Hansenocaris demodex, Olesen & Dreyer & Palero & Eibye-Jacobsen & Fujita & Chan & Grygier, 2022

Olesen, Jørgen, Dreyer, Niklas, Palero, Ferran, Eibye-Jacobsen, Danny, Fujita, Yoshihisa, Chan, Benny K. K. & Grygier, Mark J., 2022, Integrative taxonomy of crustacean y-larvae (Thecostraca: Facetotecta) using laboratory-rearing and molecular analyses of single specimens, with the description of a new vermiform species, Zoological Journal of the Linnean Society 196, pp. 549-592 : 570-578

HANSENOCARIS DEMODEX OLESEN, DREYER, PALERO & GRYGIER [SENTENCE CASE CAPS]. SP. NOV.

( FIGS 3–13 , 14A )

Z o o b a n k r e g i s t r a t i o n: u r n: l s i d: z o o b a n k. org:act: 9FADB53F-9068-424E-9AC2-223C834E87A5.

Diagnosis: Last-stage nauplius (LSN) with markedly elongate body tapering gradually towards bluntly rounded caudal end, with no distinct narrowing behind posterior end of cephalic shield in dorsal view. Dorsal region of cephalic shield posterior to poorly-defined ‘window’ plate smooth, wholly unfaceted, and devoid of ridges; surfaces between ridges on rest of shield also smooth. Plate I-1 of cephalic shield becoming subdivided by LSN − 3(?) instar. Anterior part of faciomarginal area with at least one obvious pair of pores. Labrum triangular or slightly bell-shaped, longer than wide, lacking any pattern of ridges; its posterior margin a shallowly indented declivity lacking any free posterior plate-like extension or labral spine. First antennae with four setae. Second antennae and mandibles devoid of feeding structures (lecithotrophic); natatory setal formulae of their exopods/endopods 1:1:1:1:2/0:1:1:1:2 and 2/2, respectively. Maxillules absent. Pair of reduced furcal spines situated ventrally, forward from base of short and blunt, terminal dorsocaudal spine. Dorsocaudal organ and lateral trunk spines absent.

Y-cyprid with long, fully faceted cephalic shield with nearly smooth surfaces between ridges. Small, bifid appendix in anterior midline between first antennae and anterior margin of cephalic shield. Labrum extended as linguiform process with multiple hooks (17 in holotype) on posterior side, arranged in three irregular rows. First antenna with gracile, curved hook. Second antennae and mandibles rudimentary. Thoracopods with unsegmented exopods. Tergites of thoracomeres V and VI with free pleural extensions, those of former with rounded ends, those of latter trapezoidal. Abdomen three-segmented, all segments short and lacking pleural extensions. Telson long and lacking serrate spines along posteroventral margin, with about 19 pores, including two each on anteriormost plates of upper and lower lateral rows. Furcal rami short and cylindrical.

Etymology: The specific name, a Latin noun in apposition to the generic name, refers to the resemblance of the elongate body form of the naupliar instars of this species to that of mites of the genus Demodex Simon, 1842 (Chelicerata: Trombidiformes : Demodicidae ), which are tiny parasites that live in or near the hair follicles of mammals. The name in turn is derived from Greek δηµός (dēmos), fat, and δήξ (dēx), a woodworm. The taxonomic description and supporting molecular diagnosis of this new species were prepared by JO, ND, FP and MJG, who are thus responsible for making the specific name demodex available.

Type locality: Japan, Okinawa, Sesoko Island, pier at the University of the Ryukyus Tropical Biosphere Research Center, Sesoko Station, 26°38’09.3"N 127°51’55.2"E.

Holotype: Cyprid with exuvium of corresponding last-stage nauplius (LSN), considered as two separate parts of same individual specimen (cyprid: Figs 7A, B, D–I , 8B, C , 9A, B, D, E ; LSN exuvium: Fig. 3A , top). Collected on 15 October 2018 (detailed sampling data in Table 3 ), fixed on 21 October 2018, following final moult after six days in culture. Cyprid stored in dried condition on SEM stub, LSN exuvium as glycerine jelly microscope slide, both at the Natural History Museum of Denmark (NHMD-916629).

Paratypes: Eight larvae from same locality as holotype but collected on different dates. Two cyprids mounted for SEM with corresponding LSN exuviae on glycerine jelly slides (NHMD-916630, NHMD-916632), exuvium of one LSN on glycerine jelly slide with corresponding cyprid preserved in ethanol for molecular work (NHMD- 916631), exuvium of one LSN on glycerine jelly slide (NHMD-916639), one LSN mounted for SEM (NHMD- 916636), one LSN (nested failed moultings, outermost cuticle is LSN − 2) mounted for SEM (NHMD-916638), two early nauplii mounted for SEM (NHMD-916633, NHMD-916635). Detailed sampling data in Table 3 .

Other material: Sesoko Island (same locality as holotype): One early nauplius collected 21 September 1991 mounted for SEM (NHMD-916640); six early nauplii (JA-2018-111, JA-2019-001, -100, -321, -322, -378-0), three LSN (JA-2019-165, JA-2019-107, -136) and one cyprid (JA-2018-108), all preserved in ethanol for molecular work (no vouchers retained). Green Island (Taiwan): One LSN (NHMD-916641) and one cyprid (NHMD-916642) collected early September 2018, and mounted for SEM; three nauplii preserved in ethanol for molecular work (TA-2018-066, -101, -166, no vouchers retained) Detailed sampling data in Table 3 .

Description

Last-stage nauplius (LSN) ( Figs 3A , 4–6 , 11D, E, K, L , 14A ): Mainly based on paratype NHMD-916636 ( Figs 4 , 5 ). Body markedly elongate, slightly depressed dorsoventrally. Total length (TL) 380 µm (alive) or 375 µm (after critical point drying), including dorsocaudal spine; lengths of other live specimens from Sesoko Island 352–390 µm (N = 8) ( Fig. 3 ). Greatest width 140 µm and greatest dorsoventral thickness 100 µm. Cuticle transparent with nearly fully developed, yellowish/brownish cyprid clearly visible inside, with median nauplius eye and pair of compound eyes. In dorsal view, frontal margin evenly rounded, lateral margins tapering gradually towards bluntly rounded caudal end, with no sharp border between cephalic shield and trunk (slight indentation visible in most other specimens), ending in blunt dorsocaudal spine. In lateral view, body not entirely straight, but bent c. 35° ventrally behind naupliar appendages. Three pairs of naupliar appendages (a1, a2, md) arising close together on ventral side at about 25% of body length, flanking triangular, moderately bulbous labrum. Large parts of ventral side of body (posterior to labrum) and dorsal side of trunk ornamented with transverse cuticular ridges. Cephalic shield with ridge-bounded facets, except for smooth central area flanking dorsal (but not anterior) midline. Entire body displaying bilaterally symmetrical pattern of variety of diverse pores and sensilla, all fully mapped and numbered herein (see detailed description below). No ‘ghost-like’ image of part of the cyprid thorax, particularly the thoracopods and their setae, visible inside any LSN exuviae (as previously detected in many types of y-larvae; Grygier et al., 2019).

Anterior part of faciomarginal area almost featureless except for pair of closely-set pores with oval openings (#34, Fig. 4B, D ) and pair of depressions (#33), latter resolved as pores with slit-like openings in Green Island specimen NHMD-916641 ( Fig. 6C ). Large, triangular elevation (labrum) present between naupliar appendages ( Figs 4B , 5K–M ), slightly bellshaped in mounted exuvium of paratype NHMD- 916630 ( Fig. 11K ), with shallowly indented posterior margin. Labrum with five pores, including three unpaired pores in midline (#29* posteriorly, #30* and #31* near midlength), and one lateral pair positioned level with these latter two (#32), but no free posterior labral extension. Openings of pores #30* and #31* oriented obliquely, but in slightly different ways in this and the above-mentioned Green Island specimen (compare Figs 5M , 6C ).

Naupliar appendages placed immediately adjacent to labrum in diagonal rows, with first antennae (a1) closest to midline, mandibles farthest from it ( Figs 4B , 5K ). Each limb arising from separate (a1) or partly continuous (a2 and md) outpocketings of cephalic cuticle, these probably serving to enhance appendage flexibility while swimming. First antennae short and digitiform (slightly shorter in above-mentioned Green Island specimen; Fig. 6C ), consisting of two distinct segments and, embedded in cephalic outpocketing, sclerite possibly representing additional proximal segment ( Fig. 5K ). Distal segment twice as long as proximal segment, bearing two long apical setae, one apicolateral seta of intermediate length and one small (rudimentary) medial seta. Second antennae and ‘LSN − 2’ and ‘LSN − 3’, respectively, refer to instars two and three molts earlier than the ‘LSN’; see Material & Methods, ‘Naupliar development’, for explanation. Some of the specimens from Sesoko Island shown in Figure 3 are not listed here as they died and were discarded without being preserved

# Japan: Okinawa, pier of Tropical Biosphere Research Center Sesoko Station.

¤ Taiwan, Green Island, Gongguan Harbour.

*Formalin-fixed exuvium, on glycerine jelly slide.

† Formalin-fixed, on SEM stub.

§ Ethanol-fixed and processed for molecular work (resulting in lack of voucher).

** Collected and processed by DEJ, MJG, YF, ND, JO.

†† Collected and processed by DEJ, MJG, YF, ND, FP, JO.

‡‡ Collected and processed by MJG.

§§ Collected and processed by ND, DEJ, JO.

mandibles devoid of any feeding structures (including gnathobases or naupliar processes) ( Figs 4B , 5K, L ). Coxa and basis of second antenna both broad, ringlike, with rudimentary serration along distal margin; endopod narrowly inserted on medial part of end of basis, being small, cylindrical, and undivided with two distal setae (one long and one short); exopod more broadly inserted on lateral part of end of basis, being composed of five successively smaller annuli, the first bearing one small medial seta, the next three each carrying one long medial seta, and the small distal segment bearing two setae of intermediate length (setal formula 1:1:1:1:2); in Green Island specimen, a sixth rudimentary segment visible more proximally in exopod. Segmentation and setation of mandible similar to those of second antenna, except basis shaped differently and proximal annulus of exopod smaller and lacking any setae (setal formula 0:1:1:1:2).

Cephalic shield approximately 200 µm long, 140 µm wide, its posterior margin not clearly demarcated from trunk, but indicated indirectly by shift in cuticular ornamentation from cephalic shield’s longitudinal ridges to trunk’s transverse striations ( Figs 4C , 5A ). Lateral and frontal parts of shield bent ventrad and bearing cuticular reticulations (facets or plates) and lineations, while dorsal surface smooth, with weakly defined mid-dorsal ‘window’. Ridge-defined reticulation most complicated anteriorly, with large diversity of small facets, some squarish and others oblong, while more lateral facets fewer and longer, transitioning into long, uninterrupted ridge-bounded belts reaching to shield’s posterior margin. Arrangement of ridges and facets roughly bilaterally symmetrical, but symmetry of size, shape and degree of subdivision of contralateral facets imprecise. Arrangement not described in detail, but full pattern of two LSN from Sesoko Island and Green Island as shown in Figures 4C , 5A, B and 6 , respectively, similar in both specimens but with some differences: (1) arrangement of anterior facets more strictly symmetrical in Green Island specimen; (2) some oblong frontal facets of Green Island specimen, most notably those above central pore #1 ( Fig. 6D ), corresponding to pairs of smaller facets in Sesoko specimen ( Fig. 5B ); and (3) all facet-bounding ridges less distinct in Green Island specimen, sometimes even absent (especially dorsally).

Cephalic shield ornamented with large number of other types of structures (pores, setae), these being fully mapped on paratype NHMD-916636 from Sesoko Island ( Table 4 ; Figs 4 , 5 ): 31 structures in total, mostly in pairs and concentrated in anterior third of shield. Most common type consisting of 23 relatively large (3–4 µm) pores, among which just one unpaired and situated on midline close to anterior margin (#1, Fig. 5B ), remainder distributed anteriorly and laterally in pairs ( Table 4 ; Figs 4 , 5 ). A few other kinds of structures present dorsally: two widely separated pairs of anterior setae (#7 and #8; Fig. 4E ), one middorsal pair of small, circular pores (#12) and one pair of small sensilla near posterior margin (#16, Fig. 5F ). Pore/sensilla pattern of specimen NHMD-916641 from Green Island ( Fig. 6 ) the same, except for minor differences in precise position of some structures (e.g. pore pair #19, located between different transverse lines in the two specimens).

Elongate trunk comprising about 45% of TL in dorsal view, 62% in ventral view. Behind slight indentation at posterior end of cephalic shield, trunk tapering smoothly with its lateral margins subtending angle of c. 20°, then somewhat rounding off at posterior end. Dorsocaudal spine robust, short, blunt in NHMD- 916636 ( Fig. 4 ), somewhat longer and more pointed in other LSN specimens ( Fig. 6 ). Pair of reduced furcal spines situated ventrally, about 30 µm forward from base of dorsocaudal spine. Lateral flanks and posterodorsal part of trunk bearing about 25 paired or dorsally continuous, regularly spaced and annular cuticular ridges with posteriorly directed, serrate crests ( Figs 4 , 5 ). In specimen NHMD-916641 from Green Island, these ridges less distinct dorsally ( Fig. 6B ). In both specimens, nine pairs of large pores distributed mostly laterally and ventrally on trunk (#17–#25; Figs 4–6 ), including one pair (#25) flanking dorsocaudal spine. In addition, pore pair #13 of cephalic shield possibly actually belonging to outer border of faciomarginal area. Pore arrangement generally bilaterally symmetrical, but precise positions of many pores far from symmetrical. For example, in NHMD- 916636 left member of pairs #17, #21 and #24 situated far more anteriorly than their right-side counterparts (most pronounced for most posterior dorsal pair, #24), and in both this and Green Island specimen, one member of pair #24 situated significantly more dorsally than its contralateral partner ( Figs 4F , 6B ).

Large, convex, medioventral region of trunk, reaching from point immediately behind labrum to point immediately anterior to base of dorsocaudal spine, this region being broadest anteriorly, reaching posterolateral margins of cephalic shield on both sides, then gradually tapering posteriorly to median pore (#26*) between furcal spines. In NHMD-916636 this region crossed by transverse cuticular ridges with posteriorly directed, serrate crests ( Fig. 4B ), these ridges being fewer and less distinct in Green Island specimen NHMD-916641 ( Fig. 6C )

Cyprid ( Figs 3 , 7–10 ): Mainly based on holotype (NHMD-916629); minor variation found in a few other examined cyprids from Sesoko Island and Green Island (Taiwan) mentioned directly in description with indication of specimen identity (museum number). Body elongated, consisting of head with large but not all-enclosing cephalic shield (carapace), six-segmented thorax, three-segmented abdomen and telson. Three specimens measured in life 283–305 µm long, two measured after fixation in ethanol both 360 µm long and four measured after critical point drying 280– 320 µm long. Total length of individual specimens greatly different when measured in life, in preservative (longer, perhaps due to relaxation of musculature) and after critical point drying (8–20% shrinkage).

Cephalic shield (or carapace) covering head anteriorly, dorsally and laterally and also covering anterior part of thorax dorsally and especially laterally. Small nauplius eye lying anterodorsally to pair of large compound eyes. Labrum and first antennae situated on ventral side of head, beneath compound eyes. Each of six thoracomeres bearing a pair of biramous thoracopods, unclear whether tergites of thoracomeres 1 and 2 separate dorsally (this area not visible by SEM). In live specimens, main body and anterior part of cephalic shield deep brown, lateral parts of cephalic shield largely transparent, non-functional gut orange (yolk?), and, in both fixed and living specimens, area beneath cephalic shield approximately at point of separation from main body with dorsolateral concentration of orange-coloured droplets (oil, yolk?) ( Figs 7A , 8A ).

Cephalic shield relatively long, univalved, only partially covering dorsal and lateral sides of main body of larva, with only thoracomeres V and VI being exposed dorsally and with lateral margins of shield reaching telson in live specimens ( Fig. 8A ). Shield resembling inverted boat with posterolateral parts much produced, altogether about 175 µm long along mid-dorsal line and 210 µm along lateral margins. Long longitudinal and short transverse and oblique cuticular ridges present, outlining plates (or facets) and longitudinal bands, these together occupying entire shield surface but being most distinct anteriorly and laterally; more anterior facets generally smaller. Arrangement of facets and bands not strictly bilaterally symmetrical; overall pattern in dorsal view almost so (see holotype, Fig. 8C ), but significant asymmetry apparent in anterior view ( Fig. 8B ), with no clear left–right correspondence of facets around pore/ sensilla pairs #9 and #10. In another paratype cyprid from Sesoko Island (NHMD-916630; not shown) and a cyprid from Green Island (NHMD-916642; Fig. 10B ), anterior face of shield showing more symmetry than in holotype.

Surface of cephalic shield with numerous pores, seta-bearing pits and other cuticular structures (total number 84, counting both members of pairs) in semi-symmetrical pattern ( Fig. 8B, C ) comprising five distinct types of structures ( Table 5 ). First type (19 in number) with slit-like pore surrounded by conspicuous circular rim. Except in one case (pore #1* on midline), these pores always paired, being concentrated anteriorly and laterally ( Figs 7B , 8B, C ). Oblique opening of pore #1* oriented differently in specimens from Sesoko Island (holotype, Fig. 8B ) and Green Island ( Fig. 10B ). Second type (28 in 14 pairs) a deep pit with round mouth and single short, protruding seta ( Fig. 8B, C ); these pits scattered all over shield surface, but more highly concentrated anteriorly. Third type, all with round mouths and neither cuticular rim nor seta ( Fig. 8B, C ), including eight pairs of small pores and three larger, unpaired, so-called central pores sensu Kolbasov & Høeg (2003), two situated anteriorly and one posteriorly (#14*, #20* and #39*; Fig. 8C ). Fourth type comprising four pairs of small groups of micropores (two or five per group), all situated anteriorly on cephalic shield ( Fig. 7D, G ).

Fifth type of structure on cephalic shield identified as lattice organs, grouped into two anterior and three posterior pairs flanking dorsal midline ( Figs 8B, C , 10B, C ). Anterior pairs (#13 and #15) distinguished from general cuticle by their situation 50–60 µm from anterior end of shield in four weak depressions surrounding most anterior of unpaired central pores (#14*). Their cuticle smooth and lacking any small pores (thus no pore field). First pair (#13) teardroplike, about 10 µm long and 7 µm wide, strongly converging anteriorly and each narrowing posteriorly towards small terminal pore. Second pair (#15) elongate, 12 µm long and about 2.5 µm wide, slightly converging anteriorly and weakly narrowing towards tiny, posterior terminal pore. Third pair of lattice organs (#38) situated in front of posterior unpaired central pore (#39*), almost round with diameter of about 3 µm and with barely visible posterior terminal pore. Final two posterior pairs of lattice organs (#40 and #41) situated behind unpaired pore #39*, # 41 in flattened posterior marginal area of shield. These two pairs, respectively, 10 µm long and about 1.5 µm wide and about 7.5 µm long and 2 µm wide, lack visible terminal pores. Lattice organs largely organized the same way in above-mentioned Green Island specimen (NHMD-916642), but additional rudimentary pair possibly present anterior to counterparts of abovedescribed two anterior pairs ( Fig. 10B, C ).

Proximal parts of first antennae not visible in SEM preparations. Segmentation of distal parts also unclear, but distal armament consisting of conspicuous curved hook (claw), large aesthetasc and, between these structures, one short seta with scattered setules and one smaller simple seta ( Fig. 7B–D, H ). Claw remarkably gracile, semicircular. Small, bifid, thin-walled appendix, possibly homologous to frontal filaments in nauplii, present in anterior midline between first antennae and anterior margin of cephalic shield ( Fig. 7E ). Labrum extended as linguiform process with 17 hooks on posterior side, arranged in three irregular rows of five, six and six hooks ( Fig. 7C, D, F ). Posterobasal part of labrum with five pores: one lateral pair with slit-like openings (#64) and three in midline, among which two with oblique slit-like openings (#62, #63) and one partly hidden proximally (#61; vestigial mouth opening?). Vestiges of second antennae and mandibles present lateral to labrum, showing remains of exopods and endopods in one specimen (NHMD-916630, Fig. 7C ), but small, rounded and wrinkled in another (NHMD-916629, Fig. 7B ). Small pair of so-called bifurcate paraocular processes present anterior to these, with anterior and slightly thicker posterior branches both 15 µm long and situated parallel and adjacent to lateral margin of cephalic shield ( Fig. 7B–D ). No pair of postocular filamentary tufts seen in these preparations.

Among thoracomeres I–VI ( Figs 7B , 9A , 10A, E ), posterodorsal margins of tergites V and VI serrate ( Figs 7I , 10E ). Tergite VI also equipped with two or three other transverse, serrate cuticular ridges and bearing widely spaced pair of setae close to posterior margin (#45; Fig. 7I ). Cuticular ridges less distinct on Green Island specimen ( Fig. 10E ). Tergites V and VI with free pleural extensions, those of former with rounded ends, those of latter trapezoidal with sharp posteriolateral ends (less trapezoidal in Green Island specimen) ( Figs 9A , 10A ).

Each thoracomere bearing pair of biramous thoracopods ( Figs 9A, B , 10A, D ). Each thoracopod consisting of lateral basal array of sclerites, coxa (distinct only in thoracopods 1–5), basis and pair of rami (exopod and endopod). Proximal sclerites not described further as this flexing zone appears variable between specimens. Two-segmented endopod of first thoracopod with short proximal segment and elongate (three times longer than wide) distal segment with two long apical setae; endopod slighty shorter in Green Island specimen ( Fig. 10D ). Unsegmented exopod slightly shorter than endopod, significantly wider proximally than distally, and bearing two apical setae: short outer one and long inner one. Thoracopods 2–5 composed of the same elements as thoracopod 1, but with distal segment of endopod generally longer and carrying long medial seta (dotted arrows on Figs 9B , 10D ) in addition to two terminal setae; and with exopod larger and bearing three terminal setae: short outer one and two long inner ones. Thoracopod 6 generally similar to preceding thoracopods but shorter, and with unsegmented protopod (coxa and basis fused?).

Abdomen consisting of three short segments, all lacking pleural extensions but possessing serrate transverse cuticular ridges and posterior margins; first segment with dorsolateral pair of setae (#46), third segment shortest, tapering ventrally and sometimes strongly intercalated between second segment and telson (7B, I, 9A, 10A, E). Telson long with dimensions varying somewhat among specimens, 1.5–1.6 times as long as greatest width in a specimen from Sesoko Island ( Figs 7E , 9C ), but 1.3 times as long as wide in one from Green Island ( Fig. 10E ). Telson with dense reticulation of serrate ridges roughly outlining two dorsal longitudinal rows of broad plates ( Figs 7I , 10C ) (anterior two or three pairs only weakly or not divided at midline), two lateral rows on each side and five ventral rows ( Figs 7B , 9B–D , 10A ). Number of plates in each dorsal row approximately 13 (11 in Green Island specimen), 11 in each lateral row and ten in each ventral row, with those of mid-ventral row set off half a step from those of other rows.

Total of 19 pores and setae present on telson ( Table 5 ). Four pairs (#48–#51) placed in characteristic pattern anterolaterally ( Figs 9A , 10A ). Pair of pores with slit-like opening (#52) present in third plate from front in upper row of lateral plates ( Figs 7B , 9C , 10A, E ); similar pore (#53) in fourth plate from rear in lower row of lateral plates ( Figs 7B , 9C, D , 10A ). Another similar pair of pores and pair of setae in pits (#54 and #55, respectively) situated either in same contralateral pair of posterior dorsal plates ( Fig. 7I ) or with pores and setae in successive pairs of plates ( Fig. 9C, D ) or, in Green Island specimen, pore pair #54 absent ( Fig. 10E ). Terminally, one pair of dorsal pores (#56) and one ventral central pore (#57*). Three pairs of ventral pores ( Figs 9B, D ) located far anteriorly in outer row of ventral plates (#60) and in two adjacent posterior plates in same row (#58 and #59). Furcal rami short and cylindrical, perhaps even disclike, with three setae each: two unequal lanceolate setae with serrate margins, and one irregularly denticulate short seta ( Fig. 9D, E ).

Earlier naupliar stages

Number of naupliar stages: An outline of the naupliar development of H. demodex is provided herein based on a combination of high-resolution photographs in life of different instars of the same individual ( Figs 3 , 11 ) and SEM images ( Figs 12 , 13 ) of several specimens representing three distinct instars younger than the laststage nauplius (LSN). Four nauplii developed to cyprids while in culture (NHMD-916629, NHMD-916630, NHMD-916631, NHMD-916632), transitioning 3–6 days after the date of sampling ( Table 6 ). Of particular importance are four specimens – three of them LSN as shown by presence of the developing cyprid’s compound eyes within – that failed to moult properly, resulting in a nested set of unshed exuviae with the most recent stage innermost ( Fig. 11F, G, I, J ). These specimens provide direct evidence of the last four stages of naupliar development, LSN, LSN − 1, LSN − 2 and LSN − 3. In each series, the dorsocaudal spine generally becomes thinner and less blunt as development progresses, with the thinnest spines being found in the LSN. There appear to be even earlier instars in our material. One of these, NHMD-916635 ( Fig. 13A, B ), has a dorsocaudal spine that is blunter than that of LSN − 3 (cf. Fig. 11F ); it most likely belongs to an earlier instar, perhaps LSN − 4. Another specimen ( Fig. 12 ), the earliest-stage specimen examined during this study, has much weaker developed cuticular reticulation and other cuticular armature than the specimen mentioned above and appears to be one, or more likely two, instars earlier than it, i.e. LSN − 5 or LSN − 6. The naupliar development of H. demodex thus comprises at least five to six instars, more if instars have been missed (see Discussion).

Colour, yolk and cyprid morphogenesis during the naupliar phase: Coloration clearly shown and yolk boundaries distinct in our photographs of living nauplii at various moult stages ( Figs 3 , 11 ). Anterior and posterior parts of body brownish in earliest stages ( Fig. 11A, B ). Bright-coloured cylindrical region – orange in specimens from Sesoko Island but yellow in those from Green Island – extending from behind nauplius eye to dorsocaudal spine, partly subdivided into droplets or granules and assumed to be at least partly made of yolk. Yolky region dividing into two parts early in naupliar development, these corresponding to future thorax and abdomen of the cyprid ( Fig. 11B ). Cyprid achieving its final form within last-stage nauplius (LSN) and capable of movement (e.g. thoracopodal beating, abdominal dorsoventral flection), while still inside naupliar cuticle. Before final moult, abdomen of cyprid becoming gradually thinner and yolk becoming concentrated in dorsal midline, as also seen in free-swimming newly moulted cyprids (orange area in Figs 7A , 8A ).

Naupliar stage LSN − 5 or LSN − 6? ( Fig. 12 ): Earliest stage among all nauplii of H. demodex examined here with SEM. Body short (345 µm long), dorsocaudal spine blunt. Overall body shape much like that of LSN, but markedly more compact, less elongate and distinctly inflated (due to greater quantity of yolk?). Large facets (plates) on anterior and lateral parts of cephalic shield few in number and separated by faint ridges. Anterior field of large facets marked with red overlay in Fig. 12A–C in order to trace their fate in following instars. Assuming only one row of ‘brim’ plates anteriorly, red overlay encompassing all axial ‘frontal’ plates (F-1 to F-4; definitive boundaries uncertain, so labelled as F*) in addition to small parts of ‘elongate’ plate pair E-1 (i.e. E-1*) in upper corners and both members of ‘intercalary’ plate pair I-1 and ‘polygonal’ plate pair P- 1 in lateral areas, none of these being yet delineated from their adjoining ‘frontal’ plates (cf. fully delineated state in later instars). Overlay area flanked on each side from top to bottom by four plates identifiable under Itô’s (1990b) and Kolbasov et al. ’s (2021a) expanded systems as ‘intercalary’, ‘elongate’, ‘polygonal’ and ‘marginal’ plates E-1* + I-3(a), I-2, P-2 (bordering pore #4 posteriorly) and M-1. ‘Marginal’ plate M-2 and ‘superlateral’ plate S, adjoining M-1 posteriorly and posteroventrally, with S bordering pore #2 anteriorly and adjoining plate M-3(e) posteriorly. Other plates, especially dorsally, too poorly delineated to identify with confidence except for M-2 + M-3(c) behind M-1 and lateral band behind S consisting of M-3(e) and combined M-4 to M-7.

Labrum similar to that of LSN in general form and pores, but preceded by distinct median elevation reaching to pore pair #34, and pore #29 relatively larger than in LSN and positioned significantly farther forward, away from posterior margin. Naupliar appendages differing from those of LSN in minor ways: all limbs relatively shorter and more robust than in LSN; exopod of second antenna six-segmented owing to presence of rudimentary proximal segment ( Fig. 12G , asterisk). Setation of appendages as in LSN, but medial seta of first antenna longer. Trunk long and gradually tapering, but relatively shorter than in LSN and with sides more parallel; many fewer rows of serrate vertical cuticular ridges/scales laterally than in LSN and even fewer transverse ridges dorsally. Both cephalic shield and trunk with pores and setae of essentially same kind as in LSN, but fewer in number. Most such structures on cephalic shield identifiable with counterparts in LSN by form and position, and thus numbered the same in Figure 12 , but a few pores of unclear identity on trunk labelled only as ‘?’. On cephalic shield, pore pairs #2, #10 and #14 or #15 present anterolaterally, laterally and posterolaterally near margin; pore and seta pairs #4–#8 present anterodorsally (#8r and #8l flanking plate W), and pore pair #9 present dorsally posterior to midlength of shield. On trunk, dorsolateral pore pairs #17, #19, #21 and #24 more-or-less identifiable, but interpretations complicated by left/right asymmetry in pore distribution. Pore pair #25 flanking dorsocaudal spine at posterior end of body and single mesial pore #26 situated between blunt, rudimentary furcal spines, these spines being closer to terminal end than in LSN. Anterior central pore of cephalic shield (#1) and pore pair #3 flanking it not yet apparent. Posteriorly on shield, seta pair #16 and pore pairs #11, #12 and either #14 or #15 not yet present. Ventrolateral pore pair #13 also not seen.

Naupliar stage LSN − 3? ( Figs 11B , 13A–C ): This stage at least one and probably two instars later than preceding specimen and probably one instar earlier than LSN − 2 specimen described below. Facet arrangement of cephalic shield more complicated than in preceding specimen, with greater number of longitudinal bands and division of anterior region into smaller facets. For example, anterior face of shield ( Fig. 13C , red overlay) now subdivided into three rows of clearly delineated, ridge-bounded facets in three rows: axial row extending from anterior margin of shield to approximately pore pair #6, comprising part of ‘brim’ and all derivatives of ‘frontal’ (F-1 to F-4) plates, and pair of shorter, eight-facet rows flanking it to left and right, consist of derivatives of certain ‘polygonal’ (P-1), ‘intercalary’ (I-1) and ‘elongate’ (E-1) plates. Unpaired far-anterior pore (#1) and a few other pores (e.g. pair #3 on anterior face of shield and pair #23 on ventral side of trunk) now present. In general, pores more distinctly delineated and with more pronounced slit-like openings than in above-described earlier stage. Segmentation and setation of naupliar appendages identical to those of that specimen, but trunk with greater number of more distinct vertical and transverse cuticular ridges, including narrow row of such ridges along ventral midline ( Fig. 13A ).

N a u p l i a r s t a g e L S N − 2 (F i g s 1 1G, 1 3 D– G): Incompletely moulted LSN/cyprid specimen with cephalic shields and faciotrunk integuments of two previous instars still attached in nested fashion (LSN − 1, yellow overlay, and LSN, green overlay). Moulting zone of both LSN − 1 and LSN − 2 cuticles running along dorsal transverse seam between rear margin of shield and trunk dorsum, then continuing ventrally around border between faciomarginal area and lateral and anterior margins of cephalic shield. Mainly outermost LSN − 2 stage observed by SEM ( Fig. 13D–F ), but unmoulted cyprid y observed and photographed in life within three surrounding layers of naupliar cuticle ( Fig. 11G ). LSN − 2 differing from stage described above (LSN − 3?) in having about twice as many and narrower elongate, band-like facets posterolaterally on cephalic shield and generally more complex facet arrangement anteriorly. For example, brim plates crossing and flanking anterior midline of shield margin in previous larva ( Fig. 13C , arrow), and there comprising three facets in total, now comprising two transverse rows of four smaller facets each ( Fig. 13D, F , arrows). Pores and setae of this nestedcuticle specimen not examined in detail, but evidently not appreciably different from those of LSN. Segmentation and setation of naupliar appendages apparently identical to those of earlier stages. Trunk more slender than in previously described stages, weakly tapering terminally with longer, thinner dorsocaudal spine and with less distinct vertical and transverse cuticular ridges.

Figures

Figure 3. Overview of most specimens of Hansenocaris demodex sp. nov. collected at Sesoko Island during fieldwork in 2018 and 2019. A, 19 specimens, four of which reached the cyprid stage, collected in the plankton at the naupliar stage and reared individually, with length, dish number, sample number, and museum registration number indicated for each specimen as well as a colour code (red, blue, yellow) showing its ultimate treatment/fixation (respectively, molecular work, SEM or exuvium on slide); some larvae died before preservation and are only referred to by their dish number; B, overview of setup for individual rearing of larvae; top view of green tray containing 31 2.5-cm-wide dishes with tracking information written on lids, each containing one to five live larvae. Live video of H. demodex and other y-larvae can be seen here: https:// youtu.be/seo-63AK10E.

Figure 4. Hansenocaris demodex sp. nov., paratype, last-stage nauplius (LSN) from Sesoko Island photographed live before fixation (A) and in SEM after fixation and mounting (B–H). A, ventral view; B, ventral view; C, dorsal view; D, pair of ventral pores situated in front of labrum; E, close-up views of pores and sensilla of cephalic shield; F, caudal end in dorsal view; G, caudal end in ventral view; H, caudal end viewed from behind. Abbreviations: a1, first antenna; a2, second antenna; arthro membr, arthrodial membrane; ce, compound eye; dc sp, dorsocaudal spine; en, endopod; ex, exopod; fur sp, furcal spine; la, labrum; md, mandible; ne, nauplius eye; rud, rudimentary. Small Arabic numerals, many annotated with l or r for left and right, respectively, refer to cuticular structures (see Table 4). Yellow overlay, cephalic shield; blue overlay, faciotrunk; red overlay, anterior field of facets. Square with dotted outline: sample number, museum number and type status.

Figure 5. Hansenocaris demodex sp. nov., paratype, last-stage nauplius (LSN) from Sesoko Island in SEM. A, lateral view; B, cephalic shield, anterior view; C–J, close-up views of pores and sensilla of cephalic shield and faciotrunk; K–M, labrum and naupliar limbs (a1, a2, md). Abbreviations: a1, first antenna; a2, second antenna; md, mandible; la, labrum; scl, sclerite of a1. Small Arabic numerals, many annotated with l or r for left and right, respectively, refer to cuticular structures (see Table 4). Yellow overlay, cephalic shield; blue overlay, faciotrunk; red overlay, anterior field of facets. Square with dotted outline: sample number, museum number and type status.

Figure 6. Hansenocaris demodex sp. nov., last-stage nauplius (LSN) from Green Island in SEM. A, lateral view; B, dorsal view. C, ventral view; D, frontal view. Abbreviations: a1, first antenna; a2, second antenna; dc sp, dorsocaudal spine; fur sp, furcal spine; la, labrum; md, mandible. Arabic numerals, many annotated with l or r for left and right, respectively, refer to cuticular structures (see Table 4). Square with dotted outline: sample number and museum number.

Figure 7. Hansenocaris demodex sp. nov., holotype (A, B, D–I) and paratype (C), cyprids from Sesoko Island photographed before drying (A) and in SEM after drying and mounting (B–I). A, lateral view; B, lateral view; C, cephalic region, lateral view; D, cephalic region, ventral view; E, bifid appendix (frontal filaments?); F, labrum; G, pores and sensilla; H, close-up of hook of left first antenna; I, abdomen and telson, dorsal view. Abbreviations: a1, first antenna; a1 hk, first antenna hook; a2 rud, second antenna rudiment; ae, aesthetasc; en, endopod; ex, exopod; la, labrum; md rud, mandible rudiment; par occ pro, paraocular process. Roman numerals I–VI – thoracic segments. Large Arabic numerals 1–3 – abdominal segments. Small Arabic numerals, many annotated with l or r for left and right, respectively, refer to cuticular structures (see Table 5). Square with dotted outline: sample numbers, museum numbers and type status.

Figure 8. Hansenocaris demodex sp. nov., holotype (B, C) and paratypes (A, D), cyprids from Sesoko Island in SEM. A, live specimen in four different positions; B, cephalic shield, frontal view; C, cephalic shield, dorsal view; D, cephalic shield, oblique posterior view. Abbreviations: par occ pro, paraocular process. Small Arabic numerals, many annotated with l or r for left and right, respectively, refer to cuticular structures of the cyprid (see Table 5). Squares with dotted outlines: sample numbers, museum numbers and type status.

Figure 9. Hansenocaris demodex sp. nov., holotype (A, B, D, E) and paratype (C) cyprid from Sesoko Island in SEM. A, posterior thoracic segments and anterior abdominal segments, lateral view; B, thoracopods, ventral view; C, telson, dorsall view; D, telson, terminal view; E, right furcal ramus, posterior view. Abbreviations: fur ram, furcal ramus; ba, basis; co, coxa; en, endopod; ex, exopod; pro scle, proximal sclerites of thoracopods; thp 1, thoracopod 1. Roman numerals I–VI – thoracic segments. Large Arabic numerals 1–3– abdominal segments 1–3. Small Arabic numerals, many annotated with l or r for left and right, respectively, refer to cuticular structures (see Table 5). Square with dotted outlines: sample numbers, museum numbers and type status.

Figure 10. Hansenocaris demodex sp. nov., cyprid from Green Island in SEM. A, lateral view; B, cephalic shield, frontal view; C, putative lattice organ, close-up; D, thoracic limbs 1–6; E, thorax, abdomen and telson, dorsal view. Abbreviations: ba, basis; co, coxa; en, endopod; ex, exopod; la, labrum; pro scle, proximal sclerites of thoracopods; thp 1, thoracopod 1; thp 6, thoracopod 6. Small Arabic numerals, many annotated with l or r for left and right, respectively, refer to cuticular structures (see Table 5). Square with dotted outline: sample number and museum numbers. Dotted arrows in D point to long medial setae of thoracopodal endopods.

Figure 11. Hansenocaris demodex sp. nov., nauplii from Sesoko Island photographed alive (A–J) or as slide-mounted exuvium (K, L). A–E, different naupliar instars; F, penultimate naupliar instar with exuviae from two prior moults still attached posteriorly; G, cyprid with exuviae from three prior naupliar moults still attached posteriorly; H, last-stage nauplius in lateral view; I, posterior end of cyprid with three prior naupliar moults still attached; J, posterior end of cyprid with three prior naupliar moults still attached; K, L, exuvium of last-stage nauplius, ventral and lateral views.Abbreviations:a1, first antenna; a2, second antenna; ce, cypris eye; md, mandible; ne, nauplius eye; LSN, last-stage nauplius; thx, thorax. Squares with dotted outlines: dish numbers, sample numbers and museum numbers; some larvae died before preservation and are only referred to by their dish

Figure 12. Hansenocaris demodex sp. nov., early nauplius (LSN − 5 or LSN − 6?) from Sesoko Island in SEM.A, ventrolateral view; B, lateral view; C, dorsal view; D, frontal view; E, labrum and naupliar appendages, right side; F, naupliar appendages, right side, median view; G, first and second antennae, right side, lateral view, asterisk indicating rudimentary segment of a2 exopod; H, dorsocaudal spine and furcal spines, terminal view. Abbreviations: a1, first antenna; a2, second antenna; ba, basis; co, coxa; dc sp, dorsocaudal spine; en, endopod; ex, exopod; fur sp, furcal spine; F-1 to F-3, frontal plates 1–3; la, labrum complex; md, mandible; rud, rudimentary; W, window plate. Small Arabic numerals, many annotated with l or r for left and right, respectively, refer to those cuticular structures that could be matched with those of the LSN (see Table 4). Yellow overlay, cephalic shield; blue overlay, faciotrunk; red overlay, anterior field of facets. Square with dotted outline: museum number.

Figure 13. Hansenocaris demodex sp. nov., paratypes from Sesoko Island in SEM:, an early nauplius (LSN − 3?) (A–C) and a later nauplius (LSN − 2) with unmoulted later larval stages still inside. A, ventral view; B, lateral view; C, cephalic shield, frontal view; D, ventrolateral view; E, lateral view; F, cephalic shield, frontal view; G, cephalic shield, dorsal view. Abbreviations: a1, first antennae; a2, second antennae; dc sp, dorsocaudal spine; ex, exopod; fur sp, furcal spine; la, labrum; md, mandible. Small Arabic numerals, many annotated with l or r for left and right, respectively, refer to those surface structures that could be matched with those of the LSN (see Table 4). Green overlay, LSN; purple overlay, LSN − 1; red overlay, anterior field of facets. Squares with dotted outline: sample numbers, museum numbers and type status.

Figure 14. Morphology of four types of y-larvae from Sesoko Island used for molecular analyses (see overview in Table 6), all photographed in life. A, Hansenocaris demodex sp. nov., lecithotrophic LSN (last-stage nauplius) with cyprid inside. B, ‘Bumblebee’ type, lecithotrophic LSN with cyprid inside. C, ‘Big brown’ type, lecithotrophic LSN with cyprid inside. D, planktotrophic nauplius, identified as Itô’s (1986a) Pacific type I. Squares with dotted outline: sample numbers.