Tardigrades, or Water Bears, are a distinctive group of small (usually
less than 1 mm) invertebrates related to Arthropods, Nematodes and
Velvet Worms. They have a simple segmented body with four pairs of
limbs, and are remarkably resilient to environmental stress, being able
to withstand extremely high and low temperatures, complete desiccation
and even exposure to vacuum. Marine Tardigrades, specifically Arthrotardigrades, exhibit remarkable morphological diversity. The Styraconyxidae is one of the Arthrotardigrade families and it is currently comprised of 38 species and subspecies placed withingten genera: Angursa (eight species), Bathyechiniscus (one species), Lepoarctus (one species), Paratanarctus (one species), Pleocola (one species), Raiarctus (five species), Rhomboarctus (three species), Styraconyx (15 species and subspecies), Tetrakentron (one species), and Tholoarctus (three species and subspecies). In addition to these ten genera, an undescribed genus related to Styraconyx and Tetrakentron has been reported from a submarine cave in Japan, although no formal description of that species has ever been made and two voucher micrographs of a specimen used for a molecular phylogenetic study have been published.
In a paper published in the journal Zoosystematics and Evolution on 23 March 2020, Shinta Fujimoto of the Research Center for Marine Biology at Tohoku University, and Naoto Jimi of the Japanese National Institute of Polar Research formerly describe this submarine cave-dwelling Tardigrade.
Specimens were collected from Daidokutsu, a submarine cave off Iejima Island in the Okinawa Islands, part of the Ryukyu Archipelago, Japan, by Koshin Yasumura and Shinta Fujimoto in 2013 and 2019. For extraction of meiofauna, the cave sediment samples were stirred with tap water and the supernatants were concentrated using a 30 μm opening mesh net to separate coarse sediment and to wash away seawater. Subsequently, the meiofauna and fine sediment were separated using LUDOX HS-40 colloidal silica and a 32 μm opening mesh net. The type material was sorted under a stereomicroscope and fixed in 2–4% buffered formaldehyde. Specimens for light microscopy were mounted in distilled water for brief observation and mounted in glycerol. Differential interference contrast microscopy was conducted using an Olympus BX53 and phase contrast microscopy was conducted using an Olympus BX41. One specimen for scanning electron microscopy was post-fixed in 2% OsO₄ for 2 hours, dehydrated through a series of ethanol and acetone washes, critical point dried, osmium coated, and observed using a JEOL JSM-7001F Schottky Emission Scanning Electron Microscope. Type material was deposited in the Zoological Collection of Kyoto University. Adobe Illustrator CS6 and Photoshop CS6 were used to prepare figures and to obtain morphometric data.
The species is placed in a new genus, named Cyaegharctus, which is a combination of 'Cyäegha-', a deity of darkness and caves in the writings of Eddy Bertin, and '-arctus', a Latinised Greek word meaning 'bear', commonly used as a suffix when naming Tardigrades, and given the specific name kitamurai, in honour of Akihisa Kitamura of Shizuoka University, who has been studying Daidokutsu Cave and its Bivalve assemblage. The species is described from four specimens, three adult females and a juvenile, all collected from Daidokutsu Cave.
The holotype of Cyaegharctus kitamurai (when describing a new species
one specimen is designated the holotype; all future specimens
determined to belong to the same species as this holotype therefore
bellong to the species) is an adult female with a dorso-ventrally flattened body 202 μm in length and 117 μm wide at level of leg III. The cephalic region (head) has an unpaired median cirrus (tendril), paired internal cirri, paired external cirri, paired lateral cirri, paired primary clavae (clublike structures), paired secondary clavae and an antero-ventral directed mouth. Paired spine-like cirri (38 μm) on cirrophores (stalks) arise from round lateral processes at level between legs III and IV. A rosette-like gonopore (genital opening) is 9 μm anterior to anus. There are four pairs of legs, each with an usual leg sensory organ on the dorsal side of the femur’s proximal portion, a pocket organ on the dorsal side of femur’s distal margin and four digits terminating in claws.
Drawings of Cyaegharctus kitamurai, holotype KUZ Z2624. (A) Habitus (ventral view). (B) Leg IV pocket organ. Abbrreviations: an, anus; bt, buccal tube; ca, cavity; cE, cirrus E; db, dense body; ec, external cirrus; go, female gonopore; ic, internal cirrus; lc, lateral cirrus; mc, median cirrus; pc, primary clava; pl, placoid; op, opening; sc, secondary clava; soₗ, ₗᵥ legs I and IV sensory organs; sr, seminal receptacles; ss, stylet support; st, stylet. Fujimoto & Jimi (2020).
Fixation of specimens using formaldehyde seems to have introduced an artefact in the cuticle, i.e. the detached (or loose) outer epicuticle. When the specimens were sorted in distilled water before fixation, the outer epicuticle did not look loose at 63× magnification (all four specimens) and also at 400× magnification (only observed for KUZ Z2627) as it would appear in Tholoarctus. Although these are only brief observations and Fujimoto and Jimi did not conduct any experiment to test this artefact, they consider the outer epicuticle’s detached state as an artefact and excluded this character state from the diagnoses of this genus and species.
The adult female paratype KUZ Z2625 revealed the presence of a cuticular ring surrounding the rosette gonopore and the presence of the spine-like leg III sensory organs with no subdivisions. In the adult female paratype KUZ Z2626, the legs I–III sensory organs, claws and peduncles were orientated better than the holotype and the other paratype for observation. However, the pocket organs were not recognised for this specimen, probably due to the excessively-squeezed state.
DIC and PhC micrographs of Cyaegharctus kitamurai, adult female. (A) Habitus (dorsal view), (B) cephalic region (ventral view), (C) lateral cirrus. (D) buccal apparatus, (E) caudal region (ventral view) (epicuticle pillars visible), (F) leg I sensory organ and pocket organ, (G) leg II pocket organ (arrowhead indicates protruding portion), (H) leg II digits and claws, (I) leg III pocket organ, (J) leg IV pocket organ, (K) female gonopore, (L) legs I–III sensory organs, (M) leg III digits and claws. (A)–(J) holotype KUZ Z2624, (K) paratype KUZ Z2625, (L), (M) paratype KUZ Z2626. Abbreviations: an, anus; bt, buccal tube; ca, cavity; cE, cirrus E; db, dense body; ec, external cirrus; go, gonopore; ic, internal cirrus; lc, lateral cirrus; mc, median cirrus; pc, primary clava; peᵢ,ₑ, peduncles of internal and external digits; pl, placoid; poₗ–ₗᵥ, legs I–IV pocket organs; pp, proximal pad; sc, secondary clava; soₗ–ₗᵥ, legs I–IV sensory organs; sr, seminal receptacles; ss, stylet support; st, stylet. Fujimoto & Jimi (2020).
The scanning electron micrographs of a four claw juvenile confirmed the results of light microscopy and also provided further detail. However, this scanning electron micrograph specimen also seems to have its outer epicuticle detached. If the outer epicuticle is attached to the underlying layer, a pattern is recognised on the surface of the body due to the pillar layer, but no such indentations were found, suggestive of the detached state of the outer cuticle. The proximal part of each leg has an inflated appearance not recognised in light microscopy. The view of the cephalic region revealed the three-dimensional morphology and the arrangement of the cephalic appendages and also confirmed the presence of terminal pores on the cephalic cirri and the primary clavae. The conical shape of the secondary clavae seems not as evident as in light microscopy probably due to the overlying outer epicuticle. The large anus is not on the ventral surface and rather direct posteriorly. The leg sensory organs were recognised and those of legs I and IV revealed to have terminal pores. The pocket organs were recognised on all legs, however, with slightly different degrees of protruded appearances. The protruded state might be an artefact caused during specimen preparation since specimens on microscope slides do not always have these appearances or the pocket organs are capable of moving. In the latter case, since no muscles seem to be attached to the structures, a passive movement is likely. Regarding claw/digit morphology, the peduncles (internal structures) were not recognised but the three hooks of the internal claws and the single-pointed external claws were recognised.
Scanning electron micrographs of Cyaegharctus kitamurai, four-claw juvenile paratype KUZ Z2627. (A) Habitus (lateral view), (B) cephalic region (frontal view), (C) cephalic region (ventral view), (D) anus, (E) leg I sensory organ, (F) leg II sensory organ, (G) leg III sensory organ, (H) leg IV sensory organ, (I) leg IV pocket organ, (J) leg III digits and claws. Abbreviations: ah, accessory hook; an, anus; cE, cirrus E; ec, external cirrus; fe, femur; ic, internal cirrus; lc, lateral cirrus; mc, median cirrus; pc, primary clava; ph, primary hook; poₗ, ₗᵥ, legs III and IV pocket organs; pp, proximal pad; sc, secondary clava; sh, secondary hook; soₗ–ₗᵥ, legs I–IV sensory organs; ti, tibia. Fujimoto & Jimi (2020).
Fujimoto and Jimi consider the dense body inside the pocket organs of Cyaegharctus kitamurai to be related to the van der Land’s body, often situated at the base of the primary clavae and leg IV sensory organs and suggest that the pocket organs are chemoreceptors. However, only one previous study, in 1981, investigated the function and ultrastructure of Arthrotardigrade sensory organs by transmission electron microscopy and, with our poor knowledge on Arthrotardigrade sensory organs, this remains a matter of speculation. Another possibility for the new structure is a secretory organ. However, no gland was recognised in its vicinity and there is no evidence supporting this hypothesis. To understand the true functions and evolutionary significance of the pocket organs, comparative ultrastructure studies of Arthrotardigrades (including Cyaegharctus kitamurai) are necessary.
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