Neoechiniscoides aski, 2020

Møbjerg, Nadja, Jørgensen, Aslak & Kristensen, Reinhardt M., 2020, Ongoing revision of Echiniscoididae (Heterotardigrada: Echiniscoidea), with the description of a new interstitial species and genus with unique anal structures, Zoological Journal of the Linnean Society 188 (3), pp. 663-680 : 669-671

NEOECHINISCOIDES ASKI MØBJERG, JØRGENSEN & KRISTENSEN SP. NOV.

Diagnosis: Interstitial, medium-sized Neoechiniscoides with six to nine claws on each leg and 8,8,8,7 as the most common claw formula. Large black to dark brown eyes. Dorsal cuticle with star-like, polygonal structures arranged in transverse rows that continue ventrally on the head. Abdomen with a smooth cuticle, but polygonal structures are present on lateral anal lobes.The mouth is subterminaland lacksdorsalmouthplates; instead, three circular structures are present surrounding the mouth opening. The stylets and buccal tube are relatively short. Stylet supports are not observed using light microscopy. Calcium carbonate incrustations are found only in the anterior part of the pharyngeal placoids. Cirrus Aand E have a single spiral sculpture at the basis. Internal and external cirri have swollen bases. Spine-shaped setae are present on leg III. Legs Iand II have small domeshaped sensory organs, and leg IV has a papilla similar to the primary clava. Females have a flat and males a more raised, nearly papillary secondary clava. The male gonopore is a five-lobed, nearly penis-like structure (not ovoid). The female gonopore is a six-lobed rosette located on a ring-shaped protrusion. Asmall ventral plate is present in front of the female gonopore, and two small depressions (interpreted as muscle attachment sites) are present lateral to the female gonopore. Males lack this ventral plate; the muscle attachments are present, but placed more anteriorly with respect to the male gonopore. Both sexes have enlarged lateral anal lobes (neochiniscoidid anal system).

Etymology: Named for Ask Møbjerg Jørgensen, who in turn is named for the Nordic mythological ‘Tree of Life’, Ask Yggdrasil.

Holotype ( Figs 2D, F , 4 ; Table 1 ): Female, 301 µm long, with mature ovary (three oocytes) and welldeveloped six-lobed gonopore. The animal is in moult. Deposited at The Natural History Museum of Denmark ( NHMD-230293 ) .

Type locality: Collected 17 July 2013 in the L’Aber de Roscoff, Brittany, France (48°43′17.251″N, 04°00′26.522″W) by Nadja Møbjergand Aslak Jørgensen. Collected from oxic, sandy sediment with few stones .

Allotypic paratype ( Figs 2E , 5 ; Table 1 ): Male, 228 µm long, with large secondary clavae, mature spermatozoa in the seminal vesicles and well-developed papillary gonopore. Deposited at The Natural History Museum of Denmark ( NHMD-230297 ). Collected 6 July 2012 in the L’Aber de Roscoff , Brittany , France (48°43′17.251″N, 04°00′26.522″W) by Nadja Møbjerg and Aslak Jørgensen. Collected from oxic, sandy sediment with few stones .

Other paratypes ( Figs 2C, 2G, 2H , 3 ; Table 1 ): In total, 14 additional paratypes were collected at the same locality as the holotype and allotype in the periodfrom 6 July 2012 to 17 July 2013. Seven specimens (six females and one male) were mounted on microslides in glycerol and sealed with Glyceel ( NHMD-230294 to 230302 ). Seven paratypes (four females and three males) were mounted on three SEM-stubs ( NHMD-230303 to 230305 ) and used for scanning electron microscopy. All specimens were deposited at The Natural History Museum of Denmark. Additionally, digital images of four voucher specimens that have been used for molecular work exist ( Fig. 2B ; see Møbjerg et al., 2016: Echiniscoides sp. B / T 16, GenBank accession numbers KX 363644 and KX 363655 ; Echiniscoides sp. B/T18, GenBank accession numbers KX363645 and KX363656 ; Echiniscoides sp. B/T20, GenBank accession number KX363657 ; and Echiniscoides sp. B/ T21, GenBank accession number KX363658 ) .

Description ( Figs 2–6 ; Table 1 ): The collected specimens have a measured body length ranging from 207 (smallest males) to 301 µm (holotypic female). The most common claw formula of N. aski seems to be 8,8,8,7, but the number of claws varies significantly between and within specimens, as illustrated by the holotype, which has a different number of claws on the left (8,9,8,7) and the right sides (8,8,8,8), respectively ( Table 1 ). The dorsal cuticle is characterized by a polygonal granulation that continues ventrally on the head. The polygons are between 2 and 5 µm in size, with the size of the granulation decreasing laterally and in depressions of the cuticle. Each polygon has several fibres that connect to neighbouring polygons. The dorsal cuticle is modified in connection with muscle attachment sites ( Figs 3F , 5 ). Specifically, five dorsal cuticular attachment sites (without polygon structure) were observed on the caudal segment. The buccopharyngeal tube is relatively short, and the buccopharyngeal apparatus appears similar to that of Echiniscoides sigismundi sigismundi (M. Schultze, 1865) . The sensory structures are also of E. s. sigismundi type: black to dark brown eyes, two small domes (p1 and p2) on leg Iand II, respectively, and p3 is a seta. The sensory structure on leg IV is similar to the primary clava; both are a papillary structure with a tiny spine. Cirri Aand Eare short, with a few spiral striations at the base. The secondary clava appear different in males compared with females. In females, they seem flatter and are surrounded by a ring of unsculptured cuticle ( Figs 3C , 4 ), whereas the male has a slightly more protruding secondary clava that can be seen from the dorsal side ( Figs 3H , 5 ). In both sexes, the secondary clava attach directly on the head (not as a projection as in E. s. sigismundi ). The gonopore of the female is unique within the Echiniscoididae and is formed of six large units positioned on a ring-shaped cuticular protrusion. Two small depressions ( Figs 3B , 4 ) are situated close to the gonopore; we interpret these as muscle attachment sites. Asmall cuticular plate is present in front of the gonopore ( Figs 3B , 4 ); males lack this structure. The anal system consists of three lobes: two lateral and a terminal lobe. The anus appears as an opening at the junction between the three lobes. The lateral lobes of the anal systems appear as a double structure, with two wings ( Figs 2A, F , 3B, G , 4 , 5 , 6 ). We have no transmission electron microscopy of these lobes, but hypothesize that they might be glandular.

Figures

Figure 2. Light microscopy of Neoechiniscoides aski. A–C, images of live, moving specimens. A, ventral view of a live adult female, showing the anal system with lateral wings. In live animals, the conspicuous anal complex moves from side to side when the animal walks/runs. Scale bar: 50 µm. B, dorsal view of live adult female used for DNA extraction (28S GenBank accession number KX363645; COI GenBank accession number KX363656). Note the eight to nine mature oocytes. Scale bar: 50 µm. C, dorsal view of live adult male. Scale bar: 50 µm. D, holotype of N. aski (female). Scale bar: 50 µm. E, paratypic allotype of N. aski (male). Scale bar: 50 µm. F, ventrocaudal view of holotypic female showing anal system and the gonopore, which is raised on an elevation (note that seminal receptacles are lacking). Scale bar: 20 µm. G, ventral view of paratypic

Figure 3. Scanning electron microscopy of Neoechiniscoides aski. A–C, female. A, habitus of mature female. Scale bar: 10 µm. B, close-up of gonopore with associated cuticular plate and anus. Note muscle attachment sites located lateral to the female gonopore. Scale bar: 10 µm. C, close-up of head region. Note the cuticular sculpture of the head. Scale bar: 10 µm. D–H, male. D, second leg, with dome-shaped sense organ (p2). Scale bar: 1 µm. E, habitus of adult male. Scale bar: 10 µm. F, prominent dorsal cuticular attachment sites of the caudal segment. Scale bar: 1 µm. G, posterioventral part of a male, showing the fourth leg pair, anal system and gonopore. Anterior is to the left. Scale bar: 10 µm. H, close-up of head region of male. Note the relatively large secondary clavae. Scale bar: 1 µm. Abbreviations: an, anus; cE, cirrus E; cl, claw; ec, external

Figure 4. Neoechiniscoides aski.Drawing of holotypicfemale (NHMD-230293),ventral view.Scale bar:50 µm.Abbreviations: an, anus; bt, buccal tube; cE, cirrus E; cm, claw membrane; ec, external cirrus; ey, eye; go, gonopore; ic, internal cirrus; lc, lateral cirrus (cirrus A); ma, lateral attachment sites associated with the female gonopore; mg, midgut; nc, new claws forming inside claw gland; oo, oocyte; ov, ovary; p1–p3, sensory structures of legs I–III, respectively; pa, ventral cuticular plate; pb, pharyngeal bulb; pc, primary clava; pl, placoid; sc, secondary clava; wl, lateral wing of the anal structure.

Figure 5. Neoechiniscoides aski. Drawing of allotypic male (NHMD-230297), dorsal view. Scale bar: 50 µm. Abbreviations: as, anal system; cE, cirrus E; cl, claw; ds, dorsal cuticular attachment sites; ec, external cirrus; fu, furca of the stylet; ic, internal cirrus; lc, lateral cirrus (cirrus A); mg, midgut; p1–p4, sensory structures of legs I–IV, respectively; pc, primary clava; sc, secondary clava; sp, spermatozoa; sv, seminal vesicle; te, testicle.

Figure 6. Drawing of anal system in Isoechiniscoides, Echiniscoides and Neoechiniscoides based on investigations of live animals and preparations on slides for light microscopy. In all three genera, a three-lobed anal system is present and consists of two lateral lobes and a terminal lobe of variable size. The anal opening is indicated in black. In Neoechiniscoides, two wings are present on the lateral lobes. These wings are easily visualized in live animals and using light microscopic preparations, whereas this double structure of the lateral lobes is difficult to visualize in specimens prepared for scanning electron microscopy. Scale bar: 10 µm.