Gammarinema scyllae sp. n. and Monhystrium mangrovi sp. n. (Nematoda: Monhysteridae) from land crabs from New Caledonia

Two new species of nematodes associated with crabs are described from New Caledonia. Gammarinema scyllaesp. n. from the gill chambers of Scylla serrata (Forsskål) is characterised by 3–4 mm long body, small outer labial and cephalic sensilla, distinct ocelli, short straight spicules and sub-cylindrical tail. Monhystrium mangrovisp. n. from the gill chambers and body cavity of mangrove crab Neosarmatium sp. is characterised by 1–1.4 mm long body; outer labial sensilla longer than cephalic sensilla, amphid located at level with posterior stoma chamber, denticles in posterior stoma chamber and five pairs of genital papilla on tail. Phylogenetic relationships of two new species and other nematodes from the family Monhysteridae are analysed based on 18S and partial 28S rDNA sequences.

Monhystrium includes only five known species: M. wilsoni (Baylis, 1915) Cobb, 1920, M. transitans Cobb, 1920, M. inquilinus Riemann, 1969, M. brevis Yoshimura, 1990 and M. tenuis Yoshimura, 1990 which are found exclusively in gill chambers of land crabs in different parts of the world. The first species was discovered in 1910 in the gills of a purple land crab Gecarcinus ruricola (Linnaeus) in Jamaica (Baylis, 1915). The second species was described in 1920 by N.A Cobb from the gill chambers of the same purple land crab and subsequently also from the gills of the blackback land crab G. lateralis (Guérin) from Jamaica (Cobb, 1920). In 1969 F. Riemann described the third species of Monhystrium, M. inquilinus; this new species was also found in the gill chambers of a land crab, but this time in a blue land crab Cardisoma guanhumi caught in Colombia (Riemann, 1969). The last two species to be added to the genus were Monhystrium tenuis and M. brevis, described by K. Yoshimura (1990); M. tenuis was found in the gills of Parasesarma plicatum (Latreille), P. pictum (De Haan) and Clistocoeloma merguiense de Man while M. brevis was found in the gill chamber of the redclawed crab Chiromantes haematocheir (De Haan), C. dehaani (H. Milne Edwards) and Chasmagnathus convexus (De Haan) and also in the gills of flower crabs Orisarma intermedium (De Haan).

Materials and Methods
Sampling and specimen preparation. Nematodes were found during parasitological inspection of land crab specimens obtained from the local inhabitants.
Nematodes were carefully removed from the gills of crabs and immediately preserved in 5% formaldehyde solution, 95% ethanol solution and RNAlater. For light microscopy, specimens were transferred to pure glycerine using Seinhorst's (1959) rapid method as modified by De Grisse (1969). Permanent nematode mounts on glass slides were prepared using the paraffin wax ring method. All curved structures were measured along the curved median line. Terminology follows Maggenti et al. (2005). Abbreviations are according to Hunt & Palomares-Ruis (2012).
Molecular analysis. DNA extraction was performed on two individuals for each species. Individual nematodes were each placed in 1.5 ml microcentrifuge tubes containing 20 ll buffer ATL (Qiagen, Sweden) and stored at -20°C until all samples were ready for extraction. During the extraction, 160 ll of buffer ATL was added to each sample. This was followed by the addition of 20 ll proteinase K, vortexing and incubation in an incubating microplate shaker at 56°C and 300 rpm overnight. The lysed samples were further processed to obtain pure DNA following the manufacturer's instructions for genomic DNA extraction using the Qiagen QiAmp DNA Micro kit. Two regions of the rDNA gene, the nearly full-length of the 18S and the D2-D3 expansion segment of 28S, were amplified. The approximately 1800 bp region of the 18S rRNA gene was amplified as two overlapping fragments using the primer sets 988F-1912R for the first fragment and 1813F-2646R for the second fragment (Holterman et al., 2006). Polymerase chain reaction (PCR) for both fragments was performed in 25 ll reaction mix using Illustra Hot Start Mix RTG 0.2 ml reaction kit (GE Healthcare Life Sciences, Sweden). The reaction mix consisted of 1 ll (0.4 lM) of each primer, 2 ll template DNA and 21 ll nucleasefree water. The reaction conditions were 5 min at 95°C; 5 cycles of (30 sec at 94°C, 30 sec at 45°C and 30 sec at 72°C); 35 cycles of (30 sec at 94°C, 30 sec at 54°C and 30 sec at 72°C); and a final extension for 5 min at 72°C. The D2-D3 segment of the 28S rRNA gene was amplified using the primers D2Af and D3Br (Nunn, 1992). PCR was performed in 25 ll reaction mix containing 1 ll (0.4 lM) of each primer, 2 ll template DNA and 21 ll nuclease-free water. The PCR conditions were 4 min at 94°C; 35 cycles of (94°C for 60 sec, 54°C for 90 sec and 72°C for 2 min); final extension for 10 min at 72°C. Enzymatic PCR clean-up was performed on the PCR product using Exonuclease I and Shrimp Alkaline Phosphatase (New England Biolabs, MA, USA). The purified PCR products were sent out to Macrogen Europe B.V. (Amsterdam, the Netherlands) for sequencing. Each amplicon was sequenced in both directions using the forward and reverse PCR primers. The trace files of the individual sequences were visualized inside BioEdit (Hall, 1999) and trimmed to high quality. The trimmed forward and reverse sequences were then assembled using Fragment Merger online tool (Bell & Kramvis, 2013). The two fragments of the 18S rRNA gene were also assembled into contigs using the Fragment Merger online tool.
Phylogenetic analysis. Alignment from Ahmed and Holovachov (2020) for 18S rRNA gene was used as template for alignment and annotation. New sequences were aligned to a fixed template alignment using AliView (Larsson, 2014). Partial 28S rDNA sequences were aligned de novo in AliView. Phylogenetic trees were built using RAxML ver. HPC2 (Stamatakis, 2014) via the CIPRES portal (Miller et al., 2010) for the Maximum Likelihood inference of the partitioned dataset. The GTR nucleotide substitution model was used for non-paired sites, whereas the RNA7A (Higgs, 2000) substitution model was used for paired sites. Bootstrap ML analysis was performed using the rapid bootstrapping option with 1000 iterations.
Genus diagnosis: Body length medium to long (0.7-4.0 mm). Inner labial sensilla papilliform; outer labial and cephalic sensilla in one circle, same or different in length, with either outer labial or cephalic sensilla being longer than the others. Amphids round; less than 1.5 head diameters from the anterior body end. Ocelli present or absent. Buccal cavity funnel shaped, with weakly to moderately cuticularized anterior chamber of stegostom, and narrow posterior chamber of stegostom; posterior chamber with 3 or 6 denticles or without any. Pharynx cylindrical. Progaster present. Ventral gland well-developed and visible, excretory pore opens along the anterior region of the pharynx or within the labial region. Ovary and testis on the right-hand side of intestine. Spicules simple and narrow, straight to arcuate. Gubernaculum platelike, with or without apophysis. Precloacal supplements may be present. Tail conoid to subcylindrical. Caudal glands opening via common spinneret. Usually found associated with crustaceans.
Type host and locality: The nematodes were found in the gill chambers of the mud crab Scylla serrata (Forskål) collected near Wagap, Poindimié commune, Northern Province, New Caledonia (HYNC4666; July 12, 2018).
GenBank acc. numbers: Sequences obtained are deposited in GenBank under the accession numbers MZ274175 and MZ274176 for the D2-D3 segment of the 28S rRNA gene and MZ274171 and MZ274172 for the nearly full-length 18S rRNA gene.

Description
Diagnosis: Gammarinema scyllae sp. n. is characterised by 3-4 mm long body, small outer labial and cephalic sensilla, distinct ocelli, short straight spicules and sub-cylindrical tail. In gill chambers of the mud crab Scylla serrata. Adult. (Figures 1, 2, 3, Table 1). Body cylindrical, ventrally curved upon fixation in females and in males, tapering slightly towards both extremities along pharyngeal region and on tail. Cuticle smooth. Somatic sensilla present, small setiform. Body pores absent. Lateral alae absent. Cephalic region flattened. Six equal lips surrounding mouth opening. Inner labial sensilla small, located on anterior surface of lips.
Outer labial sensilla small setiform, located at the base of lip region. Cephalic sensilla papilliform, located at the same level as labial sensilla. Amphideal opening round, located at level of stoma base. Ocelli present. Buccal cavity conoid in general, cheilostom and gymnostom both cylindrical and very short; stegostom funnel-shaped (conoid anteriorly and tubular posteriorly), with weakly cuticularised walls. Posterior chamber poorly defined, denticles indistinct, edges of pharyngeal radii visible. Pharynx uniformly muscularized along its entire length, gradually widening posteriorly but without any valves or bulbs. Cardia small and conical. Intestinal lumen well developed, progaster present. Secretory-excretory system and secretory-excretory pore present, renette cell located along anterior part of intestine, excretory pore located about 1.2 times labial region diameter from the anterior end. Tail cylindrical, with broadly rounded terminus. Caudal glands present, opening towards extension via a common spinneret. Caudal gland cells located in the tail. Spinneret not cuticularised.
Female. Reproductive system monodelphic. Ovary branch outstretched, extends anterior but not reaching the cardia, on the right-hand side of intestine. Postvulval uterine sac absent. Vagina directed anteriorly.
Vulva located posterior to midbody, a transverse ventral slit, not cuticularized.
Male. Reproductive system monorchic, on the right-hand side of intestine. Spicules paired and symmetrical, straight and relatively thin conoid, equal to 0.7-0.8 anal body diameters in length. No pre-or post-cloacal sensilla or supplements. rRNA. Sequences include two nearly full length 18S rRNA gene and two partial 28S rRNA gene representing D2D3 domain. Sequence variability of the 18S rRNA gene was small, less 1-7 bases difference; sequences of D2D3 domain of 28S rRNA gene were identical. This is the first species from the genus Gammarinema to be sequenced.

Relationships
None of the previously described species was sequenced, therefore the differentiation of new species is based on morphological characters (see also Table 2), host and geographic distribution.
Gammarinema ampullocauda is smaller than G. scyllae sp. n. with body length of 1.88-2.16 mm compared to 2.97-3.74 mm in G. scyllae sp. n. The a-value of the G. ampullocauda is 46-48 and in G. scyllae sp. n. it is 55-76. The tail is shorter in G. ampullocauda (175-180 lm) than in G. scyllae sp. n.
Gammarinema cambari is described very superficially. The new species is much bigger than G. cambari with body length reaching 2.97-3.74 mm in new species compared to 0.77 mm in G. cambari. The shape of the tail differs between two species, the tail of G. scyllae sp. n. is sub-cylindrical and the tail of G. cambari is conoid. The spicules are arcuate in G. cambari but straight in G. scyllae sp. n. G. cambari was found in North Carolina in the USA and uses Cambarus acuminatus and C. blandingii as a host, while G. scyllae sp. n. was found in New Caledonia.
Gammarinema scyllae sp. n. is more than twice as big as G. cardisomae that has body length of 1.12-1.16 mm compared to 2.97-3.74 mm in G. scyllae sp. n. Moreover, G. scyllae sp. n. has higher values of many ratios than G. cardisomae: a-value is 44-50 in G. scyllae sp. n. compared to 55-76 in G. cardisomae, b-value is 5.8-6.0 in G. scyllae sp. n. compared to 7.1-8.7 in G. cardisomae and c-value is 8.8-9.1 in G. scyllae sp. n. compared to 10.8-15.1 in G. cardisomae. Exception is a c-value where G. cardisomae has higher value of 6.0-7.9 compared to 4.7-6.3 in G. scyllae sp. n. The tails of the two species are the same in shape but differ in length: 128 lm in G. cardisomae and 201-270 lm in G. scyllae sp. n. G. cardisomae has three teeth while teeth are absent in G. scyllae sp. n. The spicules are different in shape in the two species, the spicules in G. cardisomae are arcuate and in G. scyllae sp. n. the spicules are straight. G. cardisomae has a gubernaculum with apophysis which is absent in the G. scyllae sp. n. G. cardisomae was found in Colombia, South America.
Gammarinema scyllae sp. n. is somewhat bigger than G. gammari with a body length ranging from 1.62-2.99 mm in the latter and 2.97-3.74 mm in the former. The a-, b-c-ratios are similar/overlapping, but the c-value is different, 6-10 in the G. gammari compared to 4.7-6.3 in G. scyllae sp. n. The amphid is located 9-12 lm from the anterior end in G. gammari compared to 6-8 lm in G. scyllae sp. n. G. gammari has six small teeth while teeth are absent in the G. scyllae sp. n. G. gammari has slightly larger (39-57 lm) and weakly arcuate spicules while G. scyllae sp. n. has shorter (32-36 lm) and nearly straight spicules.
G. gammari has a small plate-like gubernaculum, whereas gubernaculum is absent in G. scyllae sp. n. G. gammari uses multiple species of Gammarus as host and is found in Northern Europe.
Gammarinema ligiae is smaller with a body length of only 0.87-1.18 mm while G. scyllae sp. n. is 2.97-3.74 mm long. Body proportions are also different: a-value is 28-43 in G. ligiae and 55-76 in G. scyllae sp. n., b-value is 5.8-7.0 in G. ligiae and 7.1-8.7 in G. scyllae sp. n., c-value is 8.3-10.8 in G. ligiae and 10.8-15.1 in G. scyllae sp. n. The tail is shorter (90-130 lm) in G. ligiae than in G. scyllae sp. n. (201-270 lm). The amphid in G. ligiae is positioned more posterior from the anterior end (13 lm) than in G. scyllae sp. n. (6-8 lm). G. ligiae has six small teeth that G. scyllae sp. n. lacks. The spicules are straight and slightly larger (32-36 lm) in G. scyllae sp. n. while they are weakly arcuate and smaller (28-31 lm) in G. ligiae. The gubernaculum is present and has apophysis in G. ligiae but is absent in G. scyllae sp. n. G. ligiae stands out from other species of the genus by having cuticularised vulva. G. ligiae is Gammarinema scyllae sp. n. is bigger than G. mesidoteae with a body length of 2.97-3.74 mm compared to 2.31-2.95 mm in G. mesidoteae. The cephalic sensilla are slightly longer (4-5 lm) in G. mesidoteae than in G. scyllae sp. n. (1.5-3.5). The amphid is positioned 10-13 lm from the anterior end in G. mesidoteae and 6-8 lm in G. scyllae sp. n. The spicules in G. mesidoteae are both longer 36-39 lm and have a different shape (weakly arcuate) compared to G. scyllae sp. n. that has 32-36 lm long straight spicules. G. mesidoteae has gubernaculum in the shape of a small plate while gubernaculum is completely absent in G. scyllae sp. n. G. mesidoteae lives in Far East and is known to have Mesidotea entomon as a host.
Gammarinema paratelphusi is described very superficially, with few measurements given, and with some unusual morphological features, such as cephalic sensilla arranged in pairs or pre-and postcloacal sensilla present in males. The new species can be easily separated from G. paratelphusi in having shorter (32-36 lm) and differently shaped (straight) spicules, which are weakly arcuate and 55-58 lm long in G. paratelphusi, absence of teeth in stoma (present in G. paratelphusi), absence of pre-and postcloacal sensilla in male (present in G. paratelphusi). G. paratelphusi was found in India in a freshwater crab Paratelphusa sp.
Gammarinema prilepskyi has 2.4 mm long body while G. scyllae sp. n. is 2.97-3.74 mm long. The a-value differs between the two species, 35-48 in G. prilepskyi and 55-76 in G. scyllae sp. n. Same with c-value which is 16.1-22.1 in G. prilepskyi and 10.8-15.1 in G. scyllae sp. n. The tail is cylindrical with digitate tip in G. prilepskyi and sub-cylindrical in G. scyllae sp. n. The cephalic sensilla are longer in G. prilepskyi (6-9 lm) than in G. scyllae sp. n. (1.5-3.5 lm). The length and shape of spicules are different with G. prilepskyi having weakly arcuate 52 lm long spicules, while the spicules in G. scyllae sp. n. are Table 2 Comparison between different species of the genus Gammarinema straight and 32-36 lm long. G. prilepskyi has small plate-like gubernaculum and G. scyllae sp. n. has none. The geographic distribution of G. prilepskyi is limited to the Barents Sea and the host is Mesidotea entomon.
Genus diagnosis: Body length medium to long (0.8-1.7 mm). Inner labial sensilla papilliform; outer labial and cephalic sensilla in one circle, same or different in length, with either outer labial or cephalic sensilla being longer than the others. Amphids round; less than 1.5 head diameters from the anterior body end. Ocelli present or absent. Buccal cavity cuticularized and distinctly divided into two chambers: anterior chamber conoid; posterior chamber spherical, with sharp tooth-like anterior edges, with or withour denticles. Pharynx cylindrical. Progaster present. Ventral gland present or absent, excretory pore opens along the anterior region of the pharynx. Ovary and testis on the right-hand side of intestine. Spicules simple and narrow, weakly arcuate. Gubernaculum platelike. Precloacal spine may be present. Bursa present. Paired subventral papilla present, pre-and postcloacal, within or outside bursa. Tail conoid to subcylindrical, digitate. Caudal glands opening in via common spinneret. Usually found in gill chambers of crustaceans.
Note: The genera Monhystrium and Diplolaimelloides Meyl, 1954 are close morphologically, with Diplolaimelloides delyi Andrássy, 1958 being also found in the gill chambers of land crabs, the only clear morphological difference between them is the more strongly developed and cuticularized posterior stoma chamber with inward-pointing tooth-like anterior edges in Monhystrium. Sequenced species from two genera do not form a monophyletic lineage (Figure 7), however, the limited taxon sampling seriously undermines our understanding of the phylogeny of this group in general.

Monhystrium mangrovi sp. n.
Type host and locality: The nematodes were found in the gill chambers of the mangrove crab Neosarmatium sp. collected near Poya, Poya commune, west coast of the Northern Province, New Caledonia (HYNC4625; July 3, 2018).
Type material: Holotype male and eleven juvenile paratypes on slides MNHN-BN512 and MNHN-BN13 are deposited in the meiofauna collection of the National Museum of Natural History in Paris, France (MNHN). Three females and 12 male paratypes on slides SMNH Type-9351 and SMNH Type-9352 are deposited in the Invertebrate type collection of the Department of Zoology, Swedish Museum of Natural History, Stockholm, Sweden.
Etymology: The species name mangrovi refers to the host of this species, a species of a mangrove crab.
GenBank acc. numbers: Sequences obtained are deposited in GenBank under the accession numbers MZ274177 and MZ274178 for the D2-D3 segment of the 28S rRNA gene and MZ274173 and MZ274174 for the nearly full-length 18S rRNA gene.

Description
Diagnosis: Monhystrium mangrovi sp. n. is characterised by 1-1.4 mm long body; outer labial sensilla longer than cephalic sensilla, amphid located at level with posterior stoma chamber, denticles in posterior stoma chamber and five pairs of genital papilla on tail. In gill chambers and body cavity of mangrove crab Neosarmatium sp.
Adult. (Figures 4, 5, 6, Table 3). Body cylindrical, nearly straight upon fixation, tapering slightly towards both extremities along pharyngeal region and on tail. Cuticle smooth. Somatic sensilla present, small setiform. Body pores absent. Lateral alae absent. Cephalic region rounded. Six equal lips surrounding mouth opening. Inner labial sensilla papilliform, located on anterior surface of lips. Outer labial sensilla setiform, located at the base of lip region. Cephalic sensilla small papilliform, located at the same level as outer labial sensilla. Amphideal opening round, located at level of posterior stoma chamber. Ocelli absent. Buccal cavity composed of two chambers. Anterior chamber of buccal cavity conical. Posterior chamber broad spherical with multiple teeth on the anterior part of its ventral surface. Pharynx uniformly muscularized along its entire length, gradually widening posteriorly but without any valves or bulbs. Cardia small. Intestinal lumen well developed, progaster present. Secretory-excretory system and secretory-excretory pore not visible, absent. Tail conical, with digitiform terminus. Caudal glands present, opening towards exterior via a common spinneret. Caudal gland cells located in the tail. Spinneret not cuticularised. Female. Reproductive system monodelphic. Ovary branch outstretched, extends anterior but not reaching the cardia, on the right-hand side of intestine. Postvulval uterine sac absent. Vagina directed anteriorly. Vulva located posterior to midbody, a transverse ventral slit, not cuticularised.
Male. Reproductive system monorchic, on the right-hand side of intestine. Spicules paired and symmetrical, arcuate, very long, curved ventral, with angular manubrium and cylindrical shaft, thin velum and lateral projections near their tips equal to 2.1-3.0 anal body diameters in length. Gubernaculum platelike. No midventral pre-or post-cloacal supplements. Precloacal spine present, short distance in front of cloacal opening. Bursa well-developed, starts anterior to cloaca and extends to the posterior third of tail. Five pairs of genital papillae present: one pair just posterior to cloaca, three pairs near posterior end of bursa and one pair just behind bursa rRNA. Sequences include two nearly full length 18S rRNA gene and two partial 28S rRNA gene representing D2D3 domain. Sequence variability of 18S rRNA gene was small, less than 1-3 bases difference; sequences of D2D3 domain of 28S rRNA gene were identical. This is the first species from the genus Monhystrium to be sequenced.

Relationships
None of the previously described species was sequenced, therefore the differentiation of the new species is based on morphological characters (see also

Phylogenetic position of Gammarinema and Monhystrium
Two genes were used to build two different phylogenetic trees, 18S (Fig. 7) and 28S rDNA (Fig. 8), with members of the family Linhomoeidae being used as outgroups. The phylogeny based on 18S rDNA was the one most reliable with reasonably high bootstrap support values overall. It also covered a broader taxonomic diversity. The 28S rDNA tree included fewer taxa and showed lower bootstrap support for many clades. Both trees suggest Gammarinema scyllae sp. n. is most closely related to Monhystrium mangrovi sp. n., both belonging to the family Monhysteridae. Current phylogeny indicates that both genera, Gammarinema and Monhystrium originate from a recent common ancestor. Diplolaimella and Diplolaimelloides were recovered as the closest relatives to the two genera and the four form a very well supported clade, in agreement with morphology-based theories. Moreover, the fact that all known species of Monhystrium are found exclusively in gill chambers of different land crabs and eight out of nine known species of Gammarinema are associated with crustaceans suggests that the common ancestor was also associated with crustaceans. Future studies should  focus on sequencing other species from Gammarinema, Monhystrium and also of closely related commensalistic (Odontobius, Tripylium, Diplolaimelloides delyi) and free-living species to better understand the phylogeny of this group. Although the phylogeny did not support the subfamilies Diplolaimellinae and Monhysterinae, the family Monhysteridae received maximal support.
Availability of data and material All studied specimens are deposited in permanent and accessible repositories: National Museum of Natural History in Paris, France and Swedish Museum of Natural History, Stockholm, Sweden. Sequences are deposited in GenBank.
Code availability Not applicable.

Declarations
Conflict of interest The authors declare that they have no conflict of interest.
Ethics approval All applicable institutional, national and international guidelines for the care and use of animals were followed.
Consent to participate Not applicable.

Consent for publication Not applicable.
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