Anatomical study of Pycnophyes robustus (Kinorhyncha: Allomalorhaghida: Pycnophyidae): Evaluation of microcomputed tomography in comparison with other microscopic techniques

ABSTRACT

Micro-computed tomography is a non-invasive technique that allows 3D rendered images with anatomical information, which facilitates functional anatomy interpretations. Its usefulness has already been proven in several animal groups of relatively small size, but it has rarely been applied for the anatomical and morphological investigation of meiofaunal organisms. Here, we tested micro-CT as a potential technique for anatomical studies on Kinorhyncha, a hard-bodied meiofauna phylum. The morphological information obtained from our micro-CT images regarding the cuticle, epidermis, digestive, excretory, and reproductive systems fits with the known anatomical information on kinorhynchs through classical techniques such as Light Microscopy (LM), Transmission Electron Microscopy (TEM), Scanning Electron Microscopy (SEM) and Confocal Laser Scanning Microscopy (CLSM), thus presenting a valid option when few specimens are available for study, as it is practically a non-invasive technique for the morphological study of these animals.

KEYWORDS:
Anatomy; kinorhynchs; micro-CT; microscopic organisms; morphology

INTRODUCTION

Animal anatomy has long been a central subject of scientific inquiry. Since its inception, light microscopy has revolutionized biology and remains one of the most essential tools in the life sciences. Advances in contrast techniques have greatly enhanced the resolution and accuracy of observations-both through physical methods, such as phase contrast and Differential Interference Contrast (DIC) microscopy, and chemical approaches, ranging from traditional staining to fluorescence microscopy and confocal techniques like Confocal Laser Scanning Microscopy (CLSM). Additionally, the development of electron microscopy, including Transmission Electron Microscopy (TEM) and Scanning Electron Microscopy (SEM), has enabled detailed structural analysis at the cellular level.

However, these techniques also have limitations: TEM requires the specimen to be sectioned, while CLSM often depends on freshly collected animals and involves multiple staining and incubation steps. As a result, the search for less invasive methods to study animal anatomy continues to be a significant challenge for researchers.

In recent years, the development of computed tomography techniques has provided excellent anatomical results (e.g., Stauber and Müller 2008, Metscher 2009, Jeffery et al. 2011, Faulwetter et al. 2013, Gross et al. 2019, Ferstl et al. 2020), as it is a comparatively less invasive method that enables 3D image rendering and reveals morphofunctional relationships between organs and structures. Originally developed for medical diagnostics, computed tomography was later applied to various small-sized invertebrate groups, including several classes and orders of Insecta (Hörnschemeyer et al. 2002, Ribi et al. 2008, Garwood et al. 2009, Verdú et al. 2012, Lautenschlager 2013, Alba-Tercedor et al. 2016, 2017, Alba-Tercedor and Marcos-García 2024), Mollusca (Golding and Jones 2007, Alba-Tercedor and Sánchez-Tocino 2011, 2012), and Annelida (Paterson et al. 2014). Notably, excellent results have been achieved even for small, soft-bodied mollusks ranging from 2 to 30 mm in size (Candás et al. 2016). However, applying microcomputed tomography (micro-CT) with sufficient resolution to very small organisms, such as those in the meiofauna, remains challenging due to their minute size (Ferstl et al. 2020).

Meiofauna comprises small-sized organisms that can pass through a 1.00 mm mesh but are retained by a 0.32 µm mesh. These tiny animals have gained increasing attention in research due to their suitability as model organisms for a wide range of studies, from morphology to ecology and phylogeny (Sørensen and Giribet 2006, Worsaae et al. 2012, Bekkouche et al. 2014, Kerbl et al. 2015, Laumer et al. 2015).

Kinorhyncha is a ubiquitous meiofaunal phylum and an excellent candidate for testing the potential of micro-CT, as its overall body plan and organ systems are relatively well understood. The internal and external anatomy of kinorhynchs was first described by Reinhard (1885, 1887) and Zelinka (1928) based on light microscopy. In the last quarter of the 20^th century, more advanced techniques such as Transmission Electron Microscopy (TEM) (Moritz and Storch 1972a, 1972b, Brown 1983, Neuhaus 1988, 1994, 1997, Adrianov et al. 1989, 1990, Adrianov and Malakhov 1990, 1991, 1999, Kristensen and Higgins 1991, Nebelsick 1992a, 1992b, 1993) and Scanning Electron Microscopy (SEM) (Higgins 1983, Pardos et al. 1998) were applied to the group. Among these, the TEM study by Kristensen and Higgins (1991) stands out for its detailed characterization of the nervous, muscular, excretory, and reproductive systems. More recently, in the 21^st century, several studies using CLSM have provided further insights into the nervous and muscular systems of kinorhynchs (Müller and Schmidt-Rhaesa 2003, Rothe and Schmidt-Rhaesa 2004, Schmidt-Rhaesa and Rothe 2006, Herranz et al. 2013, 2014, 2019, 2020, 2021a, 2021b, Altenburger 2016).

The objectives of the present study are: (1) to describe, for the first time, the internal anatomy of Pycnophyes robustus Zelinka, 1928 (Allomalorhagida: Pycnophyidae); and (2) to assess the potential of microcomputed tomography (micro-CT) as a non-destructive imaging technique for anatomical studies of Kinorhyncha, through comparison with existing morphological data obtained using traditional methods. To this end, we also examined the anatomy of the same species using histological sections and light microscopy (LM), and compared our findings with published data on other kinorhynch species obtained through LM, scanning electron microscopy (SEM), transmission electron microscopy (TEM), and confocal laser scanning microscopy (CLSM). This integrative approach provided a broad comparative dataset, allowing us to evaluate the advantages and limi tations of applying micro-CT to this group of hard-bodied meiofaunal organisms.

MATERIAL AND METHODS

Specimens were obtained from sediment samples at Ceuta (Spanish north-African coast) on May 22, 2013, and were extracted from the sediment using the bubbling and blot technique (Higgins 1964, Sørensen and Pardos 2008). Subsequently, all kinorhynchs were sorted alive under a stereomicroscope using an Irwin-loop and directly preserved in 100٪ ethanol. Four kinorhynch specimens were identified as Pycnophyes robustus using an Olympus BX51 microscope with non-permanent slides. This species was selected for the study because it possesses characteristics that facilitate the observation and application of the required techniques. First, representatives of the family Pycnophyidae are relatively large within the phylum (ca. 600-700 μm in length), which makes anatomical structures easier to observe; in addition, their cuticle is very robust, which ensures that the animal does not wrinkle during the drying process, preserving the shape of the internal structures.

For histological studies, two specimens were stained with OsO₄ and included in Epon embedding medium to obtain thin histological cross-sections (2 μm thick). Each section was stained with toluidine blue and mounted on permanent slides for LM examination. For the microcomputed tomography observations, the remaining two specimens were stained with 1% iodine in 100% ethanol for four hours to enhance contrast to the structures (mainly for muscles). Then, the specimens were placed in hexamethyldisilazane for one hour and air dried overnight instead of using glutaraldehyde and critical point drying (Fratesi et al. 2004, Alba-Tercedor and Sánchez-Tocino 2011, Alba-Tercedor 2014). For the scanning process, the specimens were attached to the tip of a small filament, as described in Alba-Tercedor (2014).

Specimens were scanned using a Skyscan micro-CT attachment for SEM, installed on an environmental scanning electron microscope (ESEM) mod. Quanta 400 FEI. X-rays for tomography were generated with an electron beam accelerating voltage of 30 kV. To increase the beam current and thus the X-ray signal, the ESEM final aperture was increased from 200 to 500 μm, obtaining an average gain of about 4 (parameters: frame average = 3-5, depending on sample nature; isotropic voxel size = 1.2 µm; rotation step = 0.45° until completion of 180° rotation). Samples were centered at the micro-CT attachment for SEM using the methodology described in Alba-Tercedor (2012).

The Bruker Skyscan software NRecon, CTAn, and DataViewer were used for the primary reconstruction and “cleaning” processes. Volume rendering images were obtained using two software: Skyscan’s CTVox v. 3.3 and FEI’s Amira software (v. 2019.3) (Stalling et al. 2005). These two software were used to determinate whether either of them provided a better observation of certain structures or both offered similar results. During reconstruction with NRecon, in addition to the normal tuning procedure, we performed x/y alignments, both an x/y iterative and an x/y alignment with a reference scan. To reorient and correct the position of the sample, we used both DataViewer, and/or CTVox: this facilitated the acquisition of perfectly parallel cuts of the volume renderings when using the ‘Clipping Box’ of CTVox. With this software, as described by Alba-Tercedor (2014), it was possible to discriminate the diverse structures in a gradation of colors from red (the softer, more transparent to X-ray) to dark blue (the denser and opaquer to X-ray) without a tedious manual segmentation. The built-in color filter “volrenRed.col” was applied to obtain the color of Amira’s rendered images, obtaining a color gradation depending on the opacity of the x-rays from yellow to white.

For a better understanding and identification of the structures referred to in the following, see Sørensen and Pardos (2020), Kristensen and Higgins (1991), Nebelsick (1993), and Neuhaus (1994). For specific characters of the family Pycnophyidae, see the exhaustive list of morphological characters in Sánchez et al. (2016).

RESULTS

In this section, the main structures observed are described. If not otherwise specified, the structures can be seen with both microtomography (regardless of the software used) and LM. When a specific technique or software is mentioned (micro-CT, LM, CTVox or Amira), the structure is only visible with the specified technique or software. A video of the specimen’s reconstruction using CTVox software is available at https://zenodo.org/records/14870908.

Outer gross anatomy

The trunk is elongated with a rectangular outline, bilate ral symmetry and a conspicuously triangular cross-section. The body, covered by a cuticle, consists of three main regions: head, neck, and trunk, the latter divided into 11 segments (Figs 1A, 2A). All segments have one dorsal (tergal) and two ventral (sternal) cuticular plates, except for the first segment, which is divided into one tergal and three ventral plates; one midsternal and two episternal.

Various outer cuticular structures and appendages are detectable. Nevertheless, through micro-CT observations, both setae and sensory spot insertions appear as dark, rounded areas at the surface in frontal and sagittal views (Figs 1A, 2A). These dark dots appear in paradorsal, subdorsal, laterodorsal, paralateral, lateroventral, ventrolateral, and ventromedial positions, but it is possible to determine whether a structure is a seta or a sensory spot only in a few cases. For instance, a structure can be identified as a seta if an elongated form emerges from the dark area. In addition, large glandular cell outlets can be identified in both micro-CT and LM cross-sections as round hollows at the anterior region of some segments (Figs 3B, I, 6H), although smaller gland openings (cuticular scars) are not visible with micro-CT. As for the appendages, middorsal elevations are detectable on segments 2-9 and robust lateral terminal spines are present on segment 11. Cuticular ridges are visible (Figs 1A, 2A), whereas cuticular hairs are not detectable.

Figure 1
Longitudinal micro-CT rendered image reconstructions. (A-D) parasagittal and (E) dorsal sections of a female of Pycnophyes robustus through CTVox software. (cr) Cuticular ridge, (dvm,) dorsoventral muscle, (hg) hindgut, (m) mouth, (mc) mouth cone, (mg) midgut, (ne) nephridium, (p) pharynx, (pm) pharyngeal bulb muscle, (prm) pharynx retractor muscle, (s) segment, (sp) sphincter. Numbers after abbreviation indicate the segment number. Dotted lines in A indicate sensory spots and setae. Scale bar: 50 µm.

Although the head is fully retracted into the trunk in the studied specimens, the protrusible mouth cone and the eversible introvert are visible internally between segments 1-4 (Figs 1D-E, 2D-E, 6B-D). The retracted mouth cone, where the mouth opens, appears as a conical structure pointing forward at the level of segment 4, and its opacity increases as the micro-tomograph crosses the structure (Figs 1D-E, 2D-E). Nine outer oral styles (Fig. 6C) and two rings composed of five inner oral styles (rings -02 and -03) (Fig. 6D) are clearly discerned in the LM cross-sections, whereas the ring with 10 inner oral styles (ring -01) can be observed with both techniques (Figs 3E-F, 6D). In the micro-CT observations, this latter structure appears as thickening on the surface of the mouth cone, resembling a ten-pointed star in cross-section (Fig. 4I). The introvert shows large scalids arranged into several rings (Fig. 3C), but because it is strongly retracted inside the trunk, the scalids appear as a tangled mass of intermediate opacity filaments in the micro-CT images. The retraction prevents accurate counting and discrimination of the different scalids types and rings, except for the first ring (primary spinoscalids), which is visible with both techniques. Trichoscalids are visible in LM cross-sections but not in the micro-CT images (see Fig. 6C-D).

Figure 2
Longitudinal micro-CT rendered image reconstructions. (A-D) Parasagittal and (E) dorsal sections of a female of Pycnophyes robustus through Amira software. (cr) Cuticular ridge, (dvm) dorsoventral muscle, (hg) hindgut, (ldm) longitudinal dorsal muscle, (m) mouth, (mc) mouth cone, (mg) midgut, (ne) nephridium, (p) pharynx, (pm) pharyngeal bulb muscle, (prm) pharynx retractor muscle, (s) segment, (sr) seminal receptacle, (tm) terminal muscle. Numbers after abbreviation indicate the segment number. Dotted lines in A indicate sensory spots and setae. Scale bar: 50 µm (A-E).

Figure 3
Rendered image reconstructions in cross-sections, from anterior to posterior segments, of a female of Pycnophyes robustus through Micro-CT examination. (A, D, G, J) Overview through Amira, red square marks the exact point of the cross-sections; (B, E, H, K) cross-sections through Amira; (C, F, I, L) Cross-sections through CTVox. (bs) Ball-and-socket joint, (dg) dorsal gland, (dvm) dorsoventral muscle, (ios) inner oral style, (lvm) longitudinal ventral muscle, (m) mouth, (mg) midgut, (od) oviduct, (ov) ovary, (pb) pharynx bulb, (re) rectum, (sc) scalids, (sr) seminal receptacle, (srm) λ-shaped spinoscalid retractor muscle, (vg) ventral ganglion, (vl) ventral ligament. Scale bars: 90 µm (A, D, G, J), 30 µm (B, C, E, F, H, I, K, L).

Figure 4
Micro-CT rendered image reconstructions with CTVox of a female of Pycnophyes robustus. (A) Parasagittal section of segments 1-6; (B, D) consecutive cross-sections of segment 1, showing the longitudinal dorsal muscles as whitish bean-shaped when the micro-tomograph focuses on the surface and changing to blue color when crosses through the inner; (C, E) consecutive parasagittal sections from segments 4 and 5 to segment 10 showing muscles and the digestive organs; (F) dorsolateral view of segment 1 opened dorsally from a frontal plane, showing the longitudinal dorsal muscles at the middle of the segment; (G) detail of the anterior part of segment 1 showing the introvert short retractor muscles; (H) detail of the mouth cone and an introvert long retractor muscle; (I) detail of the posterior part of the mouth cone, with the ring of ten inner oral styles (R-01). (dvm) Dorsoventral muscle, (ilrm) introvert long retractor muscle, (isrm) introvert short retractor muscle, (ldm) longitudinal dorsal muscle, (mc) mouth cone, (p) pharynx, (pc) pharyngeal crown, (pb) pharynx bulb, (plrm) placid retractor muscle, (prm) pharynx retractor muscle. Opacity scale equal to Fig. 1. Scale bars: 90 µm (A), 50 µm (B-F, H), 20 µm (G); 25 µm (I).

Inner gross anatomy

Most of the identifiable organs and systems observed in LM cross-sections and micro-CT images follow a non-segmen ted organization (e.g., digestive, reproductive, and excretory systems) (Figs 1E, 2E). Segmentation of the body is clearly reflected by the muscular system (excluding the head musculature), the nervous system, and the mucous glands (the latter mainly observed by LM). Detailed information on the observed structures of each system is provided below in specific sections.

Cuticle and epidermis

A well-developed thick wall, the cuticle, covers the entire surface of the body, including the trunk, neck, introvert, outer structures, appendages, and anterior and posterior portions of the digestive tract (mouth, foregut and hindgut) (Figs 1, 2). Within the cuticle, three layers of different opacity can be identified by both micro-CT software, but they were discriminated more easily by CTVox. These three layers correspond to the thin epicuticle (red), the intracuticle (green), of similar thickness, and the procuticle (blue), which is the most developed and opaque layer (Fig. 3I, L). Underneath and lining the interior of the body, a band of minimum opacity is observed, corresponding to the epidermal cell layer (red) (Fig. 3I, L).

In the micro-CT images, the cuticle appears thicker, conspicuously developed, and highly opaque at the anterior edge of each segment, protruding inwards from segments 2-11 to form the pachycyclus, which serves as an anchorage point for the musculature. The cuticular plates of the same segment are articulated to each other by the ventral and lateral joints, as observed by LM and micro-CT. Contiguous segments are connected by structures of a similar nature. All these articulations are folds of the thinner, flexible cuticle that appear as transparent in the micro-CT images (Fig. 3C). Additionally, LM and micro-CT observations allow the identification of two conspicuous types of cuticular inner enlargements between the tergal and sternal plates and within the sternal plates: (1) The tergal and sternal plates are joined by special tergo-sternal joints, called ball and socket joints (Fig. 3I), and (2) Within the sternal plates, there are cuticular thickenings that reach their maximum development at the posterior segments, appearing as highly opaque, bean-shaped structures on segments 9 and 10, known as apodemes (Fig. 5D).

Figure 5
(A-G) Micro-CT examination of the anterior segments of a female of Pycnophyes robustus through Amira and H without software treatment. (A) Frontal section of segments 1-6; (B) frontal section of segments 1-4; (C) cross-section of segment 1 with Amira; (D) frontal section of segments 8-11 with Amira; (E-F) consecutive parasagittal sections from segments 4 and 5 to segment 10 showing muscles and the digestive organs; (G) parasagittal section of segments 5-7; (H) cross-section between segments 3 and 4 showing the brain. (a) Apodeme, (dvm) dorsoventral muscle, (b) brain, (ilrm) introvert long retractor muscle, (isrm) introvert short retractor muscle, (ldm) longitudinal dorsal muscle, (m) mouth, (mc) mouth cone, (p) pharynx, (mg) midgut, (sr) seminal receptacle, (prm) pharynx retractor muscle. Dotted lines in G indicate setae. Opacity scale equal to Fig. 2. Scale bars: 20 µm (A); 50 µm (B-H); 20 µm (G).

Mucous glands

Dorsal glands located in the middle region of the plates, as well as pairs of symmetrically distributed lateral glands, are observed in many segments in the micro-CT images (low to medium opacity; Fig. 3I). These structures are also recognizable in LM cross-sections; however, the granular content within the glandular vesicle and a pair of large, ventral terminal glands on segment 10 are visible only through LM (Fig. 6C, H).

Figure 6
Histological (LM) series of cross-sections of a female of Pycnophyes robustus stained with toluidine blue. (A) Segment 1; (B) beginning of segment 2 showing scalids and ventral neural cell bodies; (C) segment 3, outer oral styles and brain (dotted line marks two of the 10 lobulated regions of the brain); (D) segment 4, inner oral styles; (E) mouth and associated musculature; (F) start of the pharyngeal bulb; (G) segment 6, triradiated lumen of the pharyngeal bulb and ovaries; (H) segment 9, hindgut. (b) Brain, (dvm) dorsoventral muscle, (glr) glandular region, (hd) hindgut, (ios) inner oral styles, number after abbreviation indicates the corresponding ring, (ilrm) introvert long retractor muscle, (ldm) longitudinal dorsal muscle, (lvm) longitudinal ventral muscle, (m) mouth, (mccm) mouth cone circular muscle, (ncb) neural cell bodies, (oos) outer oral styles, (ov) ovary, (pb) pharynx bulb, (pbl) pharynx bulb lumen, (sc) scalid, (sr) seminal receptacle, (tsc) trichoscalid, (vnc) ventral nerve cord, (ncb) neural cell bodies. Scale bars: 20 µm.

Muscular system

Head muscles: The mouth cone shows a strong musculature with two concentric rings of musculature, referred to as the inner and outer mouth cone circular muscles, which are visible in LM cross-sections (Fig. 6E). The outer mouth cone circular muscle is located directly in front of the pha rynx. Both muscular rings appear inside the cuticular cap of the mouth cone. In the introvert, 10 λ-shaped spinoscalid retractor muscles are identified in both LM and micro-CT images (Fig. 3B). Because the head is fully retracted into the trunk, these muscles appear inverted (y-shaped). A total of 14 short introvert retractor muscles attach to the pachycyclus of segment 2, as observed by both techniques (Fig. 4G). Additionally, up to four pairs of long introvert retractor muscles extend toward the posterior segments and attach to the body walls of segments 4-6 (Figs 4A, C, H, 5A-B, 6B). In the micro-CT images, these long introvert retractor muscles run through the body of the animal and attach laterodorsally on segments 4, 5, and 6 and lateroventrally on segment 5.

Closing muscles: The closing system consists of six well sclerotized placids (articulated with the anterior edge of segment 1), each folded into the gap between the dorsal and ventral plates by the action of six placid retractor muscles. These structures are recognized in both LM cross-sections and micro-CT images, but the individual muscles can be traced only with micro-CT: they attach anteriorly to the placids and extend through segment 1 to the pachycyclus of segment 2 (Fig. 4B). A placid transverse muscle connecting the placids is barely observed in segment 1.

Pharyngeal muscles: The pharyngeal bulb and its muscles are identified by LM and micro-CT. The pharyngeal bulb is located between segments 5 and 7 (Figs 2D-E, 6F-G), and the pharyngeal bulb muscles appears with intermediate opacity in the micro-CT images (Fig. 1D-E). A single layer of musculature arranged as outer rings is visible at the surface of the pharyngeal bulb, formed by 13-14 strands, alternating with strands of inner radial musculature (Figs 1D-E, 2D-E, 4C, E, 5E-F). Ten pairs of pharynx retractor muscles attach to segments 4, 5, 6, 7, and 8 (Figs 1C-E, 2C-E, 4E, 5F). The retractors in segments 4 and 5 attach to the middle region of the tergal plate in laterodorsal to subdorsal position; those in segment 6 attach lateroventrally to the pachycyclus; and those in segments 7 and 8 attach at the same position as the dorsoventral muscles. Pharyngeal bulb protractor muscles are not detected in either micro-CT images or LM.

Segment muscles: The trunk contains paired dorsal and ventral longitudinal muscles in segments 1-10, which are observed in both LM and micro-CT images, and appear highly opaque in the latter. These muscles run the entire length of the segment, arising from the anterior region of the segment and attaching to the pachycyclus of the following segment or to the apodemes of segments 9 and 10. The dorsal longitudinal muscles lie subdorsally on each side of the middorsal line (Figs 2C, 4B-D, 5C), and the ventral muscles are laterally displaced (Fig. 3C). The anterior ends of the dorsal and ventral longitudinal muscles of segment 1 attach to the middle of the cuticular plates, not reaching the very anterior edge (Figs 2C, 4F). These muscles are recogniza ble as bean-like structures that increase in opacity when crossed by the micro-tomograph (Fig. 4B, D). In segment 10, the dorsal longitudinal muscles appear as a single band at the middorsal line.

Both LM and micro-CT images reveal conspicuous pairs of dorsoventral muscles in segments 1-10, attaching laterodorsally and ventromedially to the middle region of the tergal and sternal plates of the same segment. These dorsoventral muscles appear as transverse, highly opaque structures in cross-sections and as circular to oval forms in frontal sections (Figs 1C, E, 2C, E, 3C, H-I, 4B, D, 5B-C, G, 6A, G-H). In segment 1, the dorsoventral muscles are laterally displaced and notably more developed than in the other segments (Figs 4B, D, 5B, 6A). Moreover, additional muscles occur adjacent to them in segment 1, possibly oblique muscles, neck retractors, or both, forming an uninterpretable cluster in LM and micro-CT. In segment 10, the dorsoventral muscles shift towards the midline of the body (Fig. 6H). Images from segment 11 do not allow a clear identification of muscle fibers; however, a pair of muscles is present within this segment and the posterior part of segment 10, which might represent gonopore muscles or lateral terminal spine muscles (Fig. 2E).

Digestive system

LM and micro-CT show that the alimentary canal runs straight from the mouth opening to the anus. The following sections can be discriminated: the foregut (mouth, mouth cone, pharynx crown, pharyngeal bulb and short esophagus) appears retracted into the trunk and is therefore located between segments 4 and 7; the midgut occupies segments 8-9 and the anterior half of segment 10, and the hindgut is located in segments 10-11 (rectum and anus) (Figs 1E, 2E, 4A). The foregut and hindgut are visibly lined by the cuticle, which increases opacity in micro-CT images. The pharyngeal bulb has a conspicuous triradiated lumen, identifiable by both methods (Figs 3H-I, 6F-G). The musculature associated with the midgut and hindgut consist of a network of thin longitudinal and circular muscles, which are barely discernible (Figs 3K, 5D). Narrowing of the digestive canal is evident at both the anterior and posterior ends of the pharyngeal bulb and between the hindgut and rectum, as observed in LM and micro-CT (Fig. 1E). These narrowings can be interpreted as valves operated by the sphincter muscles.

Excretory system

A single pair of nephridia is observed with LM and micro-CT, located between segments 8 and 9 (Figs 1B, 2B). These protonephridia appear as club-shaped, elongated opaque canals in segment 8, from which nephridial tubes emerge and open laterally in segment 9. No further details are visible.

Reproductive system

Paired saccate gonads (ovaries) of minimum opacity are present on both lateral sides of the body between segments 6 and 9 (Figs 3I, 6G). A pair of oviducts is also visible posteriorly, specifically between segments 9 and 10 (Figs 3K-L, 6G). Each oviduct forms a diverticulum or seminal receptacle, which appears to be filled with material of medium to high opacity that likely represents retained sperm (Figs 3K-L, 6H). A pair of muscles between segments 10 and 11 may represent the gonopore muscles. Moreover, some muscles appear to be associated with the seminal receptacles. All these structures are identifiable in the LM cross-sections and in the micro-CT images, but the gonopore and the fine fibers associated with the gonads are not detectable by micro-CT.

Nervous system

The brain is identified by both methods as 10 lobulated areas around the mouth cone, forming a ring between the dorsoventral muscles of segments 3 and 4 (Figs 3E-F, 5B, H, 6C). High concentrations of neuronal cell bodies in the brain and ganglia of the ventral nerve cord are detectable in the LM cross-sections (Fig. 6B, D, H). Through micro-CT, only the ventral ganglion of segment 6 is visible (Fig. 3I).

DISCUSSION

Cuticular structures and diagnostic characteristics

Regardless of the microscopy technique used for kinorhynch identification, correct interpretation of the presence and nature of cuticular features often requires a deep knowledge of the animal’s anatomy. In the case of micro-CT, a priori knowledge of the species-specific arrangement of certain features is particularly necessary. For instance, the presence and location of many external morphological characters (such as setae, sensory spots, or glandular cell outlets), must be rendered with this background knowledge; otherwise, they could be easily misinterpreted. Consequently, micro-CT should not be the technique of choice for taxonomic or identification purposes (with other considerations of cost), only in case of very limited availability of specimens to be used in different techniques (micro-CT, SEM, histology).

Body wall and inner organs

Observations of the cuticle and internal systems generally agree with those of previous studies. No striking differences are found in the cuticle, epidermis, mucous glands, digestive, excretory, and reproductive systems (Kristensen and Higgins 1991, Neuhaus 1994, 2013, Herranz et al. 2013, 2014, Hirose and Yamasaki 2015, Altenburger 2016). However, only the densest structures of the nervous system (brain and some ventral ganglia) are perceptible by micro-CT. Components smaller in length or thickness (such as prolongations of the ventral nerve cord toward the sensory organs and lateral terminal spines, or organ innervations) are not detectable, likely because of the fixation process or the scanning voxel size. The same reasoning may explain why shorter and/or thinner muscles, such as the short spinoscalid muscles, the circular muscles of the introvert, and the lateral terminal spine/gonopore muscles, are also not detected.

Regarding the general muscular system, most observations agree with the results reported in previous studies of the phylum (Kristensen and Higgins 1991, Neuhaus 1994, 2013, Herranz et al. 2013, 2014, Altenburger 2016). However, some mismatching arises when comparing our findings with the information available for the only two species of the fami ly Pycnophyidae whose internal anatomy has been studied in detail: Setaphyes kielensis (Zelinka, 1928) and Pycnophyes ilyocryptus (Higgins, 1961). First, the long introvert retractor muscles are detectable through micro-CT examination, but we are uncertain about their total number. This uncertainty can be partly explained by the introvert’s position. When the introvert is retracted inside the trunk, it is difficult to distinguish each muscle or unequivocally follow it to its attachment point. The same consideration may explain why we do not observe the pharyngeal bulb protractor muscles. The bulb is so retracted that the protractor muscles are hypercontracted, making it impossible to discern where the bulb ends and the muscles begin. Therefore, using speci mens with the introvert everted would likely improve the observation of both muscle types; however, this could not be tested in the present contribution, because the fixatives used make the animal tend to retract the introvert.

Second, according to the study by Altenburger (2016) using CLSM on S. kielensis, a pair of lateral longitudinal muscles runs from the pachycyclus of segment 2 to the pachycyclus of segment 3. These muscles were also found in P. ilyocryptus studied with CLSM and were interpreted as neck retractors (Herranz et al. 2020). Based on the size reported in both papers, these muscles should have been detectable in our specimen; however, we could not locate them. It cannot be completely ruled out that their absence is due to an interspecific difference, as occurs in other Kinorhyncha genera (Herranz et al. 2019), such that these muscles are present in both S. kielensis and P. ilyocryptus, but absent in P. robustus. Still, other explanations, such as a problem in the fixation of the specimen or the fact that the introvert is retracted into the trunk, making the area difficult to observe, seem far more plausible.

The last notable dissimilarity found relates to Altenburger (2016), whose study disagrees with almost all existing literature regarding the muscle composition of the foregut. According to this author, both inner and outer pharyngeal bulb circular muscles are present, whereas a set of radial muscles is described in many previous (Brown 1989, Neuhaus 1994, 2013, Rothe and Schmidt-Rhaesa 2004, Schmidt-Rhaesa and Rothe 2006, Telford et al. 2008, Budd and Telford 2009, Herranz et al. 2014) and posterior (Herranz et al. 2020) studies, which agree with our results.

Micro-CT usefulness for Kinorhyncha and hard-bodied meiofaunal specimens

Despite the voxel size and the staining method used (iodine only), examination with micro-CT distinguishes most of the internal anatomical structures previously described for Pycnophyidae using other techniques such as TEM and CLSM (Kristensen and Higgins 1991, Neuhaus 1994, Schmidt-Rhaesa and Rothe 2006, Altenburger 2016, Herranz et al. 2020). Thus, regardless of the software used, most of the muscular components are identifiable, as well as the most significant parts of the inner organ systems and even components of the nervous system. Therefore, micro-CT appears as a potential, non-des tructive, and less invasive technique compared to other methods applied for morphological purposes in hard-bodied microscopic animals, allowing a three-dimensional recons truction of complete organisms and enabling the study of organs and tissues in their original positions.

Understanding Kinorhyncha morphology requires both internal and external anatomical data to fully interpret their biology. While traditional techniques such as CLSM and TEM excel in capturing the fine details of internal systems (e.g., nervous system ganglia or precise muscle counts), they are invasive and often restricted to specific sections of the organism. In contrast, micro-CT allows a non-destructive, three-dimensional visualization of both the external and internal structures, providing a general understanding of the anatomical relationships. However, as our study demons trates, its resolution is insufficient for detecting thinner details such as small external features (e.g., sensory spots) or accurately counting all internal components (e.g., muscle numbers). Furthermore, the interpretation of micro-CT data often requires prior knowledge of the structures, either to detect them or to correctly assess their anatomical form, which can limit its applicability for exploratory studies or for researchers unfamiliar with the morphology of the organism. For now, micro-CT can serve as a complementary tool for studies requiring spatial context or when specimens are rare and need to be preserved for future research.

Although confocal microscopy techniques allow high-quality results at large magnifications, they require laborious sample preparation and extremely tedious post-processing with software to segment different structures and organs separately. In contrast, the sample preparation used in our micro-CT study is much simpler and less labor-intensive than the previous techniques. The separation of structures using a color gradient, avoiding individualized segmentation, has proven to be sufficient for evidencing and studying various anatomical aspects. The limitation in microtomography resolution relative to electron or confocal microscopy is being overcome by new nanotomographs (nano-CT), which have very low scanning times and voxel sizes of only a few nanometers. This development will likely allow future studies to obtain rendered images at magnifications and quality levels comparable to those of microscopic techniques. It is worth noting that, despite the limitations of the microtomograh used in this study, it has been possible to provide valuable insights into the spatial arrangement of anatomical structures and the general organization of the body plan of these hard-bodied animals. Thereafter, the micro-CT demonstrates to be a technique potentially applicable to other meiofaunal groups, specially to those with hard cuticle as its presence reduces the likelihood of collapse of internal structures during pre-treatment of specimens, preventing their correct visualization. The technique could also be applied to soft meiofauna but with appropriate fixation protocols that may involve the use of concentration gradients and longer waiting times between steps.

Finally, it is important to note that meiofaunal orga nisms deposited in museum collections are often mounted in permanent or semi-permanent preparations, usually flattened dorsoventrally, which alters the natural shape and position of the internal structures. Although this is a common problem with other morphological microscopy techniques, microtomographical techniques maintain the shape of the specimens, and, therefore, do not have these problems. Moreover, micro-CT’s capacity for three-dimensional visualization offers additional value when analyzing preserved specimens, potentially contributing to morphometric or volumetric studies of larger structures (e.g., body shape or organ systems) that are not affected by preservation artifacts.

ACKNOWLEDGEMENTS

We thank the Bruker-Skyscan staff for their effectiveness and fast support, for their constant improvements to the software, and for implementing the new options we requested. Also, for their kindness in providing J.A.T. with fast and effective suggestions and answers to queries. In this respect, we are especially indebted to Alexander Sasov (now at NeoScan, https://neoscan.com/), Xuan Liu, Stephan Boons, and Phil Salmon. AGC was supported by a predoctoral contract from the Community of Madrid (PIPF-2023). Any language proficiency issues are the sole responsibility of the authors.

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ADDITIONAL NOTES

• ZooBank register
  https://zoobank.org/28C7A01A-D10A-46AF-A37E-AC46A7C8A8EC
• Data Availability
  All data generated and/or analyzed are included in this article.
• Funding
  Community of Madrid (PIPF-2023) AGC was supported by a predoctoral contract from the Community of Madrid (PIPF-2023)
• How to cite this article
  Sánchez N, González-Casarrubios A, Pardos F, Alba-Tercedor J (2025) Anatomical study of Pycnophyes robustus (Kinorhyncha: Allomalorhaghida: Pycnophyidae): Evaluation of microcomputed tomography in comparison with other microscopic techniques. Zoologia 42: e24060. https://doi.org/10.1590/S1984-4689.v42.e24060
• Published by
  Sociedade Brasileira de Zoologia at Scientific Electronic Library Online - https://www.scielo.br/zool

Edited by

• Editorial responsibility
  Rosana M. da Rocha

Data availability

All data generated and/or analyzed are included in this article.

Publication Dates

• Publication in this collection
  27 June 2025
• Date of issue
  2025

History

• Received
  18 Sept 2024
• Accepted
  26 Feb 2025