Analysis of major sperm proteins in two nematode species from two classes, Enoplus brevis (Enoplea, Enoplida) and Panagrellus redivivus (Chromadorea, Rhabditida), reveals similar localization, but less homology of protein sequences than expected for Nematoda phylum

Major sperm proteins (MSPs) are a nematode-specific system of cytoskeletal proteins required for amoeboid sperm movement. A number of MSP genes vary in different nematode species, but encoded protein sequences reveal high homology between these proteins. However, all studies of MSP localization and functions to date are based exclusively on the representatives of the order Rhabditida belonging to the nematode class Chromadorea, while MSP-driven sperm movement in Enoplea, another major clade of the phylum Nematoda is still unconfirmed. In this study, we documented the presence of MSPs in the enoplean nematode Enoplus brevis (Bastian, 1865) (Enoplida) and compared MSP localization in sperm of this species with that of the chromadorean nematode Panagrellus redivivus (Linnaeus, 1767) (Rhabditida). Then, we analyzed the putative MSP sequences of both species. Our results indicate that MSPs are presented in E. brevis spermatozoa and form filamentous structures after sperm activation, which may be considered as the evidence of their motor functions similar to those in the spermatozoa of chromadorean nematodes. We found that E. brevis MSPs show lower homology to known proteins of rhabditids whose species exhibit hyper-conservatism in MSP protein sequences. These results reflect the more distant evolutionary relationships between Enoplea and Chromadorea than exist within Rhabditida order. Our data reveal a need to reevaluate current views of MSP evolution within Nematoda.


Introduction
Nematodes are one of several animal groups whose spermatozoa are amoeboid cells devoid of flagella (Morrow, 2004). Haploid nematode sperm that accumulated in the seminal vesicles of males are non-motile and may be considered as immature spermatozoa because the final step of spermiogenesis proceeds only after insemination and activation in the female gonoduct, where spermatozoa are drastically transformed into pseudopod bearing motile cells termed as mature spermatozoa (Shepherd, 1981).
The amoeboid mature spermatozoa move by crawling, which resembles amoeboid motility in other eukaryotic cells including some aflagellate spermatozoa. Instead of actin, motility of nematode sperm is driven by a unique cytoskeletal system based on the nematode major sperm protein (MSP) (Roberts & Stewart, 2012), although many Vladimir V. Yushin and Konstantin V. Yakovlev contributed equally to this work proteins with MSP domain exist in plants, fungi, and other animals (Tarr & Scott, 2005). In yeasts and animals, MSPdomain proteins related to VAP protein family are integral membrane proteins, which participate in vast spectrum of intracellular processes including endoplasmic and vesicle transport (Lev et al., 2008). Motility-related functions for MSPs have been found only in nematode sperm. The molecular machinery of nematode sperm motility well-studied in Ascaris suum and Caenorhabditis elegans (Smith, 2014) is similar to actin-based amoeboid movement (Ryan et al., 2012). A basis of this movement is assembly-disassembly of MSP filaments in pseudopod, where MSP is accumulated after spermatozoon activation and forms long multi-filament fibers (King et al., 1994a). MSP filaments assemble preferentially at the leading edge of the sperm pseudopod and are organized into long, multi-filament fiber complexes. These filamentous arrays are linked to the pseudopod plasma membrane and extend back to the junction between the cell body and pseudopod. As sperm crawl forward, these complexes flow back toward the cell body due to filament assembly at the leading edge and disassembly at the rear of the pseudopod (King et al., 1994a;Sepsenwol et al., 1989). Both processes are tightly regulated by set of cytosolic, membrane, and MSP filament-associated proteins (Ellis & Stanfield, 2014;Roberts & Stewart, 2000;Singaravelu & Singson, 2011;Smith, 2014). Cytoskeletal MSPs conserved between nematode species consist of 126-127 amino acids and detected as small 14-17-kDa proteins (Höglund et al., 2008;King et al., 1992;Klass & Hirsh, 1981;Strube et al., 2009). In addition to their cytoskeletal function, MSP acts as an extracellular signaling protein in C. elegans; MSP released from spermatozoa in vesicles induces oocyte maturation and ovarian cell contraction (Kosinski et al., 2005;Miller et al., 2001). Like in vertebrates, oocyte maturation in nematodes is activated by MAPK signaling cascade (Sen & Caiazza, 2013). MSP has been shown to activate MAPK pathway in C. elegans (Miller et al., 2001(Miller et al., , 2003, while in parthenogenetic nematodes Acrobeloides nanus and Diploscapter coronatus, which have no MSP at protein level, MAPK cascade is activated by another unknown pathway (Heger et al., 2010). The lack of MSP signaling functions in parthenogenetic nematodes may be recent evolutionary acquisition, as these species have functional, but nonexpressed MSP genes in genomes (Heger et al., 2010), but its role for oocyte maturation remains unclear for other nematodes and may be specific for C. elegans and closely related nematodes.
The classification based on morphological and molecular data subdivides the phylum Nematoda into two classes Enoplea and Chromadorea ( Fig. 1) (De Ley & Blaxter, 2002). The latter comprises seven orders where the movement of spermatozoa has been observed and studied in detail in the most diverse order Rhabditida due to the free-living soil nematode C. elegans and several parasitic taxa including Ascaris. The MSPs as the nematode-specific cytoskeleton proteins or MSP-coding genes have also been found in all tested species of the order Rhabditida (Fig. 1). Within this order, MSP identity of amino acid sequences is over 80% between species (Kasimatis & Phillips, 2018;Scott et al., 1989). In representatives of other orders of Chromadorea, amoeboid movement of spermatozoa confirmed by development of prominent pseudopods filled with cytoskeleton fibers reminiscent of those in the pseudopods in rhabditids (Justine, 2002;. We assume that the amoeboid movement based on MSP fibers is characteristic for entire class Chromadorea. Another nematode class Enoplea includes two welldefined subclasses Dorylaimia and Enoplia (De Ley & Blaxter, 2002). In the representatives of Dorylaimia, amoeboid spermatozoa with pseudopods filled with cytoskeleton fibers have been described in many taxa (Justine, 2002;. This specific morphology of motile sperm is confirmed by direct observation of sperm movement in the dorylaimian Gastromermis sp. (order Mermithida) (Poinar & Hess-Poinar, 1993). However, genomic status of the dorylaimian Trichinella spiralis (Trichinellida) did not show the presence of presumptive amino acid sequences with great similarity to spermatozoan MSPs of rhabditids (Kasimatis & Phillips, 2018). So, which proteins driving the amoeboid movement of sperm in species of Dorylaimia remain undetermined.
The subclass Enoplia includes taxa in which characteristic pseudopod bearing spermatozoa have been described (Justine, 2002;Lak et al., 2015;. The spermatozoa of nematodes of the genus Enoplus (order Enoplida) are amoeboid cells moving by crawling; as in many other nematodes, they are subdivided into posterior main cell body and anterior pseudopod filled with cytoskeleton fibers (Yushin & Malakhov, 1994). Similar morphology and behavior of Enoplus sperm may point to the presence of similar motor proteins, like MSPs. Nevertheless, as in the case of Dorylaimia, it is still unclear whether MSPs underlie sperm crawling in Enoplia.
The goal of this study is to determine whether MSP is present in Enoplia and whether it functions as motility protein in this taxon. In this issue, we analyzed the presence of MSP and its localization in spermatozoa of the marine enoplean species Enoplus brevis (Bastian, 1865) (Enoplida) and compared results with MSP protein sequences and localization in the chromadorean nematode Panagrellus redivivus (Linnaeus, 1767) (Rhabditida), the species which has typical spermatozoan morphology (Zograf, 2014) and genome of which contains several MSP genes (Scott et al., 1989;Srinivasan et al. 2013). Our choice of this representative of Chromadorea as reference species is also based on the ability of P. redivivus mature spermatozoa for conjugation in female gonoduct with formation of characteristic sperm chains (Duggal, 1978;Zograf, 2014). These chains can be easily isolated from inseminated females for convenient observations of mature spermatozoa.
To estimate phylogenetic relationships of both P. redivivus and E. brevis MSPs, we then carried out phylogenetic analysis using MSP protein sequences of both taxa and available sequences in databases of several Rhabditida and Dorylaimia MSPs. By using anti-MSP antibodies, we revealed that MSPs in cases of E. brevis and P. redivivus are cytoskeletal proteins that undergo reorganization during maturation that led to formation of MSP fibers in mature sperm. Then, based on antigen sequence used for antibody generation, we identified mRNA encoding cytoskeletal MSP in E. brevis transcriptome. We conclude that E. brevis spermatozoa have MSP, and its amino acid sequence is similar to P. redivivus and C. elegans MSPs (75% and 72%, respectively). Together, our results demonstrate that sperm of nematode E. brevis have MSPs, but their amino acid sequences are less similar than between Rhabditida species. Thus, we conclude that within the phylum Nematoda, MSPs evolve more rapidly in Fig. 1 Phylogeny of nematodes and MSP-based sperm motility. Phylogenetic relationships within phylum Nematoda derived primarily from SSU rDNA sequence data are given according to De Ley and Blaxter (2002). Suborders of the order Rhabditida, in which representatives highly homologous MSPs are found at DNA, RNA, or protein levels, are marked by underlining. Taxa whose species used in this study are marked with asterisks. Orders Trefusiida, Isolaimida, Dioctophymatida, Muspiceida, Marimermithida, and Desmoscolecida are not shown in this tree their protein sequences than expected, but it does not lead to loss of their function in spermatozoan motility.

Materials and methods
Animals P. redivivus cultures were kept in oatmeal and bread-based medium with addition bakery yeasts. Adult males and females of E. brevis were obtained from sand collected in the intertidal zone at the White Sea Biological Station of Lomonosov Moscow State University (Kandalaksha Bay, White Sea).

Production of anti-MSP antibodies
Polyclonal anti-MSP antibodies were raised in rabbits against synthetic peptide IKTTNMKRLGVDPPCGVLD-PKE, which corresponds to part of the MSP domain of several Rhabditida species, like C. elegans and Onchocerca volvulus (GenBank accession numbers CCD73220.1 and AAA29421.1, respectively). For immunization, synthetized peptide was conjugated with keyhole limpet hemocyanin (KLH). Then antibodies were purified using antigenic peptide by affinity chromatography. Antibody production was carried out by the Cytokine company (St. Petersburg, Russia).

Transmission electron microscopy
E. brevis males were processed as described previously . Briefly, animals were fixed with 2.5% glutaraldehyde in 0.05 M cacodylate buffer containing 23 mg/ml NaCl and post-fixed with 2% osmium tetroxide in the same buffer containing 21 mg/ml NaCl. Then, samples were en bloc stained with 2% uranyl acetate for 2 h, dehydrated in ethanol series and isopropanol, and embedded in Spurr resin. Thin sections were stained with uranyl acetate and lead citrate and observed under JEM 100S (JEOL, Japan) and Libra 120 (Carl Zeiss, Germany) transmission electron microscopes.

Immunofluorescence and imaging
Immunostaining procedure was performed on sperm isolated by dissection from males and mated females. Adult P. redivivus were directly dissected on poly-l-lysine-coated slides in PBS and then incubated for 10 min for binding extracted sperm to slides. E. brevis were dissected on poly-l-lysinecoated slides in filtered sea water. All samples were fixed with 4% PFA in PBS for 30 min. Slides were rinsed with PBS, permeabilized 0.1% Triton X-100 for 20 min and then blocked with 1% normal goat serum and 1% BSA in washing buffer (0.01% Tween 20, PBS) for 2 h. Samples were incubated with anti-MSP antibodies (2.4 µg/ml for P. redivivus, 12 µg/ml for E. brevis) overnight at 4 °C. After washing slides were incubated with secondary goat anti-rabbit antibodies labeled with Alexa Fluor 488 and Alexa Fluor 546 (1:500) diluted in 0.1% BSA for 1 h. Nuclei were stained with DAPI (2 µg/ml) for 20 min. Slides were rinsed with PBS and mounted in Vectashield medium (Vector Laboratories, Burlingame, CA, USA). P. redivivus females were fixed and immunostained with anti-MSP-antibodies according to Ding and Candido (2000) with minor modifications. Blocking procedure was performed with 1% normal goat serum and 1% BSA for 3 h. Fluorescent images were taken using LSM 510 Meta and LSM 780 confocal microscopes (Carl Zeiss, Germany) and processed using ImageJ software (National Institutes of Health, USA).
Nucleotide sequences were aligned using MUSCLE (Edgar, 2004). Protein sequences were aligned using Prob-Cons (Do et al., 2005) and visualized in Jalview (Waterhouse et al., 2009). Phylogenetic tree was created using maximum likelihood method with WAG substitution model and branch support with SH-like aLRT (Shimodaira-Hasegawa-like approximate likelihood ratio test). Multiple alignments and phylogenetic tree construction were done in Phylogeny.fr resource (Dereeper et al., 2008).

Localization of MSP in sperm
Initially, we characterized MSP in P. redivivus because this species has conserved MSPs that can be detected by our anti-MSP antibodies. In P. redivivus samples, Western blot analysis using generated anti-MSP antibodies showed two bands with approximate weight of 15 and 16 kDa in both males and females, which corresponds to mobility of nematode MSPs (Fig. 2a). These two bands of signal may indicate phosphorylated and unphosphorylated forms of the protein, as MSP phosphorylation occur in C. elegans spermatozoa (Fraire-Zamora et al., 2011). Upper weak band (between 29 and 42 kDa) in female sample is likely to be complex of MSP with other protein, because the other secondary antibodies detected the same bands (data not presented). The presence of MSP in female samples confirms that they include mated females bearing deposited spermatozoa. Analysis of young animals showed MSP only in male samples (Fig. 2b). In young females, MSP was not detected, as they were predominantly unmated. Also, upper band (between 29 and 42 kDa), which detected in mated females, was absent.
As in other Rhabditida, immature and mature spermatozoa are well-distinguishable by morphology (Fig. 3a). Immature spermatozoa are morphologically unpolarized cells, while mature spermatozoa are polarized cells and conjugated with each other forming chains (Fig. 3b). These chains supported by tight contacts between spermatozoa were previously described in the uteri of P. redivivus females using transmission electron microscopy (Zograf, 2014) (Fig. S1a). Whole-mount immunostaining with anti-MSP   (2014) with the permission from copyright holder (Russian Journal of Nematology). b Chain of conjugated mature spermatozoa in female reproductive system. Abbreviations: N, nucleus; mt, mitochondria; mo, membranous organelles; ch, nuclear chromatin; ps, pseudopodium; mcb, mail cell body antibodies also showed chains of spermatozoa in spermathecae of mated females (Fig. S1b). Immunostaining of isolated spermatozoa revealed cytoplasmic localization of MSP. In immature spermatozoa, MSP localization is both diffuse and granular with highest signal on cell periphery (Fig. 4a). Mature spermatozoa extracted from mated females retained conjugation into chain (Fig. 4b). In mature spermatozoa, MSP localizes predominantly in well-defined pseudopodia, where MSP has granulo-fibrillar pattern (Fig. 4b).
In E. brevis, anti-MSP antibodies detected a band with approximate size of 36-38 kDa (Fig. 5a), which is higher than known for MSPs in Rhabditida. Subsequent peptide competition assay using peptide antigen showed significant reduction of the signal that confirms specificity of anti-MSP reactivity in the case of the E. brevis samples (Fig. 5b). The presence of MSP in female samples points to the presence of mature spermatozoa in female reproductive tract.
Morphology of E. brevis spermatozoa resembles in general those of rhabditids with some specific features. Immature E. brevis spermatozoa are polarized elongated cells. Polarization of immature spermatozoa is marked by unequal distribution of membranous organelles and fibrous bodies (Fig. 6a). This polarization retains after sperm activation, when membranous organelles dramatically changed their morphology, fibrous bodies dissolved, and anterior part transformed into pseudopodium (Fig. S2). Thus, spermatozoan polarization in E. brevis was clearly distinguishable by morphological observations before maturation, while intracellular perturbations appearing after maturation resemble those of rhabditids. Schematic representation of both MSP has punctate and fibrillar pattern of distribution in pseudopodia that marked by arrows. Selected area is given at higher magnification (scale bar 10 µm, magnified area 2 µm)

Fig. 5
Western blot analysis of MSP in E. brevis. a MSP has unusual mobility in gel and is found as protein with weight 36-38 kDa. Both male and female samples reveal MSP signal, because the latter include inseminated females. α-Tubulin was used as a loading control (approximate weight 55 kDa). b Peptide competition assay confirms reactivity of anti-MSP antibodies with protein band of 36-38 kDa immature and mature E. brevis spermatozoan morphology is shown in Fig. 6b.
Immunostaining of isolated spermatozoa revealed different localizations of MSP. In immature spermatozoa, MSP was detected as both diffusely distributed and in granules (Fig. 7a). Incubation of immature spermatozoa in the sea water for 5-10 min led to transformation of MSP toward more filamentous manner in peripheral cytoplasm (Fig. 7b). Mature spermatozoa extracted from females had MSP signal in periphery of anterior part of the cell, where pseudopodia is known to be formed after activation (Fig. 7c).

Analysis of MSP sequences and phylogeny
For reasons given that the peptide antigen sequence is identical or highly homological to MSPs of Rhabditida species, we decided to use its amino acid sequence to find MSP sequences of both P. redivivus and E. brevis that potentially recognized by the generated antibodies. P. redivivus putative protein sequences of MSPs are available and we used them in our analysis. Blast search using peptide antigen as a query in WormBase Parasite detected six most homologous putative MSPs of P. redivivus consisting of 127 amino acids (Fig. 8a). These proteins are highly homologous to each other with identity between them 94-99%. Alignment of the found P. redivivus MSPs with C. elegans one with accession number P53017 showed identity 87-90%. In our research, we used generated transcriptome of E. brevis from available SRA data (ERX616982). Blast search among putative proteins generated from the transcriptome with peptide antigen sequence as query detected three 124-amino acid proteins called MSP124-1, MSP124-2, and MSP124-3. Independent Pfam search among putative protein sequences generated from whole transcriptome attributed MSP124 proteins to the MSP family. The presence of MSP124 transcripts was validated by RT-PCR (Fig. S3). Primers and PCR conditions are shown in Table S1. Multiple alignment of their coding mRNAs showed that these transcripts are highly homologous in their coding regions and variable in untranslated regions (Fig. S4). Amino acid sequences of MSP124-1, MSP124-2, and MSP124-3 are almost identical to each other (98-99%) (Fig. 8b). Among proteins with MSP domain of E. brevis, MSP124 is a group, which most homologous to sperm MSPs of Rhabditida. ExPASy calculations showed that all MSP124 are basic proteins with predicted weight 13.7 kDa (https:// web. expasy. org/ compu te_ pi/) (Gasteiger et al., 2005). Predicted molecular weight of MSP124 proteins did not conform with results of Western blot, when molecular weight of E. brevis MSP was much higher than expected. Though, peptide Fig. 6 Morphology of E. brevis spermatozoa. a Transmission electron microscopy of immature spermatozoon from testis. This is polarized cell, and anterior part is mostly filled with fibrous bodies edged with layer of mitochondria, whereas posterior part is filled with fibrous bodies, membranous organelles, and few mitochondria (scale bar 2 µm). b Schematic representation of E. brevis spermatozoa based on transmission electron microscopy. Immature spermatozoon (left) from testis is polarized drop shaped cell with nucleus, mitochondria, fibrous bodies, and membranous organelles. Mature spermatozoon (right) from uterus is highly polarized cell with anterior pseudopodium and posterior main cell body with nucleus, mitochondria, and membranous organelles attached to the cell membrane and open to the exterior via pores. Mature spermatozoon reproduced from Yushin and Malakhov (1994) with the permission of copyright holder (Brill publisher). Abbreviations: N, nucleus; fb, fibrous body; mt, mitochondria; mo, membranous organelles; ps, pseudopodium competition assay showed specific binding of anti-MSP antibodies to this protein band (Fig. 5). Additionally, Blast analysis of transcriptome did not find putative proteins with molecular weight 36-38 kDa that have high homology with peptide antigen. So, we concluded that MSP124 proteins may have unusual mobility in SDS-PAGE conditions due to post-translation modifications or some unknown features of these proteins.
To evaluate phylogenetic relationships of P. redivivus and E. brevis MSPs, we analyzed these proteins by multiple alignment and created phylogenetic tree. In this alignment, we used one of six found P. redivivus MSPs (Pan_g9068.t1), all three MSP124 proteins of E. brevis, and available sequences of Rhabditida and Dorylaimia species. The latter were chosen as most homological proteins to C. elegans and P. redivivus (Trichuris trichiura, Trichinella nativa, T. pseudospiralis, and T. papuae) and subsequently selected by pl value (pl > 7) by ExPASy analysis, because MSPs found in rhabditids are basic proteins. Multiple alignment, which has been done in ProbCons, is given in Fig. 9a. MSP of P. redivivus related to that of Rhabditida with 88% identity to C. elegans one. E. brevis MSP124 proteins have highest homology with filarial nematodes Brugia malayi and Loa loa (57% identity and 77% similarity). Homology of MSP124 proteins is little bit lower with MSPs of C. elegans (52% identity and 72% similarity) and P. redivivus (55% identity and 75% similarity). Similarity between MSPs of Dorylaimia and Rhabditida taxa is 48-62%, and similarity between Dorylaimia taxa and E. brevis is 48-53%. Between Dorylaimia species there is high divergence in MSP sequences. Highly similar protein sequences can be found only within the same genus, for example, in Trichinella (Fig. 9a, T. pseudospiralis and T. papuae). Unlike Rhabditida, Fig. 7 Immunolocalization of MSP in E. brevis sperm. a Immature spermatozoon from male. MSP is diffusely distributed in cytoplasm and concentrated in large granules (scale bar 10 µm). b Spermatozoon recovered from male and partially activated by 10-min incubation in sea water. MSP undergoes transformation resulting in appearance of longitudinal fibrillar structures. Selected area is given at higher magnification (scale bar 10 µm, magnified area 2 µm). c Mature spermatozoon from female. Most of MSP signal is found in pseudopod seen using DIC optics (scale bar 10 µm) 1 3 different genera of Dorylaimia, as Trichuris and Trichinella, do not have great homology among submitted MSPs. A brief screening of available genomes of at least fourteen Dorylaimia species (WormBase Parasite) showed that these species have also relatively distant MSPs to both Rhabditida and E. brevis proteins (these sequences are not included in alignment). The presented alignment was subsequently used as input for generation of maximum likelihood phylogenetic tree (Fig. 9b). As expected, P. redivivus MSP is related to the Rhabditida proteins. The most interestingly that Rhabditida and E. brevis MSPs form sister groups with significant SH-aLRT branch support (0.85). Dorylaimia MSPs are located on different branches, and more careful phylogeny requires more MSP sequences of this group.

Discussion
Aflagellate spermatozoa appeared independently during evolution in different metazoan taxa many times. Nematoda and its sister group, Nematomorpha (horsehair worms), both produce aflagellate spermatozoa (Schmidt-Rhaesa, 1997/98). It is known that spermatozoa of nematodes locomote by amoeboid movement, while spermatozoan motility in horsehair worms has not been described to date. In the most cases, amoeboid motility is driven by actin polymerization or cortical actin-myosin contraction (Miyata et al., 2020). Numerous studies on Rhabditida, an order of the nematode class Chromadorea, showed an existence of unique MSP-based sperm locomotion. MSP protein sequences among Rhabditida, as previously noted, are highly conserved and many researchers conclude that it is the case for the whole phylum Nematoda (Höglund et al., 2008;Hojas & Post, 2000;Scott et al., 1989). Kasimatis and Phillips (2018) proposed that conservation of MSP sequences should be tightly evolutionary regulated, as nonsynonymous mutations lead to lack or incorrect MSP filament assembly (del Castillo-Olivares & Smith, 2008).
Nematodes can be found everywhere; they inhabit different ecological niches and comprise both free-living and parasitic taxa. In spite of the great ecological and taxonomical diversity of nematodes, MSP sequences are postulated to be highly conserved. This suggestion originates from the fact that all tested species, both free-living and parasitic, have highly similar MSP protein sequences. The exception may be some parthenogenetic nematodes which MSPs have not been found at protein level, though their genomes contain functional MSP genes (Heger et al., 2010). Nevertheless, a hypothesis of MSP conservatism is based only on studies of species belonging exclusively to one chromadorean order Rhabditida. Evidence that MSPs in another class of nematodes, Enoplea, are identical over 80% to those of Rhabditida, has not been published to date. Important questions originate from the well-known MSPbased locomotion of spermatozoa in rhabditids and the lack of direct evidence, whether Enoplea also use MSP machinery for sperm movement. Does the origin of the MSP-based locomotion correlate with appearance of amoeboid-moving sperm of nematodes? To answer to this question, it is necessary to ascertain, whether MSP-based sperm locomotion exists in species of the enoplean clades Enoplia and Dorylaimia. To search for MSPs in E. brevis, we applied a comparative approach using P. redivivus, the Rhabditida species, which sperm have MSP. This approach combines detecting MSPs by antibodies and subsequent searching for genome-or transcriptome-encoded MSP sequences using the antigenic peptide sequence as a query.
Firstly, we tested the chosen approach on P. redivivus, whose spermatozoa are typical for Rhabditida (Zograf, 2014). MSP localization in P. redivivus showed that in the round immature sperm, MSP was found throughout cytoplasm with maximal signal in the cell periphery. It is known that in spermatocytes and early spermatids of C. elegans, MSP localizes in fibrous bodies associated with membranous organelles (Chu & Shakes, 2013). In several rhabditids, like Acrobeles complexus, immature spermatozoa retain fibrous bodies as separate component of their cytoplasm (Yushin et al., 2016). In C. elegans and P. redivivus, ultrastructural studies did not show well-defined fibrous bodies in immature spermatozoa (Ward & Klass, 1982;Wolf et al., 1978;Zograf, 2014). The lack of fibrous bodies in immature spermatozoa of P. redivivus suggests that MSP granules are local supramolecular MSP-containing complexes, which are indistinguishable by TEM and appear after dissociation of fibrous bodies. In the mature amoeboid spermatozoa, MSPs are localized in the pseudopodia with granulo-fibrillar pattern. These changes of MSP localization before and after sperm activation are typical for C. elegans (Chu & Shakes, 2013) and correlate with appearance of MSP fibers required for sperm movement (Marcello et al., 2012). These data and identification of presumptive encoded MSP genes, which are highly homologous to those of other Rhabditida species, suggest that the P. redivivus spermatozoa use MSP-based movement.
Secondly, this approach allowed us to detect MSPs in the spermatozoa of E. brevis and identify three MSP-coding sequences in transcriptome that we called MSP124-1, 2, and 3. Despite the lack of functional analysis, we suggest that E. brevis MSPs are cell motility proteins. Confirmation in this viewpoint is the existence of typical fibrous bodies in immature spermatozoa of E. brevis as viewed by TEM (Fig. 6a), which indirectly point to the presence of MSP. Fibrous bodies are distributed in a gradient with an accumulation at the anterior end. By immunolabeling, MSP was not distributed in a gradient in cytoplasm of immature spermatozoa. This fact indicates the presence of MSP throughout cytoplasm. Rear MSP granules do not also correlate with distribution of fibrous bodies and may be unknown supramolecular complex, as in case of P. redivivus. Fibers and fibrous material visible by TEM (Fig. S2) and localization of MSP in pseudopods of mature Enoplus spermatozoa are additional evidences that MSP may underlay sperm movement of this animal, because these events are characteristic for sperm activation in rhabditids (LeClaire et al., 2003;Sepsenwol et al., 1989;Slos et al., 2020;Yushin et al., 2016). MSP assembly-disassembly depends on intracellular pH. Treatment spermatids of A. suum with weak acids or bases, which affect intracellular pH, led to disassembly or assembly of MSP filaments, respectively (King et al., 1994b). Our results revealed the assembly of MSP fibers in immature E. brevis spermatozoa after incubation in sea water, which is alkalescent (pH 8.1-8.3). Evidently, sea water only partially activates E. brevis spermatozoa, since MSP is not re-located to the anterior, where the pseudopod will form. This finding suggests that complete maturation requires additional factor, presumably supplied by the females. The observed partial activation of E. brevis spermatozoa suggests that MSP assembly-disassembly in both Enoplia and Rhabditida may be regulated by similar mechanisms by alteration in intracellular pH.
Our data show that the E. brevis MSP124 proteins are less homologous to MSPs of Rhabditida than homology of MSPs within this order. All three MSP124 proteins showed less similarity (identity and positive substitutions) to those of C. elegans (72%) and P. redivivus (75%), than has been reporting within Rhabditida (83.5-97.7% identity between  (Kasimatis & Phillips, 2018). Our results showed that MSPs of the representatives of all three subclasses, Enoplia, Dorylaimia, and Rhabditida, are moderately similar. The lack of highly homologous MSPs between different genera of Dorylaimia does not allow discussing MSP phylogeny in this group and requires detailed phylogenetic study using additional MSP samples from diverse taxa of this subclass. We think that our sampling within Dorylaimia should be expanded as current studies do not represent the full diversity of this clade. The fact that the similarity of MSP sequences between representatives of three nematode subclasses is ranged from 48 to 75% shows that MSPs in Nematoda are less conserved in their protein sequences than anticipated. MSP sequences have retained high identity during approximate 500 million-year evolution within the order Rhabditida (Blaxter, 2009) revealing protein sequence hyper-conservation (Kasimatis & Phillips, 2018). Nevertheless, MSP sequence hyper-conservation does not exist within the phylum Nematoda as a whole. This difference in MSPs variability between two major clades of nematodes correlates well with sperm morphological diversity which is very wide in Enoplea but relatively low in Chromadorea, especially in the order Rhabditida where sperm patterns are enormously uniform (Justine & Jamieson, 1999;Slos et al., 2020;. In summary, our study provides the first evidence that E. brevis spermatozoa use MSP-based locomotion and suggests that it may be the case for other species of Enoplia. Though, the early evolution of nematodes is still unresolved due to controversy in different phylogenetic analyses (Smythe et al., 2019), it is known that Enoplia is one of early-branching group, which reveals presumably ancestral features among nematodes (Bik et al., 2010;Blaxter & Koutsovoulos, 2015;Felix, 2004;Holterman et al., 2006;Joshi & Rothman, 2005;Malakhov, 1994Malakhov, , 1998Rusin & Malakhov, 1998;Schulze & Schierenberg, 2011;Smythe et al., 2019;van Megen et al., 2009;Voronov, 1999;Voronov & Panchin, 1998;Yushin & Malakhov, 2004). A more basal phylogenetic placement of Enoplia in relation to Chromadorea should contribute to our understanding of origin and evolution of nematode sperm motility based of MSP function. Moreover, availability of E. brevis MSP sequences may be useful for further functional MSP analysis to determine capacity to co-polymerize MSPs from phylogenetically distant nematodes. Thereby, interactions of divergent MSP monomers will be additional argument that cytoskeletal functions of MSPs in spermatozoa are general characteristic of phylum Nematoda.