendorf filled with substitution fluid. Over a period of fivedays, nematodes were freeze-substituted as follows: −90 ◦C for27 h, 2 ◦C increase per hour for 15 h, −60 ◦C for 12 h, 2 ◦C increase perhour for 15 h, −30 ◦C for 32 h, and finally 2 ◦C increase per hour for17 h (Claeys et al., 2004). At 4 ◦C the substitution fluid was washedaway with dry acetone for at least 8 h.

2.4. Embedding, sectioning, staining and transmission electronmicroscopy (TEM)

The specimens were gradually impregnated at room tempera-ture in Spurr’s resin (EMS, Hatfield, UK), a low viscosity embeddingmedium. Each specimen was put at the bottom of a plastic cap-

sule (Leica Microsystems, Vienna, Austria), this capsule was placedin a gelatin capsule (Leica Microsystems, Vienna, Austria), filledwith Spurr resin, and placed for 8 h at 70 ◦C for polymerization.The block face was trimmed close to the nematode specimen with

B. Lak et al. / Micron 77 (2015) 25–31 27

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Fig. 2. TEM of Trichodorus similis, immature spermatozoon at higher magnification.(Abbreviations: am = amorphous bodies, FB = fibrous bodies, mc = mitochondria,N = nucleus, scale bar = 1 �m).

ig. 1. TEM of Trichodorus similis, immature spermatozoa in testis (abbrevia-ions: am = amorphous bodies, FB = fibrous bodies, Fp = filopodia, mc = mitochondria,

= nucleus, scale bar = 1 �m).

Leica EM trim (Leica, Vienna, Austria) and semithin sections50 �m thickness) were cut with a Leica Ultracut E ultramicro-ome (Reichert–Jung Inc., Buffalo, NY, USA) using a diamond knifeDiatome Ltd., Biel, Switzerland) to locate the level of reproduc-ive system. Each four to five sections were placed in a drop ofistilled water on a glass slide on a heater plate (Schott Ceran,EL, Belgium) at 60 ◦C. After water evaporation, one drop of Tolu-

dine Blue staining was added to the sections, the glass slide wasinsed with distilled water to remove excessive dye and the nema-ode was located using light microscopy. Ultrathin sections (80 nmhickness) were cut with a diamond knife using a Leica Ultracut

ultramicrotome (Reichert–Jung Inc., Buffalo, NY, USA). Sectionsere collected on formvar-coated copper single slot grids (Agar

cientific, Stansted, UK) and stained with uranyl acetate and leaditrate using a Leica EM AC20 automatic contrasting instrumentLeica, Microsystems, Wetzlar, Germany).

Further analyses of the sections were done using a JEOL JEM010 (JEOL, Ltd., Tokyo, Japan) TEM operating at 60 kV and picturesere digitized using the Ditabis system (Pforzheim, Germany).

. Results

.1. Immature spermatozoa

The male reproductive system in T. similis is monorchic, with single outstretched testis. The germinal zone is relatively short,nd followed by a longer growth zone where spermatogoniaield spermatocytes by mitosis; these spermatocytes divide mei-tically into two secondary spermatocytes and each secondarypermatocyte then completes meiosis as it divides into two sper-atids. The immature sperm cells are stored in the seminal vesicle.uring the immature spermatozoon development, the cytoplasm

ransformation is relatively significant. Immature spermatozoa arelongated cells 8–10 �m in size with a central nucleus containingighly condensed chromatin particles without nuclear envelope

Figs. 1 and 2). The nucleus is surrounded by several mitochondriand large amorphous globules embedded into filamentous matrixFigs. 1 and 2). Long parallel bundles of filaments occupying periph-ral cytoplasm may be referred to as FBs. The peripheral cytoplasm

Fig. 3. TEM of Trichodorus similis, mature spermatozoa in spermatheca. Threeneighboring mature spermatozoa (abbreviations: N = nucleus, MO = membranousorganelle, mc = mitochondria, ps = pseudopod, scale bar = 1 �m).

comprises characteristic filamentous material (Fig. 2) which alsofills pseudopodium-like protrusions and filopodia covering sper-matozoa (Fig. 1). No structures reminding MOs were observed inthe immature spermatozoa.

3.2. Mature spermatozoa

The female spermatheca comprises mature (activated) spermcells, about 10–12 �m in diameter, with amoeboid outlines and adistinct polarization into anterior pseudopod devoid of organellesand the posterior main cell body comprises a nucleus and organelles

(Figs. 3 and 4A). The centrally located nucleus is a highly con-densed oval-shaped mass of chromatin without nuclear envelope(Figs. 3 and 4A and B). The nucleus is surrounded by sphericmitochondria with poorly developed cristae and pale matrix con-

28 B. Lak et al. / Micron 77 (2015) 25–31

Fig. 4. TEM of Trichodorus similis, mature spermatozoon. (A) General view of mature spermatozoon. The mature spermatozoon has a clear polarization; an anterior pseudopoddevoid of organelles and a posterior main cell body with nucleus, MOs and mitochondria. (B) Nucleus surrounded by mitochondria. (C) Membranous organelles in the cytoplasmof a mature sperm. (Abbreviations: N = nucleus, MO = membranous organelle, mc = mitochondria, po = pore of membranous organelle, ps = pseudopod, scale bars = 1 �m).

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aining dense particles (Fig. 4B). Distinctly for mature spermatozoa,he cytoplasm comprises MOs which appear as large vesicles,.5–1.5 �m in diameter, with a content of moderate density pen-trated by finger-like invaginations of the outer cell membraneFig. 4A and C). The peripheral MOs are usually smaller and open tohe exterior via pores (Fig. 4C). FBs and filopodia were not observedn the mature sperm. A schematic representation of an immaturend a mature spermatozoon can be found in Fig. 5.

. Discussion

.1. HPF, a promising fixation method for nematodeltrastructural research

Traditional chemical methods for TEM have proven to be suc-essful to study nematode spermatozoa (Baccetti et al., 1983;

right, 1991; Justine and Jamieson, 1999), including several soil

nd plant-parasitic nematodes (Justine, 2002; Yushin et al., 2011;ushin and Ryss, 2011; Zograf, 2014; Slos et al., 2015). However,lthough similar methods have been carried out for three Tri-hodorus species (T. variopapillatus, T. sparsus and T. similis), this

resulted in uniform unsatisfying results making further studiesimpossible (Fig. 6). Hence, this bad fixation is most likely notbecause of the used methods but because of the characteristics ofthe genus Trichodorus, i.e. most likely features of the cuticle. Tri-chodorids are known for their fixation difficulties, both for lightmicroscopy and for TEM research; their body cuticle tends to swellupon fixation and fixative penetration into the nematode body ispoor (Decraemer, 1995).

In a classical approach, the starting material is chemically fixedat room temperature, usually using aldehydes followed by osmiumtetroxide, and dehydrated with organic solvents prior to infiltrationand embedding in epoxy resin. Because this method depends on thediffusion of fixative into the cell, difficulties include slow infiltrationof fixative and extraction of cellular contents. Indeed, compared tocryo-fixation, conventional methods resulted in more obvious dis-tortions of cell structure due to time required for fixative reactionand dehydration step (Fig. 6).

In contrast to chemical fixation, significant advances in sam-ple preparation have been achieved by rapid freezing of cells,tissue samples, or small organisms under high pressure (Gilkeyand Staehelin, 1986; Dahl and Staehelin, 1989; Mcdonald, 1999;

B. Lak et al. / Micron 77 (2015) 25–31 29

Fig. 5. Trichodorus similis, schematic representation of (A) immature spermatozoon and (B) mature spermatozoon. (Abbreviations: am = amorphous bodies, FB = fibrous body,F re of m

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cdonald et al., 2007). The process of vitrification in HPF causesuperior tissue preservation (Mcdonald, 1999, Claeys et al., 2004;

eimer, 2006; Mcdonald et al., 2007; Müller-Reichert et al., 2008;ischer et al., 2014). Generally, the overall appearance of the spermells in HPF-fixed of T. similis specimens show much more details,or example of FBs, MOs and mitochondria. Our study thus confirmshe value of the HPF fixation technique for ultrastructural researchf nematodes (Claeys et al., 2004; Weimer, 2006; Müller-Reichertt al., 2008).

.2. Ultrastructural characteristics of the trichodoridpermatozoon

The main ultrastructural features of spermatozoon in T. similisre: (1) absence of a nuclear envelope; (2) mitochondria arranged as

cluster around the nucleus; (3) FBs in immature sperm; (4) filopo-ia covering immature sperm; (5) late MO formation, appearingnly in mature sperm; (6) bipolarity of mature sperm comprising

main cell body and a pseudopod.The presence of a nuclear envelope in mature nematode sperma-

ozoa of taxa belonging to the order Enoplida and its reconstitutionfter the last meiotic division have been confirmed many timesJustine and Jamieson, 1999; Justine, 2002; Yushin and Malakhov,014), while other nematodes outside Enoplida are known to lackuch an envelope. We have unequivocally demonstrated in thistudy that the presence of a nuclear envelope is not an apomorphicharacter state for the Enoplia. Although, sister to the Enoplida,riplonchida resemble other nematodes with a reduction of auclear envelope after the last meiotic division. This result sug-ests the more advanced nature of sperm cells in T. similis and therder Triplonchida.

The aberrant organelles (MO and FB) are considered as onef the remarkable and unique characteristics of nematode sper-atozoa (Justine and Jamieson, 1999; Justine, 2002). In T. similis

mmature sperm, the bundles of filaments occupying the periph-ral cytoplasm may be interpreted as FBs. In the mature sperm theundles disappear and possibly transform into the cytoskeleton ofhe pseudopod as typical for FBs of nematode sperm (Justine, 2002).

embranous organelle, ps = pseudopod).

In T. similis, the MOs appear first in the mature spermatozoaand show no association with FBs. The separate and asynchronousformation of MO and FB is a specific feature of the class Eno-plea (Yushin and Malakhov, 2004, 2014). The MO–FB complexesdeveloping in spermatocytes and spermatids were described asan essential cytoplasmic component during spermatogenesis ofspecies belonging exclusively to the class Chromadorea.

4.3. MO origin in Enoplea and Chromadorea – time and source offormation

In T. similis, MOs are absent in immature sperm, when thenewly formed spermatozoon has no organelles of synthesis (roughendoplasmic reticulum and Golgi bodies). MOs only appear in themature spermatozoa after sperm activation in female gonoducts.This implies that precursors (source) of membranous organelleswhich were found in mature sperm cannot be homologous toMOs in rhabditid nematodes, where MOs are successive stage ofGolgi apparatus development (Justine, 2002; Yushin and Malakhov,2014). MOs in T. similis sperm must have an alternative origin, e.g.,invagination of outer cell membrane.

Formation of MOs without association with Golgi bodies is alsoknown for several other orders from the class Enoplea: Enoplida,Mermithida, Dioctophymatida, Trichinellida (Foor, 1970; Neill andWright, 1973; Foor, 1983; Poinar and Hess-Poinar, 1993; Takahashiet al., 1994; Yushin, 2003; Yushin and Malakhov, 2014). MOs orig-inating from Golgi bodies may be considered as an apomorphicfeature of the class Chromadorea, separating it from the class Eno-plea. These key-data on spermatogenesis have been plotted on earlybranches of the molecular-based nematode phylogeny (Holtermanet al., 2006) (Fig. 7). However, despite a whole battery of moleculartechniques, the current molecular evidence to support the pre-sented molecular-based tree is not strong (Bik et al., 2010); the deepsubdivision of the phylum Nematoda should be regarded as a ‘workin progress’ and new molecular approaches, such as a multi loci

approach, are required. However, also the presented morphologicaldata are essential for the development of a more definitive frame-work. Morphological data are an essential complementary sourceof information to weigh up alternative hypotheses, i.e., a combined

30 B. Lak et al. / Micron 77 (2015) 25–31

Fig. 6. Immature spermatozoa in testis of Trichodorus spp. fixed with the classical chemical fixation for TEM. (A) Overview of seminal vesicle in Trichodorus similis, longitudinalsection. (B) Immature spermatozoa of Trichodorus sparsus. (C) Immature spermatozoon of Trichodorus variopapillatus at higher magnification (Abbreviations: FB = fibrousbodies, Fp = filopodia, IS = immature spermatozoon, N = nucleus, VD = vas deferens, scale bars: A, B = 2 �m, C = 1 �m).

Fig. 7. Key-data on spermatogenesis plotted on early branches of the molecular-based nematode phylogeny (Holterman et al., 2006), based on the scheme by Yushin andMalakhov (2004). Note that not all characters are apomorphic on this phylogeny.

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Yushin, V.V., Ryss, A.Y., 2011. Sperm development and structure in Bursaphelenchusmucronatus (Nematoda: Aphelenchoidea: Aphelenchoididae). Nematology 13,395–407.

Zograf, J.K., 2014. The ultrastructural study of spermatogenesis of Panagrellusredivivus (Rhabditida: Panagrolaimidae). Russ. J. Nematol. 22, 39–48.

B. Lak et al. / Mi

pproach can provide an improved insight into the evolution ofematodes, at least by reciprocal illumination.

. Conclusions

This paper has focused on spermatogenesis of the nematode T.imilis. The following overall conclusions can be derived:

Our results show that cryo-fixation is a promising method fornematode fixation.In general, the spermatogenesis of T. similis is congruent to thegeneral sperm peculiarities of the Enoplea, with exception of theEnoplida.The absence of a nuclear envelope in the mature and immaturespermatozoon of T. similis illustrates a major difference betweenthe two orders of the Enoplia, i.e. between the order Triplonchidaand the order Enoplida.Asynchronous formation of MO and FB and late formation of MOin the class Enoplea, including Triplonchida (T. similis), suggest adifferent type of aberrant organelles formation in Enoplea and adistinct difference between the two nematode classes, Enopleaand Chromadorea.

cknowledgment

This work was supported by the Foundation for Scientificesearch Flanders grant FWOKAN2013001201. Dr. V. Yushinas supported in part by the grant of Russian Scientific Fund14-50-00034 (studies of Trichodorus sparsus and Trichodorusariopapillatus in the Far East Centre of Electron Microscopy, Vladi-ostok, Russia), and grants of RFBR #14-04-00334 and FEB RAS15-I-6-109.

We thank Patricia Coenen for the soil samples containing Tri-hodorus similis.

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