Introduction

In recent years, fluorescence microscopy has revitalized the field of invertebrate comparative morphology, especially with the emergence of confocal laser scanning microscopy (CLSM). In many invertebrates, different body parts naturally emit autofluorescence, which makes it possible to employ fluorescence microscopy without any need for specialized tissue fixation or the addition of fluorescent dyes (Bluhm et al. 2001; Haug et al. 2011; Kamiya et al. 2012; Olesen et al. 2013; Metz et al. 2015; Semprucci et al. 2016; Büsse and Gorb 2018; Kamanli et al. 2017; Melzer et al. 2021; Fritsch and Richter 2022). Autofluorescence has been employed successfully even to examine fossils (Haug et al. 2014, 2015; Eiler and Haug 2016; Nagler et al. 2017). Cuticular autofluorescence, often amplified with Congo red (a textile dye), has proved to be of value in some studies of the morphology and systematics of micro-crustaceans (Mercado-Salas et al. 2018; Michels 2007; Michels and Büntzow 2010; Rötzer and Haug 2015; Suárez-Morales and Mercado-Salas 2023; Wiethase et al. 2019). Traditionally, scanning electron microscopy (SEM) has been used efficiently to study the external morphology of larval crustaceans (e.g., Martin et al. 2014). To achieve optimal results with SEM, however, access to a substantial number of live specimens is desirable. This would allow a variety of special fixation techniques, micro-dissections and thorough cleaning, drying and screening of specimens, all this being necessary to reveal critical structures free from detritus or surface bacteria (Felgenhauer 1987; Olesen 2001, 2005). The application of autofluorescence using CLSM is simpler, as significant amounts of information can be extracted from the cuticle of single specimens without special fixation; furthermore, it renders invisible any (non-fluorescent) debris that might otherwise obscure the view (Michels 2007). CLSM is especially helpful when studying museum specimens that have been irreversibly mounted and are irreplaceable, such as taxonomic type material or material from vanished (destroyed) or hard-to-access sites like the deep sea, caves, etc. This technique also allows for single-specimen DNA extraction prior to morphological analysis, which is crucial in taxa characterized by high diversity and taxonomic confusion (Böhm et al. 2011; Valdecasas et al. 2011). Here we explore the potential of using CLSM along with cuticular autofluorescence in the morphological and taxonomic study of certain neglected crustaceans in their larval stage.

“Y-larvae” (Crustacea: Facetotecta) are enigmatic marine crustacean larvae that were first described more than a century ago but whose adults are still unrecognized (Hansen 1899; Glenner et al. 2008; Dreyer et al. 2023b). Two successive stages of free-living y-larvae are known, y-nauplii and y-cyprids, a situation similar to barnacle larvae, to which they are closely related (Perez-Losada et al. 2009). Only 17 species of Facetotecta have been formally described so far, as well as a rather extensive parataxonomy of unnamed “types” (Kolbasov et al. 2021b; Olesen and Grygier 2022, in press; Dreyer et al. 2023a, b). The recent discovery of a surprisingly large diversity of y-larvae in marine shallow waters of Okinawa, Japan, corresponding to at least 40 species (Grygier 1991; Dreyer et al. 2023b), has demonstrated the need for a renewed focus on y-larval taxonomy. New methods making use of molecular markers and laboratory-reared specimens have been developed that may serve to address this need (Olesen et al. 2022; Dreyer et al. 2023a; Olesen and Grygier in press). The taxonomic focus, at least for lecithotrophic y-nauplii, is currently on the last-stage nauplius (hereinafter, LSN), the molted exuvia of which can be retained as a reference/voucher on a microscope slide or in liquid, and the corresponding y-cyprid, which emerges from the nauplius at the latter’s final molt and can be conveniently preserved for molecular systematics or more detailed microscopy (e.g., SEM) (Olesen et al. 2022). Light microscopy of y-naupliar exuviae using differential interference contrast (Nomarski) optics has provided acceptable results (Olesen and Grygier 2022, in press), but because CLSM using cuticular autofluorescence is now well-established, it is desirable to test this technique as well on such material. Because y-naupliar exuviae consist, by definition, only of the cuticle, no other tissue is present to blur the autofluorescent signal, and good results can be expected.

We tested CLSM methodology on specimens from the world’s largest museum collection of facetotectan naupliar exuviae, generated over the course of almost 30 years, to explore the method’s usefulness for continued work on y-larval taxonomy. We re-examined the material of two species, Hansenocaris demodex and H. aquila, which had already been described using other microscopical techniques, to investigate the extent to which CLSM can reveal new, overlooked morphological features. We also examined a broader assemblage of morphologically distinctive y-nauplii to explore whether CLSM can facilitate a more complete taxonomic overview of y-larvae in general.

Materials and methods

Material, sampling and laboratory rearing

We examined exuviae of seven last-stage nauplii (LSNs) of y-larvae from the extensive Facetotecta holdings of the Natural History Museum of Denmark, which had mostly been obtained through intensive sampling at a marine shallow-water site at Sesoko Island (Japan: Okinawa) (26°38′09.4″N, 127°51′55.2″E) over a period spanning almost 30 years (1991, 1992, 1996, 2003–2005, 2018 and 2019) (Glenner et al. 2008; Grygier et al. 2019; Olesen et al. 2022; Olesen and Grygier 2022, in press; Dreyer et al. 2023a, b, in press). Two of the examined exuviae belong to the already described species Hansenocaris demodex and H. aquila (Olesen et al. 2022; Olesen and Grygier 2022), and the other five belong to undescribed but parataxonomically labelled “morphospecies” (Types AB, H, AF, X and U*: see Dreyer et al. 2023a; Olesen and Grygier in press). [NB: Our Latin-letter Type X is different from the Roman-numeral Type X known from Tanabe Bay and the vicinity of Manazuru Port in Sagami Bay in mainland Japan: see Itô 1987; Kikuchi et al. 1991; Watanabe et al. 2000.]

Detailed overviews of the sampling and rearing methodology have been presented in Olesen et al. (2022) and Olesen and Grygier (in press). Briefly, plankton samples were collected off a laboratory pier with conical plankton nets of 20–30 cm mouth opening and 65–100 µm mesh size. Living y-nauplii were sorted from the samples in the lab with glass pipettes under dissecting microscopes and kept in small lots in plastic Petri dishes of 35 mm diameter. Lecithotrophic y-nauplii of different kinds passed through a molt series from early nauplius to y-cyprid within a period of about three days to two weeks. Each specimen that reached the LSN stage (recognizable on account of the y-cyprid’s compound eyes developing within the nauplius) was placed individually in a dish to guarantee an unambiguous match with the resulting y-cyprid. The cyprids were then preserved for morphological or molecular study while the corresponding last-stage naupliar exuviae were generally kept as voucher specimens on microscope slides in glycerin jelly, suitable for at least medium-term museum storage.

Preparation of semi-permanent slides

Glycerin-jelly slide preparation followed the general principles outlined earlier (Kaiser 1880; Michels 2007; Glime and Wagner 2017; Neuhaus et al. 2017). The specific recipe for the glycerin jelly was as follows: after dissolving 21 g gelatine in 120 ml warm distilled water, 140 ml glycerin was added, and finally 2 ml concentrated phenol solution to hinder microbial growth. This solution was poured into suitably sized jars to await hardening. In preparation for mounting, last-stage naupliar exuviae were moved individually in seawater to shallow depression slides with a Pasteur pipette shortly after the molt to the cyprid stage (normally within a day). A drop of 2–4% seawater-based formalin was added to each slide to kill adhering bacteria and fungi, and enough anhydrous glycerin was added to at least double the volume of liquid on the slide. A small amount of glycerin jelly was placed on a flat microscope slide with forceps and melted using a cigarette lighter or a hot plate (the latter is recommended for uniformity of heating and avoidance of boiling). Each LSN exuvia was transferred to the molten medium on a slide using either fine forceps or a needle and arranged in place. After any bubbles had been removed from the vicinity of the specimen, a cover glass was lowered onto the still-molten drop with forceps; the cover glass was supported by four drops of dried nail varnish, in place of the chewing gum used by Michels (2007), to avoid crushing the specimen. After the jelly had hardened and any excess had been wiped away, nail varnish was used to seal the slide; nail varnish is convenient, but see Neuhaus et al. (2017) concerning potential problems with using it for, e.g., slides bearing type specimens.

CLSM and illustrations

One last-stage naupliar exuvia each of Hansenocaris demodex and H. aquila, along with five other LSN exuviae, each representing a different undescribed morphospecies of y-larva, were examined with a Nikon Eclipse Ti2 inverted confocal microscope by making use of the autofluorescent signal of the molted cuticle. The signal was acquired by using a laser excitation wavelength of 488 nm and an emission detection of 500–550 nm. Autofluorescence was also tested at additional wavelengths (emission 405 nm, detection 430–475 nm; emission 561 nm, detection 570–616 nm; and emission 640 nm, detection 663–738 nm), but the signal obtained was too weak to be useful. Optical sections in the z-plane were obtained through a 40 × 1.30/ oil objective. The thickness of the optical sections was kept constant across samples (0.2 µm), but the number of sections was dependent on the specimen’s size and shape, ranging from 60 to 300. A fixed Z-intensity correction curve was applied through z-stacks with more than 100 optical sections to compensate for the decrease in autofluorescent signal deeper in the stacks. Stacks obtained during each scanning session were automatically denoised by using the Denoised.ai module included in NIS-Elements software v5.30.02., saved and exported from NIS (Nikon format) to TIFF format and handled further in Fiji, ImageJ software v2.0.0 (Schindelin et al. 2012). Three-dimensional renderings based on the acquired Z-stacks were generated either with the NIS-Elements program or in Imaris v9.5.1 (Oxford Instruments).

The final images used in the figures were generated using the above-mentioned programs (Fiji, ImageJ software, NIS-Elements and Imaris) and edited for level, contrast and brightness in Adobe Photoshop v24.0.1. Various subsets of the full stack were combined into single images using Zerene Stacker, ver. 1.04. For some specimens, to avoid ventral and dorsal structures blending and becoming indistinguishable, subsets of the stack were chosen to show either the ventral or the dorsal side. In general, dorsal and ventral sides yielded different levels of autofluorescent signal, creating a need to scan these two body parts with different settings. For Hansenocaris demodex, the dorsal and ventral scans are shown separately (Fig. 1A, B), whereas for H. aquila, the two scans are blended into a single composite image (except for the separated shield) (Fig. 2A). For many specimens, alternative views, such as lateral views of dorso-ventrally mounted specimens, were obtained using the 3D visualization mode in the NIS elements software (Figs. 1C, D, 2B, D, E, H, I). For H. demodex, which had been visualized in considerable detail earlier by other methods (Olesen et al. 2022), structures that were newly detected in this study, or understood in a new way, are indicated with yellow lettering in the figures; those that had been clearly identified previously (Olesen et al. 2022) yet did not show up easily using CLSM are indicated with red lettering. The same principle was applied to H. aquila, but this species was described in less detail originally (Olesen and Grygier 2022). For the other types of y-nauplii included here, very little prior information is available for comparison (see Olesen and Grygier in press). All figures were assembled and labelled using CorelDraw v. X8. For easy comparison with previously used methods (SEM and Nomarski), a few images from earlier papers are re-used here (Figs. 1E, F, 2K). 3D visualization of all species and types presented in the paper are available in a supplementary video deposited at figshare https://doi.org/10.6084/m9.figshare.24715968.v1 and YouTube https://youtu.be/u0megFZ6OTc

Fig. 1
figure 1

CLSM of exuvia (A–D) and SEM (E, F) of last-stage nauplii of Hansenocaris demodex (Pancrustacea: Facetotecta). A Ventral view. B Dorsal view. C Dorso-lateral view. D Frontal view of cephalon. E Ventral view. F Dorsal view. A1 First antenna; A2 Second antenna; MD Mandible; Arabic numerals Pore numbering. Arrows mark the dorsal border between the cephalic shield and the posterior part of the faciotrunk. SEMs (E, F) and pore numbering from Olesen et al. (2022). The authors retain the rights to reproduce and/or distribute the original images published in this paper

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Fig. 2
figure 2

CLSM of exuvia (A–J) and DIC (Nomarski) microscopy (K) of holotype last-stage nauplius of Hansenocaris aquila (Pancrustacea: Facetotecta). A Ventral view. B Ventro-lateral view. C First antenna and mandible of right side. D Lateral view. E Labrum, lateral view. F Labrum, ventral view. G Cephalic shield, dorso-ventral view. H Cephalic shield, frontal view. I Cephalic shield, posterior view. J Posterior part of trunk, dorsal view. K Ventral view. DIC (Nomarski) image (K) from Olesen and Grygier (2022). A1 First antenna; A2 Second antenna; MD Mandible; Mx2 Second maxilla; Arabic numerals 1–6 Spines/projections of the labrum’s median keel. Arrows mark the dorsal border between the posterior part of the faciotrunk and the cephalic shield, the latter having become disconnected from the former. The authors retain the rights to reproduce and/or distribute the original images published in this paper

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Terminology of y-nauplii

The general terminology for y-nauplii used herein follows recent works (Olesen et al. 2022; Olesen and Grygier 2022, in press; Dreyer et al. 2023b). As this study deals exclusively with exuviae, only external structures are of relevance. Two terms, “cephalic shield” and “faciotrunk”, are particularly important when considering the external morphology of y-naupliar exuviae. The shed naupliar exuviae of y-larvae typically break into two parts when they molt to the next naupliar instar (Itô 1987, 1990). One part, the cephalic shield, consists of the dorsal exoskeleton of the cephalic region. The other part, the faciotrunk, consists of the entire ventral side of the nauplius together with the dorsal side of the trunk. In the last-stage nauplii, the two pieces typically stay together after the molt to the cyprid has occurred, as seen in our specimens of H. demodex and Types H, AB, X and U*; Figs. 1, 3A, B, D–F). In our specimens of H. aquila and Type AF (Figs. 2, 3C), however, the LSN exuviae have become separated into two parts. In the studied y-naupliar exuviae, the cephalic shield and the “facial”, dorsal and lateral surfaces of the faciotrunk are divided by ridges into an intricate pattern of “plates” or “facets”, characteristic for y-larvae, which is centered around an atypical shield plate, the so-called “window”. The terminology previously applied to these facets is complicated and incomplete; for the sake of simplicity, none of the three similar but different-in-detail nomenclatural systems that have been suggested so far (Schram 1972; Itô 1987, 1990; Kolbasov et al. 2021a, b) is applied here. In addition to the appendage armature of natatory setae and (for planktotrophic y-nauplii) masticatory setae and spines, several kinds of setae and pores are found on the body surface. These were fully mapped on the last-stage nauplius of H. demodex (Olesen et al. 2022), which terminology is followed here for ease of comparison to the CLSM images. Pores and other surface structures have not been fully mapped for H. aquila or any other y-naupliar types, but see Grygier (1995: Fig. 3) regarding their distribution on the cephalic shield of an undescribed Japanese form reared by the late Tatsunori Itô). Finally, the caudal end of a y-nauplius terminates in three protruding structures, viz., the medial dorso-caudal spine and a ventral pair of furcal spines, the terminology for which follows that of other crustacean nauplii (Martin et al. 2014).

Fig. 3
figure 3

CLSM of exuviae of five different types (morphospecies) of last-stage y-nauplii (Pancrustacea: Facetotecta). A Nauplius Type H, ventrolateral view. B Nauplius Type AB, ventrolateral view. Inserted scheme shows two possible hypotheses for the identity of maxillae 1 and 2 (see “Discussion” section). C Nauplius Type AF, lateral view. D Nauplius Type X, dorsal view. E Nauplius Type X, lateral view. F Nauplius Type U*, ventral-dorsal view. G Nauplius Type U*, lateral view. Arrows mark the dorsal border between the cephalic shield and the posterior part of the faciotrunk. A1 First antenna; A2 Second antenna; MD Mandible; Mx1 First maxilla; Mx2 Second maxilla; Thp1, Thp 2 Thoracopods 1 and 2. The authors retain the rights to reproduce and/or distribute the original images published in this paper

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Results

Pancrustacea Zrzavý & Štys, 1997.


Thecostraca Gruvel, 1905.


Facetotecta Grygier, 1985.


Hansenocarididae Olesen & Grygier 2022


Hansenocaris Itô, 1985.


Hansenocaris demodex Olesen, Dreyer, Palero & Grygier in Olesen et al. 2022

Material examined: One LSN exuvia left behind after the molt to a y-cyprid, mounted in glycerin jelly in a dorso-ventral orientation. Collected at Sesoko I., Okinawa, Japan (26°38′09.4″N, 127°51′55.2″E) on Sep-29–2005 by Mark J. Grygier (NHMD-916639, paratype).

A first autofluorescence scan of the examined specimen from dorsal to ventral picked up a strong signal from the dorsal side of the body, including its facets and pores (Fig. 1B), while the ventral side and the appendages yielded only a weak signal. A second scan, performed with settings ensuring maximum intensity for the ventral side, fixed this problem (Fig. 1A). The scans showed the same overall body habitus as had been found previously using other methods, i.e., a cigar-shaped body terminating in a short, blunt dorso-caudal spine (Olesen et al. 2022; Olesen and Grygier in press). A maximum-intensity 3D digital rendering, in which the specimen was turned digitally 90° to depict a lateral view, showed the same lateral body profile as had been seen previously by SEM and live video, which suggests that the exuvia retained its true form after being mounted. In contrast to SEM, CLSM also collects information about subsurface cuticular structures and here revealed the presence of previously unseen facet-like structures in the cephalic shield’s median region and on the labrum (Fig. 1A, B). In addition, at least one pore in the dorsal midline of the cephalic shield (Fig. 1B), not previously recognized, was apparent using CLSM. Many structures previously revealed by SEM did not show up in these CLSM scans (marked in red in Fig. 1B). This pertains to ventral structures, such as the general surface of the body and labrum, some transverse ridges and the naupliar appendages including the setae of the latter. It also pertains to some pores, both dorsal and ventral, while one pore (#8) appears to lack an emerging seta otherwise known to be present based on SEM. The cuticular area corresponding to the so-called window of the cephalic shield did not emit any signal, leaving an “empty” space that shows its position more clearly than SEM did; with SEM, the whole mid-dorsal region of the cephalic shield simply appeared smooth (Fig. 1F).


Hansenocaris aquila Grygier and Olesen, 2022.

Material examined: Holotype LSN exuvia left behind after the molt to a y-cyprid, mounted in glycerin jelly in a dorso-ventral orientation. Collected at Sesoko I., Okinawa, Japan (26°38′09.4″N, 127°51′55.2″E) on Sep-22–2005 by Mark J. Grygier (NHMD-1174615, holotype).

The examined specimen has become separated into two parts, the cephalic shield and the faciotrunk. The more dorsal parts of the faciotrunk emitted a stronger signal than the ventral parts, so these parts were scanned separately at different settings and combined into one composite image (Fig. 2A). The naupliar appendages, inserting ventrally, in general emitted a weak signal and could not be studied in detail using CLSM (Fig. 2C), but their segmentation could be confirmed as matching what had been described before using Nomarski LM (cf. Olesen and Grygier 2022; Olesen and Grygier in press). The CLSM scans revealed the same overall ventral habitus as had been shown previously, but also much new information of importance for taxonomy. Previously, only a dorso-ventral view of the holotype was available (Olesen and Grygier 2022: fig. 6), but by using maximum-intensity 3D digital renderings based on a dorso-ventral z-stack, other views can be shown (Fig. 2B, D, E, H, I). A projection in which the specimen has been turned digitally 90° to present a lateral view reveals the precise angles between the long axes of the cephalic shield and the trunk region (40°) and between the trunk region and the dorso-caudal spine (15°) (Fig. 2D). The shape, pores and spination of the naupliar labrum were also revealed in detail for the first time (Fig. 2F, E). In the ventral view, the labrum is an elongated trapezoid with the posterior margin extended into a large spine, and it also bears some smaller spines (Fig. 2A). To be precise, in the lateral view (Fig. 2B, D–F) the midline of the labrum is seen to be elevated into a keel, the posterior part of which extends into a total of six projections or spines: three small projections (1, 6, 7), three moderately-sized spines (2–4) and one large spine (5). A pore is present in each postero-lateral corner of the labrum (Fig. 2A, B), and two other putative pores are seen in the posterior part of the midline keel, possibly associated with spines 2 and 3 (Fig. 2F). Aside from the appendages, all other surface structures were much more clearly depicted in CLSM than with previous kinds of light microscopy. This includes the facet pattern of both the faciotrunk (including the labrum) and the cephalic shield, pores on various parts of the body and the cuticular ridges that are abundant both ventrally and dorsally on the trunk region. A full description of these structures is postponed, but the ornamentation of the ventral side of the trunk deserves special note. Flanking a long band of short, transverse ridges along the midline of the somewhat swollen and rounded anterior half of the trunk are paired rows of bumps that evidently represent either somite boundaries or the future setae of thoracopods I–VI, and, more anteriorly, another pair of such rows possibly representing the undeveloped second maxillae (see “Discussion”) (Fig. 2A, B, D).


Type H (sensu Dreyer et al. 2023a; Olesen and Grygier in press)

Material examined: One LSN exuvia left behind after the molt to a y-cyprid, mounted in glycerin jelly in an almost dorso-ventral orientation (digitally turned to a latero-ventral view in Fig. 3A). Collected at Sesoko I., Okinawa, Japan (26°38′09.4"N, 127°51′55.2"E) on Jun-10-2019 by Danny Eibye-Jacobsen, Mark J. Grygier and Jørgen Olesen (NHMD-1704894).

The previous brief characterizations of this y-naupliar type were based on video recordings of live nauplii as well as LM examination by differential interference contrast (DIC) (Nomarski optics) of a specimen/exuvia different from the one studied here.

Although it has been difficult to keep dorsal and ventral cuticular information separate due to the body shape and the closeness of the two sides of the exuvia to each other on the slide, CLSM confirms the general morphology and habitus as previously reported (Olesen and Grygier in press), including the extremely bent body with the trunk region downturned 90° relative to the cephalic region, the conical dorso-caudal spine and the large, curved furcal spines. The labral morphology is shown for the first time here: the posterior labral margin is convex and free from the ventral side of the cephalon, with a short longitudinal keel in the ventral midline and at least four pores, two in the midline posterior to the keel and one in each postero-lateral corner.


Type AB (sensu Dreyer et al. 2023a; Olesen and Grygier in press)

Material examined: One LSN exuvia left behind after the molt to a y-cyprid, mounted in glycerin jelly in an almost dorso-ventral orientation (digitally turned to a latero-ventral view in Fig. 3B). Collected at Sesoko I., Okinawa, Japan (26°38′09.4″N, 127°51′55.2″E), Sep-25-2005, by Mark J. Grygier (NHMD-1705152).

The previous brief characterizations of this y-naupliar type were based on video recordings of live nauplii as well as LM examination by differential interference contrast (DIC) (Nomarski optics) of the same specimen/exuvia studied here.

CLSM confirms the general morphology and habitus as presented previously (Olesen and Grygier in press), including the relative dimensions of the cephalic shield and trunk and their angle of bend relative to each other, as well as the general morphology of the labrum and the caudal spines. The morphological resolution of many parts of the body is greater using CLSM, however. In particular, CLSM shows much more clearly details of the spatulate labrum, including its facet pattern, while its three pores stand out particularly clearly. The facet pattern and pores of the cephalic shield are also quite distinct (not shown). The ornamentation of the ventral side of the trunk is considerably more distinct in CLSM images than has been shown before. Most notably, a longitudinal band of short transverse ridges along the ventral midline of the somewhat swollen and rounded anterior half of the trunk is flanked by more laterally situated paired rows of small, rounded spines that evidently represent either somite boundaries or the future setae of thoracopods I–VI. A pair of prominent arched ridges situated more anteriorly delineate the somite of the second maxilla, if not the rudiments of the second maxillae themselves, and most anteriorly sit another pair of cuticular lobes that may represent the first maxillae (Fig. 3B, Hypothesis 2 in the homology scheme insert; see “Discussion”).


Type AF (sensu Dreyer et al. 2023a, b; Olesen and Grygier in press).

Material examined: One LSN exuvia left behind after the molt to a y-cyprid, mounted in glycerin jelly in an almost lateral orientation. Collected at Sesoko I., Okinawa, Japan (26°38′09.4″N, 127°51′55.2″E) on Sep-24-2005 by Mark J. Grygier (NHMD-1705195).

The previous brief characterizations of this y-naupliar type were based on video recordings of live nauplii as well as LM examination by differential interference contrast (DIC) (Nomarski optics) of a specimen/exuvia different from the one studied here.

CLSM confirms the general morphology and habitus as previously reported (Olesen and Grygier in press), including the very short and compact body, the conical dorsocaudal spine and the extension of the labrum into a needle-like process. The facet pattern and the pores of the cephalic shield are shown here for the first time. The facet pattern of the lateral side of the trunk was shown previously (Olesen and Grygier in press) but is clearer in CLSM images, which is also the case for certain pores in this body region.


Type X (sensu Dreyer et al. 2023a; Olesen and Grygier in press)

Material examined: One LSN exuvia left behind after the molt to a y-cyprid, mounted in glycerin jelly in a dorso-ventral orientation. Collected at Sesoko I., Okinawa, Japan (26°38′09.4″N, 127°51′55.2″E) on Jun-9-2019 by Danny Eibye-Jakobsen, Mark J. Grygier and Jørgen Olesen (NHMD-1705030).

and


Type U* (sensu Dreyer et al. 2023a; Olesen and Grygier in press)

Material examined: One last-stage naupliar exuvia left behind after the molt to a y-cyprid, mounted in glycerine jelly in a dorso-ventral orientation (digitally turned 90° to a lateral view in Fig. 3F). Collected at Sesoko I., Okinawa, Japan (26°38′09.4″N, 127°51′55.2″E) in Jul-16–19-1996 by Mark J. Grygier (NHMD-1705026).

The previous brief characterizations of these two y-naupliar types were based on video recordings of live nauplii as well as LM examination by differential interference contrast (DIC) (Nomarski optics) of the same specimens/exuviae studied here.

CLSM confirms the general morphology and habitus of both Types X and U*, and the z-stacks also show much potential for documenting the facet pattern and other superficial ornamentation of both specimens. This task is postponed until proper species descriptions are prepared, but one additional distinct advantage of CLSM may be highlighted. Both Types X and U* are distinctly flattened dorso-ventrally, and also quite wide in comparison to their height. Until now it has been very difficult to examine either species in lateral view by LM as the LSN exuviae always become mounted in a flat position on slides. Here, however, a maximum-intensity CLSM rendering, in which specimens of both types have been turned digitally 90°, shows for the first time how flat they are. Type X is ca. 50 µm thick and Type U* is only ca. 30 µm thick after the escape of the y-cyprid.

Discussion

Taxonomic study of minute aquatic and terrestrial invertebrate taxa with numerous co-occurring species can be challenging. To avoid error, sets of morphological and molecular information derived from the same individual are desirable, but the use of destructive methodology poses a dilemma with regard to the design of analytical protocols suitable for small specimens. In studies of molting invertebrates (Ecdysozoa), particularly proturans and aquatic mites, this dilemma has been overcome by the development of non-destructive molecular extraction techniques that leave behind a cuticular exoskeleton for study by confocal microscopy (Böhm et al. 2011; Valdecasas and Abad 2011). Here, we have applied a similar procedure to crustacean y-larvae (Facetotecta), another taxonomically challenging group of micro-invertebrates with, at least locally, large numbers of co-occurring species.

Much recent work on y-larvae has taken advantage of the fact that lecithotrophic y-nauplii can be reared in the laboratory from an early stage to the cyprisy in only 4–12 days. It has been possible to make reasonably high-resolution live video recordings of particular individuals at short intervals as their larval development progresses, keep the shed cuticle of the last-stage nauplius as a voucher, usually mounted on a slide, and preserve the corresponding cypris y for either SEM or molecular barcoding (Olesen et al. 2022; Dreyer et al. 2023a; Olesen and Grygier in press). Based on this, enough morphological information has been obtained to convincingly recognize the presence of more than 30 different “morphospecies” of lecithotrophic y-nauplii in the marine shallow-water plankton at Sesoko I., Okinawa, as well as another nine or so morphospecies of planktotrophic y-nauplii, but not enough in most cases to support full species descriptions (Olesen and Grygier in press). Voucher slides of LSN cuticles have recently been recognized as crucially important for the taxonomy of y-larvae because they unambiguously link different life stages, nauplius y and cypris y, of particular species to each other (Olesen et al. 2022). Until now, such exuviae have only been studied with conventional LM, which, while offering much information, is difficult to present visually in a clear way. CLSM, however, proves to be a quite useful adjunct to LM for the taxonomic study of y-larvae.

Examination of the present diverse set of y-naupliar exuviae by CLSM usually provided an impressive level of detail, with resolution comparable to that obtainable by SEM. The option of digitally-mediated three-dimensional rotation of a single specimen is a particularly strong asset of CLSM, opening new avenues for taxonomic investigation when limited material is available. One such example is Hansenocaris aquila (Fig. 2), which was described primarily on the basis of the exuvia of a single LSN, designated as the holotype (Olesen and Grygier 2022). Reexamination of this specimen using CLSM and autofluorescence very easily revealed new morphological features, including details of the unusual spine-bearing labrum and the precise facet pattern of the cephalic shield (Fig. 2), both of which are important for nauplius-based y-larval taxonomy. Digital manipulation of the image stack furthermore permitted the presentation of a lateral view of H. aquila for the first time. Olesen and Grygier (in press) showed that lateral views of y-nauplii provide a wealth of taxonomically important information, especially regarding the degree of “body bending” and the angles between the long axes of the cephalic shield and trunk, and the trunk and dorso-caudal spine. For H. aquila, Olesen and Grygier (2022: 307) noted “no lateral view available” but nonetheless remarked in error that the axes appeared to be “nearly in [the] same plane”. The side-view CLSM image (Fig. 2D) shows that the angle between the axes of the cephalic shield and trunk is actually ca. 40°, not very different from that seen in many other y-naupliar types (Olesen and Grygier in press).

The original description of Hansenocaris demodex was much more detailed than that of H. aquila as more specimens were available for study (Olesen et al. 2022). Using autofluorescence-based CLSM of only a single specimen, it has been possible to obtain a level of detail comparable to that in the original SEM-based description (Fig. 1), especially regarding the dorsal facet pattern and distribution of pores. Even some hitherto overlooked sub-surface parts of the facet pattern could be identified using CLSM. Otherwise, in general, CLSM confirms previous findings for this species, and it could have replaced SEM if there had been only limited material.

The present CLSM images reveal aspects of the development of the thoracopods and first and second maxillae in y-larvae that are often not clear using other methods. As in Thecostraca in general (Walossek and Müller 1998), thoracopodal limb buds do not appear during the naupliar phase of development; instead, fully developed natatory thoracopods appear in the cyprid instar. In y-larvae, the six pairs of thoracopods develop inside the trunk of the nauplius. In many lecithotrophic forms, as well as the planktotrophic H. itoi, this occurs within an array of diaphanous internal “pockets” (the so-called “ghost”: Grygier et al. 2019; Kolbasov et al. 2021a). In the facetotectan LSN, traces of the thoracopods (rudiments of their terminal setae?) are usually present externally on the trunk as paired arrays of ventral bumps or spines, although the rows may not be clearly organized (e.g., Hansenocaris cristalabri; see Olesen and Grygier 2022: fig. 2A) and thus not be clearly assignable to specific pairs of thoracopods. Here, on the contrary, the naupliar trunks of at least H. aquila and Type AB (Figs. 2A, B, D, 3B) are shown by autofluorescence-based CLSM to bear clearly demarcated and paired rows of transverse spine rows that can be easily attributed to the six pairs of thoracopods if they do not merely mark somite boundaries. More anterior pairs of spine rows or protruding ridges in these nauplii (2A, B, D, 3B) may, therefore, be anlagen of the first and second maxillae or mark their somites. This is not a simple issue, however.

Metanauplii of several groups of Thecostraca have a pair of rudimentary, at most one-segmented, “maxillary” appendages, but there appears to have been no real analysis of their homologies among the relevant groups, nor any but the most rudimentary analysis within any group as to whether they represent the first or second pair of maxillae. In later-instar nauplii of thoracican cirripedes, several authors have referred to a lateral pair of seta-bearing lobes—either articulated to the body or not—as maxillae (e.g., Egan and Anderson 1989; Kado and Kim 1996; Kugele and Yule 1996); only a few authors have called them specifically first maxillae or maxillules (e.g., Dineen 1984; Moyse 1987). Several pairs of more posteriorly situated spines on the ventral thoracic process of barnacle nauplii have been matched to the six developing thoracomeres of the cypris larvae (e.g., Rainbow and Walker 1976), but never to (second) maxillae. In Ascothoracida of several families, a pair of supposed first maxillae (maxillules) is present from the second naupliar stage onward, expressed as either a pair of setae or thin spines or, in late instars, a pair of setose, basally articulated limb rudiments (e.g., Itô and Grygier 1990; Grygier 1992; Kolbasov et al. 2021a). Their identity as first maxillae is supported by the observation of Boxshall and Böttger-Schnack (1988), in a last-stage planktotrophic metanauplius with a developing cyprid-stage larva inside, that the primordia of the latter’s second maxillae were grouped with the six pairs of thoracopodal primordia and that all of these were associated with seven posteroventral pairs of external bumps (the supposed precursors of these limbs) on the nauplius. Since the second maxillae were thus accounted for, the more anterolateral pair of small limbs would have to be the first maxillae, an identification that was accepted by Walossek and Müller (1998).

As for the Facetotecta, only a single pair of setiform “maxillary” rudiments has been reported so far, in planktotrophic LSNs of nauplius y Type VI (Grygier 1987b) as well as Hansenocaris furcifera and H. itoi (Itô 1990; Kolbasov et al. 2021a), positioned like those of ascothoracidans. An assumption that these are first maxillae would imply loss of the second maxillae in nauplii of y-larvae (and in the nauplii of other thecostracans) without leaving much of a trace externally (Hypothesis 1 in Fig. 2B). Here we suggest that the purported “first maxillae” of facetotectan LSNs may be more likely to be the second maxillae based on their position immediate anterior to the first pair of thoracopods, similar to the position of the structures in question in Type AB and H. cristalabri (Hypothesis 2 in Fig. 3B). There is clearly a need for a re-evaluation of the identity of the “maxillary” anlagen in nauplii of all groups of Thecostraca based on new evidence, for example by confirming their nervous-system connections (see, e.g., Kalke et al. 2020).

CLSM has some limitations. In several specimens, we found a very uneven autofluorescence signal, especially between the dorsal and ventral sides, with the latter exhibiting a weaker signal. This could often be compensated for by adjusting the settings at intervals within the z-stack (see “Materials and methods” section), by performing scans at different settings or by constructing a composite image that combines different parts of two or more stacks (as in H. aquila: Fig. 2A). In H. demodex, though, the autofluorescence signal of the ventral side and appendages remained weak despite any adjustment of settings. This and other specimens would probably have benefitted from being stained with Congo red (cf. Michels and Büntzov 2010), something that cannot be done with the present, already slide-mounted material. If Congo red is used, however, a different anti-microbial agent than phenol might be advisable in the glycerin jelly, because phenol can cause stains to fade (Neuhaus et al. 2017).

Beside the two described species, we successfully tested the usefulness of CLSM on a series of other very different and still little-studied y-naupliar types (Fig. 3) that were briefly characterized by Olesen and Grygier (in press). CLSM with autofluorescence easily confirmed the general morphology of all these forms and, while not explored in detail here, also showed great potential for easily adding more and quite detailed information. The examined specimen of Type AB provided strikingly clear images of labral morphology and indications of incipient thoracic segmentation. As for Type H, which is an extremely bent naupliar form, details of labral morphology were not available previously but could be readily confirmed using CLSM. Observations of Type AF using CLSM provided hitherto unavailable information about the facet pattern of the cephalic shield. Two examined specimens represent y-naupliar Types X and U*, which Olesen and Grygier (in press) characterized as significantly dorso-ventrally flattened even though they were unable to view either directly from the side. For both types, digital rotation of a CLSM z-stack has allowed this description to be confirmed; indeed, for Type U*, the body is only ca 30 µm thick, rendering it the flattest (10% relative to width) y-nauplius so far found. Although some dorso-ventral compression during mounting might be expected, videos of other examined living specimens of Type U* (unpublished data) indeed give the impression of extreme flatness, a body form that may favor planktonic dispersal (Olesen and Grygier in press).

In summary, CLSM using cuticular autofluorescence is proving to be an excellent tool in studies of larval morphology and taxonomy of the neglected crustacean taxon Facetotecta. It is probably the most effective way of making use of the Natural History Museum of Denmark’s extensive slide collection of y-naupliar exuviae. The applicability of this technique should be explored further, beyond y-larvae to other autofluorescing taxa that are frequently found as whole-body or whole-exoskeleton slide mounts in museums such as loriciferans, kinorhynchs, priapulid larvae, tardigrades, etc. (Sørensen et al. 2022, 2023).