Evolutionary transformation of mouthparts from particle-feeding to piercing carnivory in Viper copepods: Review and 3D analyses of a key innovation using advanced imaging techniques
Frontiers in Zoologyvolume 16, Article number: 35 (2019) Cite this article
Novel feeding adaptations often facilitate adaptive radiation and diversification. But the evolutionary origins of such feeding adaptations can be puzzling if they require concordant change in multiple component parts. Pelagic, heterorhabdid copepods (Calanoida) exhibit diverse feeding behaviors that range from simple particle feeding to a highly specialized form of carnivory involving piercing mouthparts that likely inject venom. We review the evolutionary history of heterorhabdid copepods and add new high-resolution, 3D anatomical analyses of the muscular system, glands and gland openings associated with this remarkable evolutionary transformation.
We examined four heterorhabdid copepods with different feeding modes: one primitive particle-feeder (Disseta palumbii), one derived and specialized carnivore (Heterorhabdus subspinifrons), and two intermediate taxa (Mesorhabdus gracilis and Heterostylites longicornis). We used two advanced, high-resolution microscopic techniques — serial block-face scanning electron microscopy and two-photon excitation microscopy — to visualize mouthpart form and internal anatomy at unprecedented nanometer resolution. Interactive 3D graphical visualizations allowed putative homologues of muscles and gland cells to be identified with confidence and traced across the evolutionary transformation from particle feeding to piercing carnivory. Notable changes included: a) addition of new gland cells, b) enlargement of some (venom producing?) glands, c) repositioning of gland openings associated with hollow piercing fangs on the mandibles, d) repurposing of some mandibular-muscle function to include gland-squeezing, and e) addition of new muscles that may aid venom injection exclusively in the most specialized piercing species. In addition, live video recording of all four species revealed mandibular blade movements coupled to cyclic contraction of some muscles connected to the esophagus. These behavioral and 3D morphological observations revealed a novel injection system in H. subspinifrons associated with piercing (envenomating?) carnivory.
Collectively, these results suggest that subtle changes in mandibular tooth form, and muscle and gland form and location, facilitated the evolution of a novel, piercing mode of feeding that accelerated diversification of the genus Heterorhabdus. They also highlight the value of interactive 3D animations for understanding evolutionary transformations of complex, multicomponent morphological systems.
Key evolutionary innovations are adaptations that facilitate rapid and sometimes extensive diversification of lineages within which they arise [1, 2]. Familiar examples include insect wings , bird feathers , biting jaws of vertebrates , and pharyngeal jaws in cichlid fish . However, to be fully functional, many such key innovations require concordant changes in multiple body components, which can yield controversies about the order and integration of the evolutionary transformations that ultimately gave rise to them (e.g., ).
A less familiar, but no less fascinating, key innovation evolved in pelagic ‘Viper’ copepods (Heterorhabidae, Calanoida): mandibles bearing tubular, hypodermic-needle-like structures (e.g., Heterorhabdus, Fig. 1) that are thought to inject venom secreted from openings of enlarged glands located in the upper lip (labrum) . This fang-bearing mandible differs considerably in form from the mouthparts of typical particle-feeding copepods such as Calanidae and Paracalanidae . It also differs from primitive particle feeding heterorhabid copepods [9, 10], all of which possess mandibles with macerating or cutting teeth (e.g., Disseta, Fig. 1). These unique hollow fangs of Heterorhabdus were the first potentially envenomating structure to be reported from crustaceans [9, 11, 12].
Piercing carnivory in Viper copepods (asterisk, Fig. 1) qualifies as a key innovation because it is associated with both a) accelerated diversification, and b) expansion of ecological (depth) range. Of eight heterorhabdid genera , the two with the most derived forms of piercing carnivory (Heterorhabdus and Paraheterorhabdus) include nearly 2/3 of all heterorhabdid species [9, 10] (Fig. 1; WoRMS 2018). In addition, these two genera span the widest depth range of all heterorhabdid genera, which mostly occur in the deep sea  [mesopelagic (M) or bathypelagic (B), Fig. 1].
Previous morphological and phylogenetic studies showed that feeding habits changed from particle feeding to carnivory in the Heterorhabdidae [7, 9, 10] (Fig. 1). The basally branching genera Disseta and Microdisseta are essentially particle-feeders, while the derived genera Neorhabdus, Hemirhabdus, Paraheterorhabdus and Heterorhabdus are carnivores. Mesorhabdus and Heterostylites are intermediate between these extremes.
Mandible form changed dramatically associated with these diet changes . The ventral-most mandibular tooth of typical particle-feeders (e.g., Disseta and Microdisseta) is unspecialized and similar to that of other particle feeding calanoid copepods (Fig. 1). In intermediate taxa, the ventral tooth is enlarged (e.g., Mesorhabdus and Heterostylites; Fig. 1). In one intermediate taxon the elongate ventral tooth possesses a fine groove (Heterostylites; Fig. 1). The ventral tooth in carnivorous taxa bears a massive groove or is partly tubular in some taxa (Hemirhabdus and Neorhabdus; Fig. 1). In the most diverse and ecologically widely distributed Viper copepods (Paraheterorhabdus and Heterorhabdus) the ventral mandibular tooth forms a completely enclosed tube (Fig. 1).
The effectiveness of piercing carnivory depends not only on mandible form, but also on a) glands that secrete substances to facilitate prey capture and ingestion, and b) muscles that move the mandibular gnathobase. Three sets of gland openings in the upper lip (labrum) are associated with secretory cells in all heterorhabdid copepods [7, 9]. Each set is thought to be associated with a different set of glands. However, details of gland structure are known only for the carnivore Heterorhabdus , and nothing is known about the muscles that control mandibular motion in any heterorhabdid copepod.
To better understand the fine structure and spatial relations among glands and muscles within the mouthparts of Viper copepods, we utilized two advanced, high-resolution imaging methods — serial block face scanning electron microscopy (SBF-SEM) and two-photon excitation microscopy — to produce 3D nanometer-scale reconstructions of the external and internal morphology of the labrum (=upper lip) and paragnath (=lower lip) of heterorhabdid species from four genera: Disseta palumbii Giesbrecht, 1889, Mesorhabdus gracilis Sars, 1907, Heterostylites longicornis (Giesbrecht, 1889), and Heterorhabdus subspinifrons Tanaka, 1964. We also video-recorded mouthpart movement in live specimens of all four taxa to clarify muscle function. Finally, to reconstruct the evolutionary history of this innovative feeding mode, we compared putative homologues of component elements (muscles, glands, gland openings) among all four genera and discuss character variation across the phylogenetic tree of heterorhabdid copepods.
Both state-of-the-art SBF-SEM microscopy  and two-photon excitation microscopy , combined with associated image-analysis technologies, yielded full 3D perspectives — at nano-scale resolution — of the glands and muscles in the mouthparts of the heterorhabdid species studied. Although these two methods are based on different principles, and use different types of fixative, the results were similar for both (Fig. 2). Sections from two different individuals of Mesorhabdus gracilis (Fig. 2) show planes of four pairs of glands (dashed outlines), and planes of one pair of muscles, which correspond nicely between the two pictures. Both imaging methods clearly show the same spatial relationships of glands and muscles. Gland contents, however, appeared to differ somewhat between methods. For example, gland lg1C2 in the SBF-SEM scan (Fig. 2a) appeared to be filled with tiny and flattened disc-shaped granules, whereas in the two-photon excitation microscopy scan (Fig. 2b) the granules appeared to be rather big and more rounded in shape. Curiously, gland contents also appeared to differ between sides even within a single specimen (compare contents of gland lg1C2 on the left and right side of Fig. 2b).
Numerous muscles and glands are associated with the mouthparts examined. All of the descriptive terms used here to refer to morphological units of muscles and glands do not imply any homology hypothesis (see  for a discussion of homology-free terminology in morphological description). Homology hypotheses for these descriptive terms are outlined explicitly in Table 1 and discussed in detail in the Discussion.
Gland morphology and arrangement
We adopted labral gland terms from Nishida and Ohtsuka , where gland cells were divided into three “Types” according to the arrangement of gland openings (Fig. 3a-d). We use the same terminology here, but apply these terms differently except for Heterorhabdus subspinifrons. The arrangement of gland openings is essentially the same as reported previously , but we found an extra opening of labral gland Type 2 in Disseta palumbii: two openings were reported earlier , but we found a third (Fig. 3a).
Even though gland openings were readily identified and easy to homologize among taxa, the size, shape and configuration of gland cells differed considerably among the four genera. In the particle feeding D. palumbii, gland cells are located postero-ventrally in the labrum, and are not associated with muscles (Fig. 3a, e: see Additional file 1: Figure S1 for viewing instructions for the interactive 3D-pdf images). In Mesorhabdus gracilis (intermediate feeding mode), the labrum is almost fully packed with labral gland cells and parts of these cells intercalate between the muscles lab-eso.dM3 and u-l.labM2 (Fig. 3b and f). In Heterostylites longicornis (intermediate feeding mode), labral gland cells are located at the posterior half of the labrum, and half of the cells are stacked between muscles lab-eso.dM3 and u-l.labM2 (Fig. 3g and c). Significantly, in the piercing carnivore, H. subspinifrons, all of the labral gland cells are highly extended anteriorly: a) Type 3 gland cells are enveloped by three muscles u-l.labM2, lab-eso.dM1–4 and for-eso.dM (Fig. 3h and d: click on the view “Labral Gland Type 3 and muscles” in the interactive 3D-PDF, Fig. 4d), b) Type 2 gland cells extend up to the posterior margin of the paragnath (Fig. 4d), and c) Type 1 gland cells are inflated, and posteriorly elongated into the paragnath (Fig. 4d). The total number of cells in gland Types 1–3 also differed among these genera (Table 1). Disseta palumbii has 15 pairs of cells, but M. gracilis, H. longicornis, and H. subspinifrons have only 8 pairs (Table 1). Type 1 and Type 3 glands were largest in the piercing carnivore, H. subspinifrons (Fig. 4d), but all three types were well-developed in the intermediate feeding-mode M. gracilis (Fig. 4b).
Our observations of cell numbers and orientation in each gland differ somewhat from Nishida and Ohtsuka . They reported “Type 1 and 3 labral glands have two secretory cells...Type 2 labral glands and the paragnathal gland have one secretory cell” in Heterorhabdus abyssalis, H. pacificus, H. papilliger, and H. spinifrons. However, our observation of H. subspinifrons confirmed two cells in Type 1 glands, but revealed three cells in each of Types 2 and 3 (Fig. 4d, Table 1). Regarding cell structures, Type 2 gland cells were previously considered to be anteriorly elongate cells along the labral wall, and Type 3 gland cells as small cells located within the posterior side of the labrum . However, our observations revealed that Type 2 gland cells extend toward the paragnath, and that the dramatically inflated Type 3 gland cells were directed anteriorly, reaching all the way to the forehead.
The arrangement of gland openings also differed between the carnivore Heterorhabdus and the non-carnivore taxa. The openings line up nearly in a straight line in D. palumbii, M. gracilis and H. longicornis, but the opening for Type 1 lies far off the line in H. subspinifrons (Fig. 3a-d). Significantly, the opening for the Type 1 gland in H. subspinifrons lies directly at the posterior end of the hollow fang (Fig. 4d).
Secretory granules in the gland cells appeared to vary among taxa and among the three gland types (Fig. 5). Granules in homologous types of gland cells (based on location) were not similar in shape and size (e.g., compare “lg3c1”and “lg3c2” in Fig. 5a; “lg1c1” and “lg1c2” in Fig. 5b; “lg1c2” and “lg1c1” in Fig. 5f). However, granule form of homologous gland cells also differed between individuals of the same species (Fig. 2), and even between sides of the same individual (Fig. 2b). Therefore, these observations, combined with inconsistent resolution due to technical limitations of contrasting and resolution, greatly limited the utility of granule form as a tool for making any inferences about gland function or homology.
A small, fourth type of gland — termed here Epidermal Gland — was found by the ventral side of the epidermis, with the duct opening on the ventral side of both the labrum and paragnath in M. gracilis, H. longicornis, and H. subspinifrons (Fig. 4b, c and d: represented in yellow). No such cells were seen in D. palumbii. Cell numbers were lowest in M. gracilis (2 cells; but paragnath epidermal gland might have been overlooked because of the limited scanning field), greater in H. subspinifrons (7 cells), and highest in H. longicornis (14 cells). In addition, arrangement of the labral epidermal gland cells was erratic and not always symmetrical (e.g., Fig. 4c and d).
Epidermal gland cells in H. subspinifrons contained distinctive spindle-shaped secretory granules (“peg” cells and “leg” cells in Fig. 5g, h). Unfortunately, the contents of these epidermal gland cells were unclear in other genera due to limited contrast and resolution (Fig. 5c, d and e).
Muscle configuration and movement of mouthparts
Given the large differences in mandible form, the overall arrangement and attachment sites of muscles were surprisingly similar among the four genera examined (Fig. 4). These muscles are named based on their attachment sites or locations (Table 1). The only species-specific muscle we observed was in the highly derived carnivore Heterorhabdus subspinifrons, (“saggital labral muscle”, Fig. 4d, Table 1). This muscle was located at the posterior side of the labrum: one end attached just beside the opening of labral gland Type 1 and the other end attached near the esophagus opening (Fig. 4d: click on the view “Sagittal Labral Muscle insertions” in the interactive 3D-PDF).
In all four genera, masticatory movement of mandibles and cyclic muscular contraction within the labrum were synchronized soon after stimulation with a fine needle (Additional file 2: Movie SM1 A-D). In Disseta palumbii (particle feeder), cyclic contractions of the “Upper-Lower Labral Muscles 1” (u-l.labM1 in Fig. 3e) and the “Forehead-Esophageal Dilator Muscles” (for-eso.dM in Fig. 3e) were observed (Fig. 6a, Additional file 2: Movie SM1A). In Mesorhabdus gracilis (intermediate feeding mode), muscle bundles were not clearly recorded, but the “Forehead-Esophageal Dilator Muscles” (for-eso.dM in Fig. 3f) seemed to cyclically contract and lift up the esophagus area (Fig. 6b, Additional file 2: Movie SM1B). In Heterostylites longicornis (intermediate feeding mode), simultaneous cyclic contractions of the “Lateral-Esophageal Dilator Muscles 1” (lat-eso.dM1), the “Forehead-Esophageal Dilator Muscles” (for-eso.dM) and the “Labrum-Esophageal Dilator Muscles 1” (lab-eso.dM1 in Fig. 3g) created an expanding motion of the esophagus (Fig. 6c, Additional file 2: Movie SM1C). In Heterorhabdus subspinifrons (piercing carnivore), distinct muscles were not clearly recorded, but cyclic and coordinated contraction appeared to occur in the “Lateral-Esophageal Dilator Muscles 1” (lat-eso.dM1), the “Forehead-Esophageal Dilator Muscles” (for-eso.dM in Fig. 3h), the “Labrum-Esophageal Dilator Muscles 1” (lab-eso.dM1 in Fig. 3h) and the “Labrum-Esophageal Dilator Muscles 2” (lab-eso.dM2 in Fig. 3 h), which created an expanding motion of the esophagus (Fig. 6d, Additional file 2: Movie SM1D).
Additional file 2: Movie SM1. Mandible, muscle and esophagous motions in four heterorhabdid copepod species, all filmed at 30 frames per second. (A) Disseta palumbii, (B) Mesorhabdus gracilis, (C) Heterostylites longicornis, (D) Heterorhabdus subspinifrons. (MP4 50122 kb)
Muscle homology among the four genera, and a novel muscle in carnivorous Heterorhabdus subspinifrons
Because of the highly conserved arrangement of muscles among the four heterorhabdid genera, putative homologues could be readily identified throughout (Table 1). Therefore, muscle arrangement itself did not appear to play a major role in the evolutionary shift among feeding modes. However, one unique muscle was found in the piercing carnivore Heterorhabdus subspinifrons (“Saggital Labral Muscles (s.labM)”, Fig. 4d). Labral gland Type 1 is assumed to be the main gland that charges venomous substances into the hypodermic needle-like mandibular fang . This inference is supported by the position of the Type 1 gland opening, which is located exactly behind the charging pore of the mandibular fang (Fig. 4d). This structural arrangement of muscle and pores therefore suggests that muscle contraction enhances the efficiency of venom charging by adjusting the position of the pores relative to the base of the fang.
Revised homology hypotheses for the glands
Ohtsuka et al.  hypothesized that the Type 1 gland in Heterorhabdus subspinifrons is a specialized form of one of the two Type 3 glands that exist in other heterorhabdid species. To be consistent with our homology hypothesis, we revised this terminology, and adopt the term Type 1 for all species examined here (see Figs. 1 and 3). Our hypothesis of gland homology, consistent with traditional criteria for structural homology , is based on the unambiguous spatial relationships of gland openings: Type 2 are located at the lateral tip of the labrum and contain 2–3 openings; Type 1 are located beside the Type 2 opening complex; and Type 3 are located at the most medial part of the labrum compared to the other openings. Although we did observe differences among species in the contents of putatively homologous glands (Fig. 5), gland contents also differed significantly between individuals of the same species and sides of the same individual (Fig. 2). Therefore, apparent differences in gland contents among species in Fig. 5 are unlikely to be informative phyologenetically and do not impair our homology inferences.
As noted in the results, we did find a few differences in cell numbers and structures between our results and previous observations . These differences might be due to the different species we used, but they are more likely due to the greatly enhanced spatial resolution of our 3-dimensional analysis. Nonetheless, these differences do not affect our homology inferences.
Finally, the previously reported “paragnathal gland” (presumed to be associated with a “pore in the ventro-medial region of the posterior surface of each paragnath”; visible in Fig. 3a of Nishida and Ohtsuka ) we now consider to be a synonym of the paragnathal epidermal gland defined here.
Venom-assisted feeding in Viper copepods
Venom-assisted feeding is a complex adaptation that requires concordant evolution in two novel functional domains: novel physiology (pharmacologically active venom compounds ) and novel morphology (venom delivery system ). Despite this complexity, venom-assisted feeding has evolved multiple times in arthropods, including chelicerates, myriapods and insects . Surprisingly, despite their immense diversity and success in marine and aquatic systems, venom-assisted feeding appears to be exceedingly rare in Crustacea. The only definitive case occurs in remipedes . Envenomation is suspected in two parasitic crustacean groups (branchiuran fish lice and siphonostomatoid copepods) as well as two free-living taxa (caprellid amphipods and heterorhabdid copepods) , but pharmacological confirmation is still required.
In the piercing carnivorous copepod, Heterorhabdus subspinifrons, cells of the Type 3 labral gland are tightly enveloped by multiple muscles (for-eso.dM, lab-eso.dM 1–4 and u-l.labM2; Figs. 3h, 4d). This arrangement implies that muscular contraction squeezes the gland to eject secretions. Ejection of secretions via muscular contraction appears to be a common strategy in many animals: venom release in cone snails ; venom gland discharge in elapid and viperid snakes ; silk ejection from the antennal exopod in ostracods ; and venom ejection from the head of specialized soldier termites . Furthermore, these muscles in H. subspinifrons all contract cyclically during experimentally induced mastication (Fig. 6d, Additional file 2: Movie SM1D), so presumed venom ejection likely occurs simultaneously with mastication.
Three of our observations suggest that the piercing carnivore Heterorhabdus subspinifrons injects a poison or venom into its prey via the hollow fang on its mandible. First, one gland type (Type 1) is greatly enlarged. Second, the opening to that enlarged gland shifted to lie at the base of the hollow fang (Fig. 3d). Third, secretions from the Type 3 gland in H. subspinifrons may have a specific role in carnivorous feeding, because the enveloping arrangement of muscles around the glands does not occur in the other heterorhabdid species examined (Fig. 4). However, pharmacological evidence would be required to show definitively that the secretions are a venom and not mucus or some other salivary secretion.
Minor morphological change supported a major radiation of feeding strategies
Despite drastic functional changes — from particle feeding with a mundane mandible to carnivorous feeding with a sophisticated piercing-injection system — the overall morphological units and their arrangement are strikingly similar among the Viper copepod species examined here. This similarity implies that differential use of the mandible, for simple mastication or for venom injection, can be accomplished by a slight modification of cuticle structure and minor modification of muscle structure. Other examples of great functional innovation in pancrustaceans follow a similar principle, where minor morphological modifications facilitate significant functional change. First, in some highly derived snapping-shrimp genera, like Alpheus and Synalpheus, minor changes in muscle structure (e.g., subdivided claw-closer muscle) maximize the efficiency of the latch-releasing motion before snapping . Second, a similar evolutionary sequence of muscle subdivision to control latch release is seen in Anochetus trap-jaw ants . Third, muscles in the suction disc of adult parasitic branchiuran Crustacea are identical to those in the larva that control ordinary appendage-like motion (the larval mouthpart appendage is the anlagen of the suction disc), except for two newly acquired muscles — “circular sucker muscle” and “disc rim muscle” — that both minutely adjust the shape of the sucker to attach it to the host surface in the most efficient way . This evolutionary tendency — for seemingly minor but functionally significant adjustments of form to a novel function — is comparable to acquisition of the novel “sagittal labral muscle” in Heterorhabdus subspinifrons. Because that muscle, which adjusts the gland opening to the pore of the mandibular fang, is the only newly acquired muscle associated with piercing carnivory, it may enhance efficiency of charging the fang with liquid. Since this efficiency may have critical role in the newly acquired “fang” function of the mandible, this small muscle may play an important role in the new carnivorous feeding strategy. The evolutionary shift of the Type 1 gland opening to lie at the proximal end of the mandibular fang in H. subspinifrons (unlike other taxa), also likely enhances the efficiency of injection.
Such drastic changes of this function-adaptation complex, enhanced by minor morphological change, may facilitate invasion of wholly new adaptive zones and potentially explosive diversification in harmony with body mituarization . The remarkably high diversity of heterorhabdid copepods that utilize piercing carnivory (, Fig. 1) implies that functional transformation of feeding structures may have greatly accelerated the rate of evolutionary diversification.
Three-dimensional visualization of small animals and the “renaissance of morphology”
This study also illustrates the great power of new imaging tools, and sophisticated 3D visualization techniques, to help understand complex morphologies, particularly in the small creatures that make up the vast majority of animal diversity. These advances have led to a “renaissance of morphology” [16, 17].
The difficulties of 3D imaging in small animals were overcome by using two advanced imaging techniques: serial block-face scanning electron microscopy (SBF-SEM) and two-photon excitation microscopy. SBF-SEM uses a robotic ultramicrotome-embedded within a scanning electron microscope. It is a major advance over confocal laser scanning microscopy (CLSM: appropriate specimen thickness roughly 10–150 μm) and micro-computed tomography (micro-CT: appropriate specimen size roughly 1 mm-20 cm) because it permits 3D reconstruction of meso-scale structures (roughly 100–1000 μm) at nanometer resolution . Two-photon excitation microscopy also yields nanometer resolution of meso-scale structures up to one millimeter depth-of-field .
The 3D information contained in the high-resolution image stacks were made comprehensible and presentable by advanced 3D visualization techniques. First, each discrete morphological element (specific muscle or gland) can be segmented out of each plane of an image stack (e.g., see outlined regions in Fig. 5) so that it can be rendered in three dimensions and assigned an informative color and shading (e.g., Fig. 3e-h). But such 3D renderings can still be difficult to interpret from 2D perspective images where many component elements are involved (e.g., Fig. 3e-h). The limitations imposed by 2D representations of 3D renderings are overcome entirely by interactive 3D models that can be incorporated directly in pdf files (e.g., Fig. 4). These 3D interactive models give the viewer extraordinary viewing power: 1) virtually unlimited zoom and pan capability, 2) the ability to look at one subset of structures at a time (e.g., only muscles or only glands), and specific items in each subset, via logically structured hierarchical groupings of elements (see Additional file 1: Figure S1 for viewing tips), 3) the ability to examine specific pairs or specific sets of structures in isolation (e.g., the relations of specific muscles (for-eso.dM, lab-eso.dM1–4 and u-l.labM2)) associated with the Type 3 labral gland in Heterorhabdus subspinifrons) by excluding all other structures. Such selective viewing was vital to understanding the 3D spatial relations of component parts in the piercing carnivore H. subspinifrons. It also allows readers to judge for themselves these relations free from any author prejudice.
Materials and methods
Collection and imaging
Specimens were collected off the Nansei Islands, southwestern Japan in 2016–2017, by oblique towing of a large-diameter plankton net (ORI, diameter 1.6 m; mesh size 0.33 mm) between 0 and 728 m depth with the vessel TRV Toyoshio-maru, Hiroshima University. Detailed localities are: Disseta palumbii- east of Nakanoshima Island (29°31.412′N, 130°37.296′E); Heterostylites longicornis- east of Tanegashima Island (30°13.218′N, 131°09.252′E); Heterorhabdus subspinifrons- east of Tanegashima Island (30°52.168′N, 131°34.897′E); Mesorhabdus gracilis- east of Okinoerabujima Island (27°10.857′N, 129°03.307′E).
In preparation for observations by SBF-SEM, individuals were fixed with 2% glutaraldehyde and 2% paraformaldehyde in 0.15 M cacodylate sodium buffer with 2 mM CaCl2 (pH 7.4) for 5 h at 4 °C, then decalcified in 10% EDTA in water for 2 days at 4 °C. The specimens were post-fixed with 2% osmium tetroxide and 1.5% potassium ferrocyanide in the same buffer for 2 h at room temperature. They were incubated in 1% thiocarbohydrazide for 30 min at room temperature, and fixed again with 2% osmium tetroxide in water for 1 h at room temperature. En bloc staining was performed with 1% uranyl acetate for 3 h at room temperature and then with Walton’s lead-aspartate solution (20 mM, pH 5.5) for 60 min at 60 °C. The specimens were washed with cacodylate buffer or distilled water between each step described above. Each specimen was a) dehydrated by a graded ethanol series (30–100%) at 4 °C with 30 min for each step, b) transferred to 100% acetone for 1 h, and c) incubated in a graded Durcupan resin series (25, 50, 75, 100% using acetone as a solvent) in a vacuum chamber for 12 h at each step. The resin was allowed to polymerize at 60 °C for 3 days. Trimmed resin blocks were glued onto an aluminum SBF-SEM rivet with conductive epoxy resin (SPI Conductive Silver Epoxy; SPI Supplies and Structure Prove, Inc., West Chester, PA, USA), and coated with gold using an ion coater. Scanning electron microscopes (SIGMA/VP and MERLIN, Carl Zeiss Microscopy, Jena, Germany), equipped with an in-chamber ultramicrotome system and a back-scattered electron detector (3View; Gatan Inc., Pleasanton, CA, USA), were used to slice and image each specimen as described previously . The serial-section image stack was acquired in an automated fashion by using Gatan Digital Micrograph software.
In preparation for observations by the multiphoton microscope (Leica TCS SP8 MP), specimens were fixed in Bouin’s solution, dehydrated in an isopropanol series, and then mounted on slides using a 2:1 mixture of benzyl benzoate and benzyl alcohol for clearing. Specimens were imaged using autofluorescence, so excitation wavelength, detected emission wavelength range, etc., were adjusted individually for each specimen to obtain maximum brightness and contrast.
3D visualization and videography
Image stacks from SBF-SEM were automatically aligned using the registration plug-in “Register Virtual Stack Slices” in Fiji/ImageJ software package (http://fiji.sc/Fiji). Surface and volume renderings of the scanned data were performed using IMARIS 7.0.0 (Bitplane AG). Objects for the 3D-pdfs were exported as vrml format. File sizes were reduced by MeshLab (http://www.meshlab.net/), and then exported as u3d format. Files were arranged using Deep Exploration (Right Hemisphere) and re-arranged by Adobe Acrobat Pro (Adobe) to create 3D-pdf files.
To video mouthpart motion, living copepods were briefly semi-dried and attached to a glass dish with cyanoacrylate glue on the dorsal side of the metasoma and the dish was then filled with seawater. Positioned copepods were stimulated to move their mouthparts using a needle. A video camera EX-F1 (CASIO, Japan) was used to record mouthpart behavior.
Forehead-Esophageal Dilator Muscles
Labrum-Esophageal Dilator Muscles
Lateral-Esophageal Dilator Muscles
Saggital Labral Muscles
Transverse Labral Muscle
Upper-Lower Labral Muscles
Labral Epidermal Glands
Labral Epidermal Gland Cell Z
Labral Gland Type X
Labral Gland Type X Cell Z
Labral Gland Type X Group Y Cell Z
Paragnathal Epidermal Glands
Paragnathal Epidermal Glands Cell Z
Paragnathal Epidermal Glands Group Y Cell Z
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We thank the captain and crew of the TRV Toyoshio-maru, Hiroshima University for safe voyage. We thank the copepod research team from Hiroshima University for field assistance and the Electron Microscopy Group in the National Institute for Physiological Sciences for technical support. We thank C. S. Wirkner (Universität Rostock, UR) and J. Runge (UR) for guidance of morphological description and assistance of creating 3D-PDFs. TK especially thanks S. Richter (UR) for facilitating extended stays and imaging work in Rostock. This study was partially supported by a grant-in-aid KAKENHI (No. 16 K07825) awarded to SO. This work was supported by NIBB Collaborative Research Program for Integrative Imaging (18-526) to ARP.
This research was funded by the Cooperative Study Program of National Institute for Physiological Sciences No.2017–235, and Natural Sciences and Engineering Research Council of Canada Discovery Grant RGPIN 04863 to ARP.
Availability of data and materials
Please contact the lead author for data requests.
Department of Biological Sciences, University of Alberta, Edmonton, AB, T6G 2E9, Canada
Tomonari Kaji & A. Richard Palmer
Allgemeine & Spezielle Zoologie, Institut fur Biowissenschaften, Universität Rostock, 18055, Rostock, Germany
National Institute for Physiological Sciences, Okazaki, Aichi, 444-8585, Japan
Chihong Song & Kazuyoshi Murata
National Institute for Basic Biology, Okazaki, Aichi, 444-8585, Japan
Setouchi Field Science Center, Graduate School of Integrated Sciences for Life, Hiroshima University, 5-8-1 Minato-machi, Takehara, Hiroshima, 725-0024, Japan
Yusuke Kondo & Susumu Ohtsuka
Biosystematics Laboratory, Faculty of Social and Cultural Studies, Kyushu University, Okazaki, Hiroshima, Japan
TK and SO designed the study, TK and SO collected the specimens, YK supported specimens collection. TK, CS, KO did the microscopy, KM and SN supervised microscopy, TK did the image analysis and videography. TK and ARP conducted the review and wrote the manuscript, with input from the other authors. All authors read and approved the final manuscript.
Correspondence to Tomonari Kaji.
Ethics approval and consent to participate
All experiments involving live copepods were performed in Japan, where no ethics approval is required for the maintenance and handling of these invertebrate species.
Consent for publication
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Additional file 1:
Figure S1. Instructions for how to use the viewing functions of the interactive 3D-pdf in Fig. 4. 1) Click any panel you want to view. 2) Click model tree icon (A) to reveal operation windows (B) and (C). 3) Window B shows the heirarchical tree diagram of morphological characters defined in this paper. Click the arrowhead to the left of each to reveal subcategories, and click each checkbox to hide/unhide each specific character. 4) Click “view”s (C) to view the specified perspective of the characters selected in (B) as described and instructed in the main text. (JPG 877 kb)
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An evolution in piercing
A piercing parlor is a very misunderstood thing to a large portion of the population, partly because the industry is changing in ways that even those involved could not have imagined in the past.
On the out – the smoky back alley tattoo parlor, filled with dirty needles and bikers drinking Jack Daniels and popping Quaaludes.
On the rise – the professional studio, savvy businesses with friendly staff, clean offices and a desire to bring an air of respectability to a field to which cultural prejudice has not been kind in the past.
These differences can be seen with a visit to many of the Columbus studios, where the old stereotypes are being replaced by apprenticed professionals complete with portfolios and business cards. Many piercing studios – such as Stained Skin, Piercology and Evolved, all in the Columbus area – resemble Fortune 500 franchises more than the smoky dive bars with which they have been historically associated, becoming a more mainstream business community.
Though no sufficient, standardized training program exists for the piercing community, the best piercers adhere to a fairly strict system of apprenticeships and education.
“I took a couple of apprenticeships, which resulted in a lot of bad habits. It’s all about the money to some people. But mainly everything I learned came through BodyWorks in Cleveland. I took a lot of seminars up there. I did a lot of self-research and watching over other people,” said Chris Sherrer, a piercing artist at Stained Skin.
Piercology, easily the most prestigious piercing studio in Columbus, insists on a rigorous three-year apprenticeship program open only to employees that have worked there for at least six months. Through the program, apprentices are taught the intricacies of sterilization, biology and piercing, along with other aspects of the industry.
That it has become an industry at all shows how far body art has come and how much deeper into the mainstream it has delved. And this progress seems to be helping everyone involved.
Piercology owner Pat McCarthy notes that, overall, the practice of piercing is benefiting as its popularity grows and techniques become more refined.
As a past president of the Association of Professional Piercers – an organization formed to educate both piercers and the public about safety issues and new developments in the art – McCarthy has championed piercing as not only a legitimate art form, but as a field that needs to be viewed as a legitimate business as well.
He has spoken at numerous conferences – including some held by the Red Cross and other health institutions – insisting on the need for standards to be implemented for the piercing industry, to ensure the APP motto of “Health, Safety, Education.”
“It comes down to educating the public about which studios to go to, so each studio will be forced to raise their standards,” McCarthy said.
This work has led to much progress, he said.
“Things are changing. I never thought it would be like this,” he said, noting that piercing has still not quite gotten the mainstream acceptance it deserves.
Leading a push that surprisingly has come predominantly from inside the industry rather than from the outside, McCarthy and Dave Vidra, a Cleveland-based piercing artist, are working for laws ensuring that professionalism and safety continue in each shop in the industry.
In the future, as piercing grows even more popular and both the artistic and the scientific aspects of the practice become further advanced, Columbus piercers and many other advocates are hoping that stricter and more broad standardization will follow.
At present, there is no specific license for piercing. The APP cannot license or certify shops because it has not been accredited by any federal department. Thus, studios are only subject to inspections from the same city health inspectors who check restaurants and other small businesses and have limited knowledge of proper piercing procedures.
“Actually, last time the health department was in, we helped teach them what to look for in a shop, especially about sterilization and what should and should not be reused,” Sherrer said.
However, as industry standards take a turn toward the more refined and the market widens to include everyone from teenage girls to their middle-class and middle-aged mothers, what is being done with body art is pushed to greater extremes, as artists include scarification and other over-the-top modifications in both their portfolio and their studios.
Perhaps that correlation is the result of a shift to professionalism in the piercing community. As the network for sharing information grows stronger by the day, as knowledge begins to be shared rather than hoarded, and as scientific techniques are perfected, the industry allows for the envelope to be pushed further than ever.
This is evident with Shawn Lower, a piercing artist and the manager at Evolved. A longtime enthusiast of body art, Lower looks like what the soldiers in Battlefield Earth might have looked like if the movie had been directed by Genghis Khan.
He knows – even enjoys – the way piercings have made him stand out and seems to respect that there is a social price to be paid when one dedicates oneself to a certain art form, and that art form transforms into a lifestyle.
The number of tattoos he has increases fairly regularly, and what he has pierced changes consistently as well, though his ballpark figure remains around an estimated twenty. He, like many local artists, has studied extensively in other cultures, uniting American techniques with forms of body art that have existed for thousands of years attached to rites of passage, tests of strength and tribal membership.
The result seems to be a combination of ancient aesthetics with modern technology, making the practice of piercing safer as well as more creative.
Why piercing is enjoying widespread appeal in the U.S. remains partly a mystery, as most forms of body modification stay completely disconnected with any ritual significance in this country. And even with the more common forms of body art, the line between self-expression and self-mutilation is fine, if not blurred altogether.
In Columbus, the real motives for and appeal of getting pierced seem as varied as the recipients themselves.
“I always thought the tribal-esque aspect was pretty cool, the originality of it,” said Sherrer about his 30-or-so piercings.
For a large section of the pierced population, body art – especially the more common forms of tattooing and piercing – has a much deeper meaning. McCarthy insists piercing can be part of the healing process for many suffering mid-life or adolescent crises. He has pierced women with gold rings melted down after failed marriages in places that may have been deemed unacceptable by ex-husbands.
Many insist their piercings are partly rebellious statements, a boundary-breaking anti-aesthetic allowing many to set themselves apart from others by sporting unconventional forms of body art.
But the broader aspects of self-expressive artistic value still seem to lie at the heart of body modification.
“Our generation has always been influenced by art,” said Giovanni, a tattoo artist at Stained Skin. “A lot of tattooing and piercing now has brought a lot of artists to the foreground. Since you can’t make money doing visual art anymore, body art has become an outlet for a lot more real creative artists.”
The world of body piercing has evolved as fast as tattooing. New fashions, new trends, retro fads and innovations abound. At evolvInk, we draw on fifteen years of piercing experience. Quality body jewelry and a clean environment to give all your piercings the greatest head start in the healing process. We also offer a wide selection of jewelry for existing piercings and are happy to assist with changings.
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Ear Piercing, It’s History And Evolution
Ear piercing practice dates back to about 5,000 years ago. This is backed by evidence of a mummy known as Ötzi the Iceman, a man who lived during the 4th millennium. It was discovered that his ears had piercings on them.
It is also proven, that the Egyptians of ancient times had ear piercings as evidenced by the holes found on the ear lobes of King Tutankhamen’s mummy – a pharaoh of ancient Egypt. They also found earrings in his tomb.
Ear piercing has cultural and spiritual significance. In some primitive societies, when a child approaches puberty, their ears are pierced in celebration. Spiritually, some communities in the past believed that evil spirits possessed humans through the ears, so the earlobes were pierced for them to be able to leave when they enter while some believed that metals kept the evil spirits away, so they wore earrings.
In Africa, in a clan known as Samburu, their fighters are made to pierce their ears and put on earplugs made of ivory to signify that they have become men and are brave. The father and mother of a child in Borneo would each pierce an earlobe of their child, to show that the child depends on them.
Also, the Borneo people wore barbells on their ears. The Dayak of Borneo was known to wear them, many years ago. In the 70s, the man who founded the first body piercing shop in the United States, Jim Ward, was the one who made barbells popular.
Around the 16th century, in Europe, men who worked in the King’s court were known to pierce their ears. Sailors also usually wore an earring on one ear to signify that they have sailed far and wide. They also wore them in case they died at sea, so whoever found them would sell the earring and give them a decent burial with the proceeds.
However, towards the 18th century, clip-on earrings started replacing earrings that required piercing. Piercings were left to the lowly of birth, till the 50s, after the war. It was as if people, women especially, wanted to make up for looking disheveled during the war.
Ear piercings became fashionable once again. Then, there were no piercing shops like can be found everywhere today. What young women did was to get their lobes numb using ice block cubes, before piercing. As time went on, it became necessary to get ear piercings in hospitals to avoid infection.
Ear piercing shops began gaining popularity around the 80s, and by the 90s, fewer people had reservations about getting their ears pierced. Today almost every country and culture have people who wear earrings, mostly for adornment purposes.
Piercing of the ear has evolved so much so that different parts of the ear can be pierced. Types of piercing include the standard piercing; which is done on the earlobe. Daith piercing, conch piercing, tragus piercing, industrial piercing;(usually done on both parts of the top of the ears, for barbells to be inserted, ) and rook piercing. An individual can have more than twenty different piercings on the ear because almost every part of the ear can be pierced.
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