Muscles of the male and female copulatory organs of Bursaphelenchus mucronatus and Chiloplacus sp. (Nematoda: Rhabditida)

Abstract Male and female copulatory organs figure prominently in nematode taxonomy, but the associated musculature remains insufficiently explored. The aim of this study was therefore to further our knowledge of the musculature of the vulva and male copulatory organs in nematodes by using phalloidin staining and confocal microscopy to examine two nematode species, Bursaphelenchus mucronatus and Chiloplacus sp. The musculature of the round vulva in Chiloplacus sp. comprises three pairs of radial vulval dilators and another pair of dilators of the anterior inner vulval plate. This arrangement is similar to that of the Rhabditida, but in Chiloplacus the anterior pair appears to have been transformed into the vulval plate muscles. The musculature of the slit-like vulva in B. mucronatus includes dilators of the vulval lips and external vulval flap, constrictors of the vulval slit and posterior transverse muscle bands. The opposing pairs of vulval dilators show quadrilateral symmetry as observed in the Rhabditida, but the constrictors running along the rim of the vulva have no counterparts in other species. The musculature of the male copulatory organ in Chiloplacus sp. comprises two pairs of spicule protractors and retractors and three pairs of gubernacular muscles. In B. mucronatus, as in the other Aphelenchoididae, the gubernaculum is absent and there is one pair of spicule protractors and two pairs of muscles inserted on the saddle (angular bend) of the spicules. The arrangement of the spicule saddle muscles resembles those of the gubernaculum, which may indicate that in this family the gubernaculum has become fused to the spicules. The literature review of muscles of nematode copulatory organs are given in a table for 15 muscle groups; it can be used for phylogenetic reconstruction and classification of the order Rhabditida.

B. cocophilus (Cobb, 1919) Baujard, 1989 and its vector, the weevil Rhynchophorus palmarum (Linnaeus, 1758), are the pathogenic associates causing the "red ring disease" of Cocos nucifera L., Elaeis guineensis Jacquin and other palms in the Caribbean region (Corbett, 1959;Goodey, 1960;Griffith and Koshy, 1990;Harrison and Jones, 2003;Silva et al., 2016). Data collected by the first author indicate that the elm nematode B. ulmophilus  plays a role of an opportunistic pathogen in dieback diseases of the elm Ulmus spp. in the parks of St. Petersburg (Russia). This elm wood nematode accelerates the progression of the Dutch Elm Disease  by facilitating the spread of the fungi along the tree trunk in sapwood (Polyanina et al., 2016;. The molecular phylogeny of Bursaphelenchus (Kanzaki et al., 2015;Ryss et al., 2015;Gu et al., 2016;Ryss and Subbotin, 2017) and morphology-based evidence are only partially congruent warranting the search for new phylogenetically informative characters. The most important diagnostic characters of the nematode species are the structures of the anterior and posterior body regions, lateral fields, and copulatory organs. These structures have been studied primarily by differential interference contrast (DIC) and SEM, but recently these methods have been increasingly supplemented by confocal microscopy, with its ability to generate accurate three-dimensional (3D) reconstructions from optical sections.
The confocal study of nematodes, however, has so far been focused only on a few taxonomic groups. The favorite object of research is the nematode Caenorhabditis elegans, a model animal in neuroscience and developmental biology (Lints andHall, 2009a, 2009b). Some confocal studies have been conducted on Meloidogyne spp. (Calderón-Urrea et al., 2016) and unstained collection specimens of aquatic nematodes have been studied by autofluorescence methods, among them the nematodes of the families Tobrilidae (Zullini and Villa, 2006) and Cyatholaimidae (Semprucci and Burattini, 2015). Beyond these examples, very little confocal research has been published.
This study was conducted to compare the muscular systems of copulatory organs in two phylogenetically distant species of nematodes using phalloidin staining in conjunction with confocal microscopy, with the objectives to evaluate the possibility of using the arrangement of copulatory muscles at the anatomical level to identify phylogenetically and taxonomically informative diagnostic characters that may distinguish the families within the order Rhabditida.
Two species of saproxylic nematodes were studied: the specialized fungal and plant feeder Bursaphelenchus mucronatus Mamiya and Enda, 1979 and the bacterial feeder Chiloplacus sp.; both are species occupying decaying wood and bark, but are clearly distant from each other in the phylogenetic tree of the order Rhabditida (Megen et al., 2009).
Bursaphelenchus mucronatus infects Pinus sylv estris L. using Monochamus spp. (Cerambycidae) vectors; it combines mycophagy with parasitic feeding on plant cells in the resin canals, while Chiloplacus are commensals of longhorn beetle larvae inhabiting the bark of pines and feeding on bacteria. A further imperative for this study was to study the morphological adaptations of nematodes to the different modes of mating.

Nematode samples and extraction
Two nematode species were studied: Bursaphelenchus mucronatus Mamiya and Enda, 1979 (European type) and Chiloplacus sp., both belonging to the class Secernentea: order Rhabditida according to the classification of De Ley and Blaxter (2002). The isolates of both species are maintained in the Live Nematode Collection at the Zoological Institute of the Russian Academy of Sciences, St. Petersburg.
Bursaphelenchus mucronatus (family Aphelenchoididae) is a fungal and plant feeder using Pinus sylvestris L. as a plant host. The nematodes were extracted from larval tunnels of the pine sawyer beetle Monochamus galloprovincialis (Olivier, 1795) in the wood of Pinus sylvestris (Tomsk region of Russia, Tomsk district, Timiryazevo settlement, forest farm "Timiryazevskoye", GPS: 56°27.597'N 084°51.157'E). Wood was collected on 19 June 2015.

Nematode cultivation
To obtain mass suspensions of nematode isolates, they were propagated on a standard culture of an asporogenous mutant strain of the fungus Botryotinia fuckeliana (de Bary) Whetzel, 1945 (the name of teleomorph stage) (syn. Botrytis cinerea Pers. 1794). The fungus was grown on sterile potato sucrose agar (PA) at 20-22°C. Five days after fungus inoculation, the agar surface was occupied by the mycelium lawn. A suspension of 50-100 nematode individuals was inoculated into fungus culture. In 10-14 days, the nematodes multiplied reaching abundance of 3,000-10,000 individuals at different life cycle stages and devastating the fungus lawn .
Chiloplacus sp. is not a fungal feeder and according to its stoma structure the genus can be characterized as a bacterial feeder (Yeates et al., 1993;Wilson and Khakouli-Duarte, 2009). However, these nematodes multiplied in the B. fuckeliana culture feeding both on fungus and unidentified ectosymbiotic bacteria, which were present on the nematode surface coat and multiplied rapidly in PA media; these bacteria serve as food for their nematode hosts. The nematodes were capable to multiply on the PA medium without fungus while feeding on their ectosymbiotic bacteria; however, in the PA medium with B. fuckeliana, the population growth was faster and more stable.
The arrangement of mating pairs could be easily seen at the spots where agar was sufficiently thin and on the lids of Petri dishes.

Fixation, staining and manufacture of collection slides
The nematodes were fixed with hot 4% formaldehyde in 0.01M PBS for 1 h and then transferred to 0.01 M PBS containing 0.05% (w/v) sodium azide for storage. Permanent preparations of nematodes were made using a modification of the Seinhorst method (Ryss, 2003(Ryss, , 2017. For confocal microscopy, the nematodes were permeabilized for 2-3 hr with 0.25% Triton X-100 in 0.01 M PBS (Tr-PBS) and incubated in 20 µg/ml proteinase K in 0.01 M PBS for 5 hr. Permeabilized animals were rinsed 3 × 15 min in Tr-PBS, stained for 5-10 hr in Alexa Fluor 555 phalloidin (1:150) (Thermo Fisher Scientific), rinsed in 0.01 M PBS for 15 min and mounted on slides with Vectashield (Vector Laboratories Inc., Burlingame, California, USA). The optimal time of proteinase incubation was determined experimentally: a 5-hr incubation provided sufficient permeabilization of the cuticle to stain the whole body of the worm, but was not long enough to digest filamentous actin in the muscles.
In addition, freeze-cracking technique (Duerr, 2013) was used as an alternative to proteinase permeabilization. In freeze-cracking of nematodes, live animals were placed in a small droplet on a slide, compressed under a second slide placed perpendicular to the first, and immediately transferred into a refrigerator at -25°C. After 1 h, the slides were taken from the refrigerator and their ends were quickly pulled together to bring the slides in parallel position thus cracking the body cuticle. The animals on the lower slide were fixed with 4% formaldehyde in 0.01M PBS for 1 hr. The subsequent steps for phalloidin staining were performed as described above starting from permeabilization in Triton-PBS, but without proteinase incubation. While nematodes species were cultured, about 1000 specimens of each species were stained with phalloidin, from which the adult nematodes were selected for confocal microscopy observations (three and two specimens for vulval muscles in B. mucronatus and Chiloplacus sp., respectively, and six and eight specimens for male copulatory organs in B. mucronatus and Chiloplacus sp., respectively); other individuals were also checked to avoid occasional artifacts and contradictions in the results.

Morphological study and reconstructions
Confocal images were collected using a Leica TCS SP5 confocal laser scanning microscope at the "Taxon" Research Resource Centre (Zoological Institute of the Russian Academy of Sciences, St. Petersburg, Russia; http://www.ckp-rf.ru/ckp/3038/?sphrase_id=8879024). Three-dimensional reconstructions of confocal Z-stacks were made by manual data segmentation and automatic volume rendering using Avizo 8.1 (FEI Visualization Sciences Group, Burlington, MA). Cuticular structures and their relationships with muscles were visualized in the reflection mode of the confocal microscope as described in Petrov et al. (2016).
Where muscle homologies were evident, the muscles were named and labeled according to their names for C. elegans as summarized in Wormatlas (Lints andHall, 2009a, 2009b).
The vulval musculature of the Chiloplacus sp. females comprises three pairs of radial vulval dilators (ar, pr1, pr2) and one pair the anterior vulval lip dilators (dav, Figs. 3B; 4D). The radial dilators include one pair of anterior muscles (ar) and two pairs of posterior muscles (pr1, pr2). Each radial muscle originates on the body wall dorsal to the vBWM and inserts on the rim of the round vulvar opening. The dilators of the anterior lip (dav) are a single pair of muscles inserted on the arcuate cuticular plate of the anterior vulval lip (vp) (Figs. 3B; 4D). Each dav muscle runs anteriorly to end on the vBWM muscle of the corresponding body side (Figs. 3B; 4D).

Male copulatory organs
Bursaphelenchus mucronatus 2. Longitudinal muscles (ail, pil, cdl, pol, pm). The inner longitudinal muscles, one anterior (ail) and one posterior (pil), extend posteroventrally from their origin on the subventral body wall to insert at the anterior rim of the cloaca. The ail lies dorsal to the pil and the muscles remain in tight contact with each other along most of their length (Figs. 5A, B; 7B, F, G). The paired caudal muscles (cdl) originate broadly at the ventrolateral sides of the body wall just anterior to the cloaca and continue posteriorly to the mb muscles (Figs. 5A, B; 7A-C). At the level of the cloaca, the cdl muscle forms a contact with the posterior longitudinal muscle (pol, Figs. 5A; 7 A). The posterior outer longitudinal muscle (pol) lies ventrolaterally between the cdl and vBWM. From its broad anterior attachment to the lateral body wall above the dgl muscles, it runs posteriorly toward the tip of the tail to terminate narrowly at the anterior end of the mb muscle (Figs. 5A, B; 7A, B, G). Near its posterior end the pol muscle gives off a narrow dorsal branch that ends on the lateral side of the tail (Fig. 5A). The papillary muscles (pm) arise internally from the ventral parts of the ob muscles on each 3. Precloacal copulatory diagonal muscles (dgl). Diagonal muscles (dgl) are a pair of precloacal muscles lying ventral to the pol and superficial to the inner longitudinal muscles (ail and pil) (Figs. 5A, B; 7A, B). These muscles extend posteriorly from their attachment on the ventrolateral body wall and end submedially at the lateral borders of the cloaca.
4. Postcloacal copulatory oblique muscles (ob). The oblique post-cloacal muscle is a broad muscle band stretching between the bursa and the cloaca to the inside of the vBWM and cdl muscles (Figs. 5A; 7A, C, D, F, G). Ventrally, the posterior part of the muscle is anchored on each side of the body midline and its anterior part is attached to the body wall lateral to the cloaca; dorsally, the muscle is attached to the dorsolateral body wall. The muscle is a single continuous sheet in its posterior third, but anteriorly is split into a series of 11-15 parallel myofibers (Figs. 5A; 7A, G).

Spicule and spicular muscles.
Spicules are paired cuticular copulatory hooklike structures inside a Y-shaped proctodeum (cloacal sac). The spicule capitulum is flattened anteriorly and bears two processes: ventral rostrum (r) and dorsal condylus (c) (Fig. 6A). The spicule is strongly curved; the posterior third of its blade is conical and forms an angle with its anterior part. The dorsal angular point is denoted as the spicule saddle (sa). The conical tip of the spicule bears a slightly widened extension called the cucullus (cu). A special rostral ligament (rl) joins the rostrum of each spicule to the ventral body wall (Figs. 6A; 7 D, E). The proctodeum lacks a gubernaculum, whose absence is the diagnostic feature of the fam. Aphelenchoididae.
The dorsal spicule protractor (dsp) is a bilateral pair of very thick longitudinal muscles that originate broadly from the dBWM muscles in the posterior portion of the tail and extend anteriorly toward the spicule. The contralateral muscles run together in close contact, but as they reach the spicule saddle,  Chiloplacus sp. 8,9).
The arrangement of the four body wall muscles is generally similar to that described for B. mucronatus, except that the vBWM are significantly longer than in the latter species and their posterior endings are located more anteriorly, at the lateral sides of the cloaca (Figs. 5E, F; 9A, C, E).
Although the overall arrangement of the longitudinal muscles is similar to that of B. mucronatus, there are some significant differences. The pol are much shorter than in B. mucronatus; they end anteriorly at the lateral sides of the cloaca and lie ventrally to the mb muscles, with which they appear to be in tight contact through much of their length (Figs. 5E, F; 9A, B, D, E). The pm muscles are lacking and only one inner longitudinal muscle (il) is present rather than two as in B. mucronatus (Figs. 5F; 9 B, C).

Discussion
Phalloidin staining for actin, in conjunction with confocal microscopy, is a powerful technique that provides detailed information on morphology and spatial arrangement of muscles. Its use in nematode research, however, has been limited, primarily due to the difficulty in rendering the cuticle sufficiently permeable for staining. This difficulty can be partially eliminated by using prolonged proteinase treatment, which is sufficient for adequate permeabilization of the cuticle, but does not incur excessive damage to the muscle tissue. Using this technique, the present study provided a detailed description of the vulval and male copulatory musculature in two phylogenetically distant nematode species: Bursaphelenchus mucronatus and Chiloplacus sp. The confocal microscopy studies of musculature in nematodes has so far been conducted only in C. elegans and our knowledge of morphology of copulatory and vulval musculature in other species derives exclusively from electron and light microscopy studies. Although only a few nematode taxa have so far been examined in detail, the results of these studies summarized in Table 1 may serve as a baseline for future studies on the diversity of reproductive musculature in nematodes. The following discussion provides comparison between the results of the previous research on vulval and male copulatory muscles (most notably, in C. elegans) and those obtained in the present study.
The musculature of the vulval apparatus in the females of Chiloplacus sp. comprises radial muscles (dilators of the vulva) and muscles of the anterior vulval lip attached to the anterior inner vulval plate. The four pairs of radial dilators in Chiloplacus sp. are similar to the pattern in C. elegans and other Rhabditidae: Pelodera scrofulata, Metarhabditis spp., and Hirschmanniella oryzae (Table 1; Shakil and Jairajpuri, 1976;Bird and Bird, 1991;Ahlawat, 2011), but in Chiloplacus sp. the anterior pair of vulval dilators has been transformed into the muscles of the anterior vulval plate.
The musculature of the vulval apparatus in Bursaphelenchus is more complex than in Chiloplacus and includes longitudinal and diagonal muscles of the anterior and posterior lips of the vulva (ma and dip), longitudinal muscles of the external vulval flap (flp), transverse muscles of the vulval slit (ta, ma and tp), and posterior transverse muscle bands (su). The functional role of these muscles can be readily determined from their arrangement: longitudinal and diagonal muscles of the vulval flap and vulval lips (ma, dip and flp) are dilators serving to open the vulval slit structures upward and sideways, while the transverse muscles (ta, ma and tp) act as constrictors of the vulval slip and the su muscles may function as suspensors during mating and oviposition.
In Bursaphelenchus, the two opposing pairs of vulval dilators (ma and dip) show quadrilateral symmetry similar to that of the Rhabditidae; however, the anterior vulval flap has its unique paired fld dilators. Vulval constrictors of B. mucronatus (ta and tp pairs and an unpaired mta muscle at the slit-shaped vulval rim) have no identifiable counterparts in the other nematode species studied (Table 1); although two pairs of vulval constrictors are also present in Hirschmanniella, Pratylenchus (Pratylenchidae) and Hadronchus (Mononchidae) ( Table 1; Shakil and Jairajpuri, 1976;Mai et al., 1977;Ahmad, 1980), they radiate outward from the rim of the vulval opening, rather than running along the rim as those of B. mucronatus.
The arrangement of muscles described here in the caudal body region of the males of Chiloplacus sp. is similar to that of C. elegans as reviewed by Lints andHall (2009a, 2009b, Table 1). The significant differences concern the morphology of gubernacular muscles and there is also uncertainty about the identity of the anal depressor muscle. In C. elegans, the gubernacular muscles comprise two pairs attached to the dorsal body wall and functioning as erectors and retractors of the gubernaculum; Chiloplacus has a similarly arranged pair of erectors, but the second pair of muscles is attached to the body wall near the posterior cloacal lip also apparently acting as erectors rather than retractors. Chiloplacus sp. also has a third pair of gubernacular muscles (gsp) lacking in C. elegans. These muscles are connected to the spicules and their arrangement suggests that they may serve as additional spicule protractors pulling the spicular capitula toward the proximal part of the gubernaculum.
The anal depressor (adp) is a sexually dimorphic muscle that assists in defecation in the females but acts as an auxiliary spicule protractor in the males of C. elegans (Lints andHall, 2009a, 2009b). In the males, its ventral contractile portion connects the gubernaculum and the posterior cloacal lip, while the non-contractile dorsal portion is H-shaped and is attached to the dorsal roof of the tail. No H-shaped muscle has been observed in the males of Chiloplacus sp., but the ventral gubernacular erectors (vge) of this species may be homologous to the ventral part of the anal depressor (adp) in C. elegans, as both have similar attachment to the gubernaculum and the posterior cloacal lip. Since the dorsal H-shaped part of the adp lacks contractile elements in C. elegans, it cannot be visualized by phalloidin staining and it is likely that in Chiloplacus sp. this part of the muscle is also present but cannot be detected by the methods used in the present study.
The sphincter (sp) muscle of both Chiloplacus sp. and C. elegans appears to be modified in the males to assist in insemination by closing the midintestine and drawing it dorsally from the vas deferens thus expanding the cloaca for sperm passage (Lints and Hall, 2009b). The sphincter of Chiloplacus sp., however, has two dorsal arms, while only one arm persists in the males of C. elegans (in the juveniles the sphincter has four arms). The copulatory muscle system of Bursaphelenchus differs significantly from those of Chiloplacus sp. and C. elegans. Like the other Aphelenchoididae, Bursaphelenchus lacks a gubernaculum and has strongly curved spicules. Only three pairs of muscles are associated with the spicules: two pairs of spicule protractors (the dorsal pair inserted on the condylus and the ventral pair attached to the spicular saddle), and the third pair of transverse muscles (tsm) extending from the saddle laterally. No apparent retractor muscles have been identified.
Although in B. mucronatus the gubernaculum is lacking, the arrangement of muscles attached to the spicular saddle bears certain resemblance to that of the gubernacular muscles in Chiloplacus sp. and C. elegans. The vsp muscles of B. mucronatus, like the vge muscles of Chiloplacus and the adp muscles of C. elegans, are attached to the posterior cloacal lips and have a similar orientation; the tsm muscles of B. mucronatus are positioned essentially like the gubernacular erectors in Chiloplacus and C. elegans except that their origin on the body wall is located more ventrally than in the two latter species. This indicates a possible homology between the conical endings of the spicules of B. mucronatus and the gubernaculum of Chiloplacus and Caenorhabditis. It is possible that the gubernaculum has become fused with the anterior body of the spicule in the ancestral line of the Aphelenchoididae and the gubernacular muscles have transformed into the musculature of the spicular saddle.
In Chiloplacus sp., the female and the male are oriented parallel to each other during mating (Fig. 1 G) and the male embraces the vulval region of the female with the caudal bursal alae. When the spicules are inserted into the female's vulval opening, the gubernaculum serves as a support for the spicular tips, as it was shown for C. elegans (Lints andHall, 2009a, 2009b) and Pelodera strongyloides (Wagner and Seitz, 1981); in Chiloplacus sp. it probably also assists in spicule protraction serving as attachment for the gubernacular spicule protractors lacking in C. elegans. It is unlikely that in Chiloplacus sp. the ventral gubernacular erector (presumably homologous to the anal depressor) functions as an auxiliary spicule protractor as does the anal depressor in C. elegans (Lints and Hall, 2009b), because in the latter species the contractile part of the anal depressor is aligned and coupled with that of the spicule protractors, while in Chiloplacus sp. these muscles are positioned at a distance from each other. Spicule protraction in Chiloplacus sp. is therefore accomplished by three muscles: two pairs of spicule protractors and one pair of gubernacular spicule protractors with the gubernaculum raised by two pairs of gubernacular erectors, and retraction is performed by two pairs of spicule retractors inserted on the anterior spicular ridges.
In Bursaphelenchus, the mates are oriented perpendicular to each other during copulation; the male clasps the female vulval region by coiling its tail around the female's body (Figs. 1E, F;2;Vieira et al., 2009). The bursa of Bursaphelenchus is different from the male bursa of Chiloplacus and other Rhabditidae. It is a terminal flap-like appendage bordered by glandular papillae, its morphology being specifically adapted for holding the female during mating in perpendicular orientation (Ryss et al., 2005(Ryss et al., , 2021. Spicule protraction in B. mucronatus is apparently accomplished by two muscles: the dorsal and ventral protractors (the latter presumably being homologous to the anal depressor as discussed above). The ventral protractors may also function as spicule rotators similar to the muscles attached to the mid-portion of the spicules in the family Xyalidae (order Monhysterida) and in Hirschmanniella oryzae (Paratylenchidae) ( Table 1: Character 12;Shakil and Jairajpuri, 1976;Vincx and Furstenberg, 1988). It is not clear how spicule retraction is achieved as no apparent retractors have been described in B. mucronatus, but it is possible that spicules are retracted by a combined action of the transverse saddle muscles (tsm) and the rl ligaments attached to the spicular rostra. The rostral ligaments are evidently stretched and tensed during spicule protraction and it may be assumed that once the spicule protractors are relaxed the ligaments would be able to pull the spicules back into the cloaca by elastic recoil. The tsm are oriented transversally in retracted spicules and may pull the spicules apart, but they assume a diagonal orientation when the spicules are extruded so that the force vector of these muscles is directed into the cloaca and their contraction may therefore assist in spicule retraction.
Differences between Bursaphelenchus and Chi loplacus in mating arrangement are reflected in different patterns of the outer muscles of the tail. In B. mucronatus, the caudal oblique muscles (ob) are a pair of broad transverse muscle sheets consisting of numerous parallel myofibers, while in C. elegans and Chiloplacus there are two distinct pairs of muscles (aob and pob). A broad bilateral muscle sheet may represent the basal condition; during mating the contraction of these muscles causes the tail to curl into a ring enabling the male to grip tightly the female's vulval region (Figs. 1 E,F;2). The long rows of ob muscles have also been reported in Spironoura chelydrae (Harwood, 1932) (order Ascaridida: family Kathlaniidae) and Hadronchus shakili (order Mononchida: family Mononchidae) (Mackin, 1936;Ahmad, 1980; Table 1). In the C. elegans and Chi loplacus males, a short postcloacal zone of two pairs of oblique muscles may be explained by the peloderan shape of their bursae provided with paired caudal alae. The male uses the alae to clasp the female's vulval region embracing it from the sides (Fig. 1G) and the oblique muscles come into play only during the 'turning phase' of mating (Lints and Hall, 2009a). It is likely that when the peloderan bursa has appeared in evolution, the zone of postcloacal ventral muscles has been reduced from a long muscle sheet (as in Bursaphelenchus and Mononchidae) to only two pairs (C. elegans and Chiloplacus). In B. mucronatus, the terminal bursa is a small sticky appendage with a pair of mb muscles.
Another adaptation to the perpendicular mating orientation in Bursaphelenchus is the vulval flap with developed fld dilators and vulval slit constrictors (paired ta and tp and the unpaired mta muscle). This unique vulval flap-and-slit muscle system wedges the inserted endings of the spicules in place during mating functioning effectively as a lock for the spicules. The terminal bursa and broad oblique muscle sheets that cause the tail to coil during mating together with strongly curved spicules act as a "key" designed for this type of vulva. Such mutual evolutionary adjustment of copulatory organs in both sexes exemplifies the "key and lock" principle in morphology of copulatory structures (Ryss et al., 2005;Behr, 2008;Vieira et al., 2009;Ryss and Subbotin, 2017).
In the females of Chiloplacus sp., the rounded vulva is not covered on the outside by an external vulval flap; the vulva is opened by contraction of radial dilators (Figs. 3B; 4E) and the spicules in the males are shaped as almost straight cones (Figs. 6B, C; 8). During mating, the function of the "key" is performed by spicules and the bursal alae with their ventrolateral longitudinal constrictors (cdl, mb and pol muscles). Such configuration may be described as a "key without a lock"; only male copulatory structures are responsible for the connection of partners during mating and insemination. Similar mating has been described for C. elegans (Lints andHall, 2009a, 2009b). Wagner and Seitz (1981) pointed out that the male bursa of Pelodera strongyloides serves as a mould for the adhesive secretion of the cloacal glands; this ensures fixation of the spicule tips opposite the vulval opening of the female.
The comparison of the copulatory musculature between the species examined in the present paper and those studied previously allows for some preliminary phylogenetic conclusions. If the gsp in Chiloplacus is indeed homologous to the anal depressor, the spicular muscle systems of C. elegans and Chiloplacus can be regarded as essentially similar differing only in the gubernacular retractors (absent in Chiloplacus) and gubernacular spicule protractors (absent in C. elegans). The gubernacular retractors may have been lost in the ancestral lineage of Chiloplacus because of the changes in the role played by the anal depressor (=gsp), while the gubernacular spicule protractors could have become reduced in the Caenorhabditis lineage, because the anal depressor took over the function of an auxiliary spicule protractor. The remaining spicular muscles may have been present in the common ancestral stock of both groups, i.e. a group that includes clades 11 ("Cephalobomorpha") and 9A ("Rhabditomorpha") in the total nematode phylogeny of Megen et al. (2009). In the phylogeny of Megen et al. (2009), clade 10B that includes Bursaphelenchus spp. and other Aphelenchoididae is more basal than clades 11 and 9A, and this clade appears to exhibit a combination of ancestral and derived traits: a broad postcloacal muscle sheet is likely to be a primitive condition, while  the system of spicular muscles has been modified as a result of reduction of the gubernaculum.