Morphology, development stages, and phylogeny of the Rhabditolaimus ulmi (Nematoda: Diplogastridae), a phoront of the bark beetle Scolytus multistriatus from the elm Ulmus glabra Huds. in Northwest Russia

Abstract The nematode Rhabditolaimus ulmi was found in galleries, adults, and larvae of Scolytus multistriatus, the vector of the Dutch elm disease, in St. Petersburg parks. This nematode co-occurred with Bursaphelenchus ulmophilus, which is another phoretic partner of S. multistriatus. Nematodes were cultured on the fungus Botryotinia fuckeliana in potato sugar agar (PA) and used for morphological analyses of adults, juveniles, eggs, and dauers. Nematode females showed a didelphic female genital tract rather than a monoprodelphic gonad as reported in the original description. Male bursa peloderan, caudal papillae include three preanal pairs and one precloacal unpaired papillae; seven postanal papilla pairs, among which one is pore-like and possibly the phasmid homolog, one subdorsal, and a pair of three closely situated posteriorly at bursa alae. The juvenile stages differ in size and structure of their sexual primordia. Sex of juveniles may be identified from the third stage. The dauer juvenile is a phoretic third juvenile stage (DJ3), which enters and remains localized in the buccal cavity of beetle adults and last-instar larvae and also under the elytra and in the ovipositor’s cavity of pupae and imagoes. The first molt J1-J2 occurred inside the eggshell. Adult females laid eggs in early stages of embryonic development or containing molted J2. The propagative non-phoretic J2 inside the egg and J3 have a long and well-developed median bulb. The phoretic dauer DJ3 has a small spherical bulb like the J1 juvenile within the egg. In a sterile fungal culture, the nematodes feed on both mycelium and their unidentified ecto-symbiotic bacteria, located on nematode surface coat and multiplying in PA. Diagnosis and tabular key to the Rhabditolaimus species are given. Phylogenetic analysis of the D2-D3 of 28S rRNA gene sequences resulted in the Bayesian consensus tree with the highly supported clade of the Rhabditolaimus species.

Among wood-and bark-inhabiting nematodes, the fungal feeders in the family Aphelenchoididae, such as the pinewood nematode, Bursaphelenchus xylophilus (Nickle, 1970;Steiner and Buhrer, 1934) and the red ring nematode, B. cocophilus (Baujard, 1989;Cobb, 1919) have been studied extensively because of the devastating diseases they cause on pines and palms, respectively, and their spreading capabilities using insect vectors (Jones et al., 2013;Mota et al., 1999;Ryss et al., 2005). In the nematode fauna of declining trees, some species are vectored by bark and longhorn beetles (families Curculionidae and Cerambycidae). These nematode phoronts use their insect vectors to colonize live trees or for transmission to new decaying wood sites to feed on fungi or bacteria. The transmissive dispersal life stage of these nematodes is the dauer juvenile (DJ), which is morphologically different from the non-phoretic juveniles of the propagative generation inhabiting wood and bark. The ecological roles of many obligatory phoronts of the bark beetles belonging to the families Diplogastridae and Rhabditidae are not known.
Only two nematode species were found most consistently in a 2014 to 2020 survey of declining Ulmus glabra and U. laevis trees in St. Petersburg parks: Bursaphelenchus ulmophilus  and Rhabditolaimus ulmi (Goodey, 1930;Susoy and Herrmann, 2012). The latter was originally described by Goodey (1930) and later redescribed by Rühm (1956) from Bavaria, Germany. However, the biology, morphology, and phylogeny of this species have not been well elucidated. Both species were detected in galleries of Scolytus multistriatus (Marsham, 1802), a bark beetle that is known to transmit Dutch elm disease (DED). Their dauer juveniles were also regularly collected in beetle adults, under elytra.
The objectives of this paper were to (i) describe and characterize morphologically all developmental stages of R. ulmi, including the dauer juvenile stage, (ii) describe R. ulmi's ecological role in relation to its vector life cycle, and (iii) provide molecular characterization of R. ulmi and reconstruct phylogeny of the genus.
In previous years, all trees that showed symptoms of dieback were found infected by the fungus Ophiostoma novo-ulmi Brasier, the causal agent of DED and vectored by the S. multistriatus (Marsham, 1802) beetle (Kalko, 2008(Kalko, , 2009. The elm bark with beetle galleries, as well as beetle larvae, imago, and pupae were sampled for nematodes in all locations. In total, 75 bark samples with live adult beetles and larvae were taken. In February 2019, additional 15 wood and bark samples were also collected during the sanitary cutting of declining elms infected by DED in the park of the St. Petersburg Forestry and Technical University (GPS coordinates: 59.991923°N, 30.342697°E). Nematodes were extracted from these samples and then cultured on the fungus Botryotinia fuckeliana (de Bary) Whetzel, 1945 (= Botrytis cinerea Pers., 1794) on potato sugar agar media (PA).

Nematode extraction and rearing
Nematodes were extracted from bark by Baermann's funnel technique modified by . To extract nematodes from beetles, the elytra of adults or a whole larvae or pupa were placed in a 0.5-ml drop of distilled water and incubated for 30 to 60 min to allow the nematodes to slowly move from insect body into the water. Cultures of B. fuckeliana fungus on the 2% potato sugar agar media (PA) were used for nematode rearing. In total, 50 g of fresh sliced potato tubers were boiled for 40 min in 200 ml of water, then smashed, and additionally boiled for 5 min, filtered through gauze. In total, 4 g of agar, 4 g of sugar, and 10 ml of glycerin were added to the hot solution, the volume was brought to 200 ml, and finally autoclaved at 3 atm and 130°C for 40 min. The hot mixture was poured into 6-cm Petri dishes, cooled down, and inoculated with mycelium of a strain of B. fuckeliana, which does not produce spores. After 5 to 7 days at 20°C, when a white mycelium lawn occupied the entire surface of the agar, 20 R. ulmi specimens that had been extracted from the bark and identified with a compound microscope, were transferred to the fungal culture. Separately, dauers taken from beetles were also reared in fungal cultures, reaching the maturity and then producing the nematodes of propagative generation.
After 10 to 14 days, nematodes had multiplied and consumed all the mycelium. Simultaneously, bacteria from the surface coat of the nematode body grew onto the agar medium. Nematode cultures were passaged 2 to 3 times until all the nematodes were R. ulmi at different stages of development: eggs, juveniles, and adults. Every 10 days, cultures were transferred to new PA media with fungus. After every five cycles in fungal culture, nematodes were passed through a superficially sterilized (30 min in a 1.0% NaOCl household bleach solution) elm branch for one month, followed again by culture on B. fuckeliana. Cultures of the nematode isolates in Petri dishes can be stored in a refrigerator at + 8°C for up to six months without recultivation. Nematodes obtained from these cultures were used for morphological analysis.

Morphological study Fixation and mounting
Nematodes were fixed with hot TAF (2 ml of triethanolamine, 10 ml of formalin, and 90 ml of distilled water) in a water bath (Ryss, 2017a), processed in a glycerol-water series, and mounted in the permanent collection slides using the method described by Ryss (2003Ryss ( , 2017b. Some juveniles fixed in TAF and processed to anhydrous glycerin, were stained with methylene blue stain by adding a 10-µl drop of saturated water solution of methylene blue to a drop of glycerin with the suspended nematode juveniles, on a 75 × 25-mm slide. After 12 hr, the stained suspension was covered with a 20 × 20-mm coverslip previously sealed on its edges with a thin film of glycerin jelly. After quick 60°C heating on a hot plate, the drop spread reaching edges of the coverslip; thus, the suspension was flattened and fixed with a glycerin jelly frame. The stained juveniles were studied and photographed using a Leica compound microscope. Following their examination and identification, a few specimens preserved in glycerin were selected for scanning electron microscopy (SEM) observations. The nematodes were hydrated in distilled water, dehydrated in a graded ethanol and acetone series, critical point-dried, mounted on stubs, coated with gold, and observed with a Zeiss Merlin microscope (Álvarez-Ortega and Peña-Santiago, 2016). The Adobe Photoshop CS2 software was used to adjust the contrast of photos, the ImageJ software for measurements (Collins, 2007), and MS Excel for calculations and statistical processing. Terminology of Fürst von Lieven and Sudhaus (2000) was used to describe the pharynx and stoma structures.

Nematode localization in beetles
The beetle larvae, pupae, and imagoes were examined for presence and localization of nematode dauers using the stereomicroscope LOMO MBS-10. Surface deworming was used to check the adhesive strength between the nematode and vector. The insect larvae and adults were immersed for 45 min in a 5% solution of acetic acid and 0.02% chlorhexidine and then washed with water on a 200-µm sieve. Beetles and most nematode dauers remained alive and attached to insect mouth parts and cavities, while other nematode stages could not stay attached and were washed out. This test was aimed to identify places in the body of the vector where nematodes could remain viable under aggressive external influences.
DNA extraction, PCR, sequencing, and phylogenetic analysis DNA was extracted from several pooled nematodes using proteinase K. PCR and sequencing protocols were as described by Tanha Maafi et al. (2003). The forward Rhabditolaimus_D2A-1 -(5′-GGC GAA GCG GAT AGA GTT GAC-3′) and reverse D3B (5′-TCG GAA GGA ACC AGC TAC TA-3′) primers were used for amplification of the D2-D3 expansion segments of 28S rRNA gene. Sequencing was done at Genewiz., Inc (CA, USA). A sequence was deposited in GenBank under the accession number: MW044951.

Detection of nematodes in trunks and branches
Of 75 samples, 20 were pieces of branches 20-cm long and 3 to 10 cm in diameter collected during sanitary cuttings. These twig segments are parts of the tree crowns contained the bark holes formed by beetles during maturation feeding. A red-brown ring in phloem was found in the cross section, which is typical for symptoms of DED. In bark, the fungal and plant-feeding nematodes B. ulmophilus and Aphelenchoides sp. were found, but not R. ulmi.
Other samples were 20 × 30-cm side parts of trunk segments, 3 to 6-cm thick, with the bark beetle galleries in the inner bark layer were consistently inhabited by both nematode species, R. ulmi and B. ulmophilus. In the period between February and June, the beetle larvae and imagoes with nematode dauers were present in the trunk samples. When the dauers from these samples were placed on the B. fuckeliana culture, they molted into preadult juveniles and then into adults of the transmissive generation, and then grew in cultures transforming to adults and juveniles of the propagative generations of two nematode species, R. ulmi and B. ulmophilus. The total number of trunk segments with living beetle larvae and adults was 25, whereas other 30 samples contained empty galleries from which the beetles had already emerged.

Nematode culture
Rhabditolaimus ulmi successfully propagated in sterile plates seeded with B. fuckeliana. Observations of nematode inoculated fungal plates revealed that nematodes grab the mycelium by the head probolae and stoma. Unidentified bacteria were also observed on the surface body coat of nematodes in the agar media and in the intestine of the nematode, suggesting that the nematodes were feeding on both fungi and bacteria.

Nematode localization in beetle
Rhabditolaimus ulmi nematodes were found under the elytra of beetle imagoes, in the ovipositor and buccal cavity, the latter containing the highest number of dauers. Dauers were observed in the buccal cavity and in developing ovipositor cavity of beetle pupae. In later stages beetle larvae nematodes were localized on the body surface and in the buccal cavity. When treated with deworming solutions, the nematodes were removed from the body surface but remained in the buccal cavity. In beetle adults and pupae nematodes remained also in the ovipositor cavity. Many of the beetles contained both B. ulmophilus and R. ulmi. The B. ulmophilus dauers were easily recognized by the stylet and very narrow body shape; they were completely flushed out when treated with deworming solutions.

General morphology
Body almost straight, with weak annulation; width of 10 cuticle annuli at mid-body 7.5 µm. Lateral field starts at level of a telostom bulb continuing to the hyaline zone of a tail tip in females and to cloacal opening in males. The field marked by four incisures: its two prominent lateral ridges separated by a hollow where width equals the width of a ridge. On the ventral and dorsal side of the lateral field bordered by 3 to 5 lower ridges, crossed by transverse annulation, the maximum number of these ridges outside the lateral field is five at mid-body; decreasing to three ridges at body extremities. Outside the ridges the cuticle annuli are crossed by longitudinal lines; thus each annulus consists of rectangles where the long sides are parallel to the axis of the body. The prominent dorsal and ventral ridges of the cuticle beginning at the level of combined procorpus and first pharyngeal bulb; the dorsal ridge ends near the hyaline zone of tail tip in female and at the level of the cloaca opening in males; the ventral ridge ends in front of cloacal opening in males and before the anterior lip of anus in females. Cephalic region continuous, low, its sides angular in lateral view and hexagonal radially symmetrical in anterior view; devoid of annulation. With SEM, the cephalic area clearly separated by the first body annulus. Six triangular identical probolae point to the center of the six-lobed oral opening, forming a conical cupola. At the anterior margin of each probola is a tubular inner lip papilla with a central hole. Cephalic papillae absent in female but four tubular cephalic papillae are distinct in male in the middle of two subdorsal and two subventral probolae. Amphids oval, located behind the lateral probolae immediately behind the level of the posterior folds of the oral opening between cephalic lobes; the long amphid axis transversal, it equals the width of the base of probolae, its longitudinal axis is half as wide. Cephalic framework moderate, its basal plate at the probolae bases. Cheilostom cup-shaped, consisting of adradial cuticular plates, devoid of denticles; its front edge is connected to the six-looped oral opening of the body surface cuticle. Tubular base of cheilostom overlaps the anterior margin of triangular flexible gymnostom, encircled by muscular collar where length equals head width. Gymnostom 8-12 times longer than cheilostom, ending in stegostom in the anterior part of a first bulb. Stegostom is anisomorphic with distinct dorsal anterior bulge; the posterior part of stegostom encircled by a cuticular ring with two distinct lateral foramens. The first bulb muscular, its width 3 to 4 times of its length; it consists of two parts weakly separated by interruption of the triangular inner canal lining: its anterior part is a procorpus and its one-third posterior part is a homolog of metacorpus (median bulb of the Rhabditida); the metacorpus part of the muscular bulb includes a pair of unicellular glands with ducts opening into pharyngeal lumen. Isthmus is muscular gradually continuing to the pyriform second (posterior) bulb; canal within isthmus triangular thin walled. Cuticle ring encircled isthmus at the base of median bulb. Nerve ring in the middle of the isthmus, with ganglia anterior and posterior to it. Excretory pore at the posterior edge of the nerve ring. Deirid pore-like, its diameter is half of that of excretory pore, situated on the central hollow of lateral field between the field ridges, 3-5 annuli posterior to excretory pore. Hemizonid three annuli wide immediately anterior to excretory pore. Second bulb without grinder or valve; two pairs of unicellular glands are distinct in the basal part of bulb. Cardium distinct, at 1/3 bulb length, dividing pharynx and mid-intestine, its length twice its diameter; consisting of three rows of three cells. Midintestine cellular, wide, its wall consisting of one layer of large granular cells, its inner lumen 1/3 intestine diam. with distinct hyaline layer. Paired phasmids small pore-like with diameter equal to that of deirid, located one anal diameter posterior to anus, in the central hollow between the field ridges.

Male
Body moderately curved to ventral side. Genital system at right side, 2/3 of mid-intestine length. Testis anteriorly reflexed to branch of 1 to 2 body diam. Spermatocytes in multiple rows in reflexed branch, posteriorly arranged in one row of large elongated cells; spermatids as 1 to 3 quartets in the middle zone of gonad; followed by the tubular vas deferens, filled with secretory granules and small spherical sperm; the gonad opening into cloaca sac ventral, at 20% of spicule length, while the mid-intestine opens via small valve into cloaca dorsally between heads of spicules; three large rectal glands at sides of valve.
anteriorly arch-shaped. Unpaired papilla-like papilla P1 two cuticle rings anteriorly to cloaca opening. It is transversely elongated, twin, with two connected tubular pores.
It is reasonable to divide the rest of the male tail papillae into three groups according to their positions in structure rows (i) outer paired papillae on the fold of bursal alae (7 pairs of papillae: OP1…OP7); (ii) inner paired papillae on the ventral tail surface (two pairs, their numeration continues that of unpaired papilla P1, abdominal: P2...P3); a pair of paired pore-like papillae, presumably homologous to phasmids of the female, locating on the tail surface at the inner base of lateral wings at the level of upper edge of triad OP4, OP5, OP6. The papilla-like papillae located on folds of bursal alae form bursal ribs. A pair of OP1 one anal diameter anteriorly to the cloacal opening mark start points of the bursal alae. Paired OP2 at the level of half the distance from OP1 to the cloacal opening. Paired OP3 at the level of mid-tail. Pairs OP4, OP5 and OP6 form a group that are close to the posterior edge of the bursa. Paired OP7 at the dorsal side of the bursal alae at a distance of two cuticle annuli from the bursal terminus, immediately behind the grouping of OP4, OP5 and OP6. The paired OP7 devoid of ribs; they form the posterior horns of the shovel-shaped end of the bursa, without ribs. Inner (abdominal) tail papillae. P2 at OP2 level at the base of the bursal alae; P3 anteriorly to the OP3 locating from the latter at the distance equal to that of OP3-OP4, i.e. three cuticular rings.

Female
Genital system paired, anterior branch at right side and posterior to left side of intestine. Ovaries reflexed (antidromous) with multiple rows of oocytes in both terminal endings. The mature oocyte enlarged at the loop of ovary flexure. Spermatheca filled with large cytoplasmic sperm, oval, its length twice of its diam. Spermatheca is the dorsally branching blind appendix of uterus that joins with ovary via narrow duct. Mature large oocyte passes the oviduct to uterus; and thus pushes the spermatheca; the latter injects sperm into uterus where the oocyte insemination occurs. Uterus paired, oval, anteriorly joined with oviduct ventrally and spermatheca dorsally; posteriorly the uterus connected with small oval uteral chamber bo rdered laterally with two pairs of dense thickwalled cells; the uteral chamber separated with two sphincters from both uteri: anteriorly from anterior uterus and posteriorly from posterior uterus. The chamber opens into cuticle vagina, perpendicular to ventral body surface; vagina opens into transversal vulva surrounded by valve, pyriform in lateral view. The valve surrounded with massive vulval muscles. The vulval hole is rounded, surrounded by dense cuticle folds that form a wrinkled oval-shaped vulval zone, the length of this zone is one and a half times more than its width. On the outside, the wrinkled zone is surrounded by a smooth round cuticle area, the width of the smooth zone equals to the width of the wrinkled zone. Outside of the smooth zone, the cuticle is annulated with a rectangular pattern typical to the rest of body surface. In front of the vulva at the edge of the smooth and annulated zones, two porelike papillae are located at the level of front edge of the wrinkle zone. Their arrangement may be asymmetrical -anterior vulval papilla may be situated by the width of one cuticle ring at front of the other one.
Rectum 1.2 to 1.5 anal body diam., consisting of capillary posterior part and broad pyriform anterior part with wide lumen, bordered by three large rectal glands.   Tail conical, 4 to 5 times its diam., gradually tapering to hyaline narrowly conical tail tip with hyaline zone of 1/6 of tail length. The anterior lip of female anus rounded, arch-shaped with marked cuticular border. Posterior anal lip is bulged, smooth in front and posteriorly annulated; annuli are crossed by longitudinal incisures breaking the cuticle rings into longitudinal elongated rectangles, just behind the posterior anal lip seven rows of rectangles form a V-shaped figure, posteriorly at a distance of one anal body width from anus the number of rows decreases to five and they are parallel.

Eggs
Egg is large, its length 58 to 65 µm; 2.5 times of its diam. Eggs are laid by a female at different stages of cleavage: from single cell stage up to the second stage juvenile after the first molt inside eggshell; in latter case it is the ovoviviparity. Eggs are usually laid in chains of 2 to 5 units. Eggshell is smooth but after a juvenile breaks the shell its surface is marked with longitudinal slit.

± 1 (3-5)
Notes: All values are given as mean ± s.d. (minimum-maximum); GP center from anterior/L: ratio, head to center of genital primordium/body length; GPL/GPW: ratio, genital primordium length to its diameter; GPL/L: ratio, genital primordium length/body length. mid-intestine part of the body. Four large germinal cells with an apical cell in posterior pole; in anterior part of primordium a small hook-like part with somatic nuclei reflexed posteriorly. This part in later stages will grow to cloaca primordium. Cloaca primordium present, encircling rectum, with its ventral side narrow and elongated anteriorly and dorsal side widely expanded.

J3 female
Except for genital structures, the J3 female's general morphology is similar to that of the adult female. Vulval primordium absent. Genital primordium is a small Z-shaped to horseshoe shaped structure with central somatic zone and two flexed branches, each of them with two large germinal cells and small apical cell, situated in the middle of mid-intestine section of the body.

J4 male
Except for genital structures, the J4 male's general morphology is similar to that of the adult male. Genital primordium horseshoe-like, in the middle of the midintestine part of the body, its length 5 to 7 times of its diam. Main part of primordium with 10 to 15 large germinal cells and with a small apical cell on posterior pole. Anteriorly primordium has a hook-like reflexed part with somatic nuclei which grows during this stage in the direction of cloaca primordium, finally joining with the latter. Cloaca primordium encircling the expanded rectum canal; its ventral side narrow and elongated anteriorly and dorsal side widely expanded. Primordium has very dense structure filled with numerous somatic nuclei.

J4 female
General morphology is the same as in adult female with smaller sizes of organs. The only difference is the genital structures. Genital primordium 4 to 5 body diam. long; it consists of two equal reflexed branches with central part with vulval primordium attached to the ventral body wall in the middle of the mid-intestine section of the body. Anterior branch at right side and posterior branch at left side of the mid-intestine. Vulval primordium 1/5 body diam., massive, containing 30 to 40 somatic nuclei, with the central transverse lens-like invagination under cuticle. Genital primordium attached to dorsal surface of vulval primordium. Reflexed part of each branch of genital primordium consists of ca 10 large germinal cells and the apical cell, almost reaching the vulval primordium invagination. Non-reflexed part of genital primordium tubular, consisting of somatic cells.

Molting individuals
The molting individuals (between stages JN and JN + 1) are characterized by the presence of shed cuticle and the genital primordia with structures which are intermediate between those typical for the nonmolting specimens of JN and JN + 1. Two preadult molting phases are described below.

J4-Adult male molting juveniles
Body in shed molting cuticle, separated from body at extremities; it is remarkable that the shed elongated conical tail cuticle envelops the rounded male tail with bursa. All structures as in adult male, including caudal bursa and its papillae, except for the weak and very transparent spicules in cloaca canals.

J4-Adult female molting juveniles
Body in shed molting cuticle, separated from body at extremities. All structures as in adult female, except genital tube. Vulval-vaginal primordium is a transparent ventral wide invagination under cuticle which dorsal bottom is shaped as provisory uteral chamber. Two branches of genital primordium equal in size, fused with vulval-vaginal primordium attaching to the provisory uteral chamber. Each branch is divided into sections of future uterus, spermatheca, oviduct and the reflexed provisory ovary of 15 to 20 germinal cells reaching the vaginal primordium from dorsal side. Anterior genital primordium branch at right side and posterior at left side of mid-intestine. Tail conical as in female, anus and rectum well developed, three rectal glands visible. All nematodes described above were initially collected from the bark and wood and then multiplied in agar cultures with the fungus Botryotinia fuckeliana. Additionally, transmissive dauer juveniles (DJ3) were collected from the undersides of the elytra of Scolytus multistriatus.
Body straight. Annulation weak, four incisures in lateral field. Deirid indistinct. Cephalic region continuous, high, conical, probolae absent, lips amalgamated; with labilal disc. Cephalic framework weak hyaline-like. Stoma slit-like, thick-walled, short, 5 to 8 its widths. Pharynx narrow. Procorpus and metacorpus joined in elongated pyriform corpus, but with distinct border between long cylindrical procorpus and compact ellipsoid metacorpus, the latter with distinct inner triangular canal and devoid of cuticle valve. Isthmus narrow, with capillary canal, posterior bulb elongated ovoid; with two paired gland ducts opening into the canal. Cardium of ¼ bulb length, collar-like, separating pharynx from midintestine. Nerve ring at mid-isthmus; excretory pore at its posterior border; hemizonid 3 annuli wide just anterior to excretory pore. Genital primordium ventral, at ½ distance of mid-intestine; in female juveniles U-shaped with terminal germinal zones each with two large cells and central somatic part with ca 10 small nuclei; in male juveniles primordium drop-like with anterior hook-like somatic part and the germinal part of 4 large cells directed posteriorly. Rectum 1.5 times anal body diam., posterior part thickened, anus distinct. Tail conical, straight, 9 to 10 times anal body diam; terminal hyaline zone equal to rectum length. Phasmid small, at anterior third of tail. Male dauer juvenile differs from female dauer juvenile in presence of cloaca primordium around the thickened posterior half of rectum; its diam. equals to ¾ of anal body diam. According to 300 to 360 µm body size and genital primordium with 4 germinal cells the dauer juveniles correspond to the J3 juveniles of the propagative generation and therefore identified as the DJ3 stage of transmissive generation. After 3 to 6 hr in water or on PA media the DJ3 molted to DJ4 and then to adult nematodes with well-developed stoma and pharynx as described above for males and females.
Diagnosis (emended): Diplogastridae. Lateral field of two ridges separated by hollow (four incisures); outside the field the additional less prominent ridges crossed annules. Cephalic region and stoma hexagonal; female with six lip sensilla and two amphids, male with additional four cephalic sensilla. Cheilostom with inner cuticular plates, without denticles; its outer wall overlaps anterior end of gymnostom. Gymnostom flexible, very long, 10 times of its width and longer, ratio: gymnostom to corpus 0.4 to 1.3; a ratio stoma (cheilostom and gymnostom) to pharynx 0.2 to 0.4. Stegostom in the anterior part of corpus, asymmetric with anterior dorsal bulge and with posterior inclined subventral plates without denticles; it is encircled posteriorly with cuticular ring with two lateral foramens. Corpus strong cylindrical muscular bulb, 2 to 5 widths long, combining procorpus and metacorpus which borders are marked by interruption of the lumen triangular lining. Excretory-secretory pore at second bulb which devoid of grinder valve, or the pore shifted to base of pharynx. Hemizonid just anterior to excretory pore. Deirid a small pore at nerve ring or shifted to the base of pharynx. Female genital system didelphic with central vulva, or monoprodelphic with vulva shifted posteriorly (V = 55 or more). In females two small pore-like phasmids in the anterior part of the long conical tail. Male with well-developed peloderan or leptoderan narrow bursa, in type-species and bursa enveloping tail but with a mucronate 2 to 10 µm spike outside the bursal flap. Generally three preanal and six postanal caudal papilla pairs (sometimes 2 preanal and 5, 7 or 8 postanal pairs); the postanal papilla located at bursa alae and ventrally, a group of closely situated 3 papilla pairs (ribs) at the posterior margins of alae; one of postanal papilla is subdorsal. In some species unpaired pre-cloacal papilla detected and one of the paired postanal papilla may be pore-like: they are the 'glandular papillae' which may be homologs of phasmids. Male spicule narrow, widely varied in size from 17 to 600 µm. Gubernaculum present, with complicated species-specific shape. Saprophages and phoronts of bark-beetles with the specific dauer juveniles of transmissive generation. Some species associated with soil and decaying matter.

Key to species
Tabular key to species of Rhabditolaimus is given in Table 4. The data were obtained from the original species descriptions and drawings. Missing values are calculated from the data in original taxonomic species descriptions and drawings, or in detailed re-descriptions.

Molecular characterization and phylogenetic position of the genus Rhabditolaimus
Sequence of D2-D3 of 28S rRNA gene from R. ulmi was obtained in the present study. The D2-D3 of 28S rRNA gene alignment (786 bp) included 47 sequences of diplogastrids, including R. ulmi and two sequences of outgroup taxa. Phylogenetic analysis resulted in the Bayesian consensus tree given in Figure 10. Representatives of the genus Rhabditolaimus (= Myctolaimus) formed a highly supported clade (PP = 100%). Rhabditolaimus ulmi sequence from Russia (GenBank no. MW044951, CD3323a) was identical to that deposited by Kiontke et al. (2007).

Discussion
Rabditolaimus ulmi is commonly found in bark of declining elms with symptoms of DED. In our sevenyear survey R. ulmi and B. ulmophilus co-occurred as phoronts carried by the beetle Scolytus multistriatus in Northwest Russia. All developmental juvenile stages, the transmissive dauer juvenile DJ3 as well as molts between stages were studied and described in this study, except DJ2 within egg shell and DJ2-DJ3 molt. Juvenile stages of R. ulmi can be easily distinguished by the size, structure, and number of germinal cells of the genital primordium. Morphological differences between male and female juveniles can be diagnosed from the third-stage juvenile. According to genital primordium structure, the dauer juvenile should be considered as the third stage (DJ3). The transmissive juvenile DJ3 differs from J3 of the propogative generation by its head structure: absence of six prominent probolae, presence of hyaline head cupola instead of cuticle skeleton, cuticle stoma tube tapering into the pharyngeal capillary canal vs broad triangular canal of the stoma and pharynx in J3. The most marked feature is the small pyriform first bulb in DJ3 vs long cylindrical and well-developed corpus with a triangular lumen in J3. Additionally, the DJ3 is very slender and devoid of annulation of the surface cuticle vs broad annulated body in J3.
The shape of the first bulb (combined procorpus and metacorpus) can be used as a diagnostic character to differentiate the DJ3 and J3. The J2 inside the egg, before hatching and even in the parent female before oviposition, already has a wide cylindrical first bulb which is a characteristic feature of the propagative J2 and J3. Both J1 in an eggshell and DJ3 differ from the J2 in the compact weak first pharyngeal bulb (combined procorpus and metacorpus) vs a first bulb muscular and elongated in J2 before hatching and after hatching from an eggshell, J3 and following stages.
Researchers believe that an environmental stimulus such as exposure to the mature adult beetle in the beetle galleries or a lack of nutrition may trigger the 2nd stage juvenile to develop into a dauer (Kanzaki, 2008). It is very possible that the DJ2 within an eggshell in the female genital tube has the compact weak first pharyngeal bulb as the first bulb of J1 and DJ3. Perhaps the stimulus to shift development into the dauer stage affects the parent female, not only the juvenile itself. To confirm this hypothesis, it is will be necessary to find the J2D (predauer) of transmissive generation in the female genital tube.
The finding of a didelphic female genital system in R. ulmi has very important taxonomic implications Figure 10: Phylogenetic relationships within Rhabditolaimus spp. and other related taxa as inferred from Bayesian analysis of the D2-D3 expansion segments of 28S rRNA gene sequences. Posterior probability values more than 70% are given on appropriate clades. New sequence indicated in bold.
opposed to monoprodelphic females with posterior vulva. Unfortunately, the specimens used by Rühm were stained nematodes on temporary slides that cannot be examined. Type specimens of R. ulmi (= Cylindrogaster ulmi Goodey, 1930) do not exist. Later, all researchers had accepted the unpaired female genital system in R. ulmi as true. Here we have shown the presence of two genital branches in females of R. ulmi. This fact will simplify the generic systematics of the Diplogastridae, because most Rhabditolaimus species are didelphic with a central vulva position. Only two species of the genus: R. goodeyi and R. dendrophilus have monoprodelphic females; both species have females with the vulva posteriorly located and males with very long spicules. Probably, this feature caused by the specific position of females during copulation. Both species may be again transferred to the genus Protocylindrocorpus Rühm, 1959 formerly synonymized by Susoy and Herrmann (2012) with Rhabditolaimus. This fact may be an argument for the future study to reestablish the genus Protocylindrocorpus. Unfortunately, no sequencing results on both species are available in GenBank.
In our study, we propose to enumerate the male papillae first as groups and then as numbers in each group. Most of them are transversal ribs of the bursa, located anteriorly and posteriorly to the cloacal opening. Grouping of papillae according to their form and position is a perspective tool to establish homologies and thus verifies the phylogenetic analysis. Besides, the cloacal opening that used earlier as a marker of papillae positions (Kanzaki and Futai, 2004) we propose to use a phasmid position to characterize the caudal papillae pattern.
In the female genital tract, the spermatheca is not axial. It is the dorsally branching blind appendix of the uterus, indicating that the spermatheca in Rhabditolaimus is a section derived from the uterus and not an expanded posterior part of the oviduct (Chizhov, 1991). Sperm via a spermathecal duct is injected into the uterus; the eggshell formation and maturation of the embryo takes place also in the uterus. The uterus has no inner differentiation into sections; the preuteral gland is not discernible. However, just opposite the vagina, there is the unpaired uteral chamber. The latter is compact and bordered by two sphincters: anterior and posterior ones. This chamber is evidently used for oviposition; in many females, it contains a fully developed egg before it moves into the vagina. The uteral chamber is a special anatomical section of the genital tract not previously described in diplogastrid adult females.
Our observations reveal the combined (omnivorous) feeding of R. ulmi. The nematodes may feed on bacteria which they vector on their surface coat, after the bacteria multiplied in sterile PA, and the Botryotinia fuckeliana mycelium in PA culture. New data expand our knowledge on a range of foods of the Rhabditolaimus spp. (Diplogastridae), which are generally characterized only as bacterial feeders (Yeates et al., 1993).
Rhabditolaimus ulmi was found in bark beetle galleries; however, this species was never detected in twigs of the tree crowns injured by the maturation feeding of S. multistraitus. The dauers of R. ulmi are situated in buccal cavity of senior beetle larvae and, additionally, in the ovipositor's cavity of beetle imago and pupae, where they adhere and remain attached also after deworming treatments, thus indicating tight relationships with the vector in comparison with the Bursaphelenchus ulmophilus dauers, which easily washed out from the same beetle vector after a treatment with deworming solutions.
Numerous nematode adults and juveniles were detected in excrements produced by the beetle larvae in galleries where they feed on bacteria and fungi. Evidently, that crawling nematodes fulfill the function of dispersal of the bacterial and fungal mass located in their surface coats. Further research will help to determine whether R. ulmi plays any role also in the pathogenesis of the Dutch elm disease, or whether its role is limited to a simple phoretic association with the beetle vectors of the causal agent of this disease. In contrast to R. ulmi, the co-inhabiting phoront nematode Bursaphelenchus ulmophilus is localized both in galleries and at the top of elm trees with symptoms of elm dieback. This indicates on its phoresy by a vector during the period when the young beetles after emergence from pupal cambers feed on young twigs to sexually mature (maturation feeding) (Santini and Faccoli, 2015). It is confirmed by the nematode dauers localization under elytra of young beetle adults .