First ultrastructural investigation of the pharynx apparatus of Scutigera coleoptrata (Chilopoda: Notostigmophora)
Gero Hilken & Jörg Rosenberg
Central Animal Laboratory, University Duisburg-Essen Medical School, 45122 Essen, Germany;
e-mail: gero.hilken@uk-essen.de; sommerhaus-rosenberg@t-online.de
Abstract
The organisation and possible function of the pharynx apparatus of Scutigera coleoptratawas first described in 1967 by Seifert. The pharynx apparatus in the foregut of S. coleoptrataconsists of a ball- shaped outgrowth of the dorsal pharyngeal wall, the pistil, and a cooperating ventral groove, the pharynx furrow. Here we present new structural details of the pistil and the pharynx furrow with its underlying epithelium, using LM, TEM, and SEM techniques.
Keywords:centipedes, foregut, pistil, pharynx furrow, fine structure
1. Introduction
Our knowledge of the digestive system of centipedes is still fragmentary. The more recent overviews (Lewis 1981, Minelli 1993, Rosenberg 2009) describe the gut as a straight tube which is clearly divided into an ectodermal stomodeum or foregut, an entodermal mesenteron or midgut, and an ectodermal proctodeum or hindgut. As ectodermal invaginations, the foregut and the hindgut are lined by a cuticle. The gross anatomy of the alimentary canals of representatives of the Lithobiomorpha (Lithobiidae), Scolopendromorpha (Cryptopidae), and Geophilomorpha (Geophilidae, Himantariidae) was first presented by Plateau (1878).
Subsequent studies focused particularly of anatomical and histological details of the gut of scolopendromorph (Willem 1889, Balbiani 1890, Kaufman 1962, Jangi 1966, Koch et al.
2009) and geophilomorph (Kaufman 1960) centipedes. The gut of Lithobius forficatus (Linnaeus, 1758) (Lithobiomorpha) was surveyed by Sograff (1880) and studied in more detail (anatomy and histology) by Kaufman (1961a) and Rilling (1968). Almost no information exists for the anatomy of the gut of Craterostigmus tasmanianusPocock, 1902 (Craterostigmomorpha), apart from Manton´s (1965) short outline. The digestive system of the Scutigeromorpha has not been described in full detail thus far. Our present knowledge is largely based on anatomical descriptions on scutigeromorph centipedes by Takakuwa (1955) and histological descriptions of Scutigera coleoptrata(Linnaeus, 1758) by Kaufman (1961b).
The digestive system as a whole has not yet been described. Verhoeff´s (1902–1925) description of the centipede pharynx also included the roof of the preoral chamber
(epipharynx) and its bottom (hypopharynx), which is subsumed as peristomatic organs (Haase 1884, ‘Schlundgerüst’, Verhoeff 1902–25, ‘peristomatische Organe’). Recent investigations of the epi- and hypopharynx confirmed considerable variability and morphological complexity of the peristomatic structures in Scutigeromorpha, Lithobiomorpha, and Scolopendromorpha (Koch & Edgecombe 2006, 2008, Edgecombe & Koch 2008). An account of the pharynx apparatus in Scutigeromorpha in more detail is still missing.
Seifert (1967) first recognised that the pharynx apparatus in Scutigera coleoptrata represents no mere prolongation of sclerotised bars of the preoral chamber extending into the pharynx. He identified a stalked ball-shaped outgrowth of the dorsal pharyngeal wall that he called the pistil (‘Pistill’) and a ventral groove of the pharynx bottom (‘Mörserrinne’, pharynx furrow). Here,we present some new structural details on the pharynx apparatus of Scutigera coleoptrata. We will describe the cuticle of the pistil and of the pharynx furrow with its underlaying epithelium, using LM, TEM, and SEM techniques.
2. Materials and methods 2.1. Animals
Specimens of Scutigera coleoptrata(Linnaeus, 1758) were collected around Banyuls-sur Mer, France. The specimens were kept in glass boxes (15 cm x 8 cm x 15 cm) filled with 1 cm garden soil and two pieces of moistened (tap-water) blotting paper (15 cm x 15 cm each).
The animals were fed with wingless Drosophila melanogaster Meigen, 1830 and Musca domesticaLinnaeus, 1758. Specimens of S. coleoptratawere anaesthetised with CO2, cut into several parts and fixed as described below.
2.2. Light and transmission electron microscopy (TEM)
For TEM investigation, heads of Scutigera coleoptratawere fixed in phosphate-buffered (pH 7.2) paraformaldehyde (4 %), containing 15 % saturated picric acid and 0.08 % glutaraldehyde. They were postfixed with 1 % OsO4 in the same buffer and, after alcohol dehydration, embedded in Epon. Semithin sections (0.5–1 µm) were stained with toluidine blue (Trump et al, 1961; modified: toluidine blue share 1 %). Sections were studied using a DMSL-Leica microscope. Ultrathin sections were stained with uranyl acetate and lead citrate and studied using a ZEISS EM 902 A electron microscope.
2.3. Maceration procedure and scanning electron microscopy (SEM)
The SEM investigation of the cuticle lining of the pharynx furrow requires the removal of the tissue. We chose a maceration technique using pepsin following Hilken (1994). The solution of 1.5 g pepsin in 100 ml HCl-solution (1.5 %) was effective with a maceration period lasting up to 10 to 30 days. SEM specimens were critical-point dried, sputter-coated with gold, and examined using a CAMSCAN DV4 scanning electron microscope.
3. Results
The foregut of Scutigera coleoptrata with its pharynx apparatus was described on a light microscopical level by Seifert (1967). He identified a stalked pistil on the dorsal pharyngeal
wall and a ventral groove on the bottom of the pharynx. Therefore, we restrict our description to some features of the pharynx apparatus mainly detected by TEM and SEM-investigations.
The pharynx apparatus (‘Pharynxapparat’, Seifert 1967) of Scutigera coleoptrata is in part a sclerotised structure on the dorsal and ventral wall of the middle part of the pharynx (Fig.
1A, D, E). It consists of a cuticular groove on the floor of the pharynx, the pharynx furrow (Fig. 1A–E). This furrow flattens at its posterior end (Fig. 1F). Its length is about 200 µm, its diameter about 70 µm. Additionally, on the dorsal wall of the pharynx a cuticular ridge that bears a stalked knob is developed, the so-called pistil (Figs 1E, 2A, 3A, B). In the area of the pharynx apparatus, the lumen of the foregut is relatively small and U-shaped in comparison to the posterior part. The roof of the pharynx is typhlosole-like invaginated in the area of the pistil (Fig. 1D, E).
A
B C
D E F
100 µm
50 µm
20 µm 10 µm
50 µm 50 µm
vi
vi
vi
dm pf
p nr po po
pf pf pf
Fig. 1 A–F: The pharynx apparatus of Scutigera coleoptrata. – A: Foregut in the area of the pharynx apparatus, from ventral (SEM); B: Porous wall of the pharynx furrow, from ventral (SEM); C: Detail of B; D: Anterior region of the foregut with the beginning of the pharynx furrow (cross section, LM); E: Middle region of the foregut with full developed pharynx furrow and an inserted pistil (LM); F: Posterior region of the foregut with a flattened cuticular section in the region of the pharynx furrow. Huge dilator muscles are attached dorsally to the foregut (cross section, LM). dm, dilator muscle; nr, Nervus recurrens; p, pistil; pf, pharynx furrow; po, pores; vi, ventral invagination.
A B C
D E F
G H I
10 µm 10 µm 2.5 µm
1 µm 1 µm 1 µm
1 µm 0.2 µm 0.2 µm
ml
po po
me me ac
eg vi
vi
c p pf
pf
pf
pf
c
c dm
c
Fig. 2 A–I: The pharynx apparatus of Scutigera coleoptrata. – A: Pharynx furrow with the inserted pistil. The nuclei of the epithelial cells are arranged lateral to the furrow (cross section, LM);
B: The two flaps of the pharynx furrow. The pistil is extracted. A dilatator muscle and the underlaying ‘multi-layered’ epithelium are visible (TEM); C: Detail of B. Numerous pore canals pass the cuticle of the pharynx furrow. Only in its basal part, the endocuticular lamellae are packed densely, whereas in the apical part, the distances between the lamellae are widened (TEM); D: Ventral invagination between the two flaps of the pharynx furrow and the underlaying ‘multi-layered’ epithelium with rounded cells in cross section (TEM);
E: Detail of the ‘multi-layered’ epithelium at a lateral region of the furrow. The projections of the cells run oblique around the furrow and are equipped with numerous mitochondria (TEM); F: In the region where the flaps are connected ventrally, the cuticle is conspicuously structured. Apically, a broad sclerotised area is observable. Horizontally arranged sclerotised stripes reaches into the underlaying cuticle (TEM). G: Perpendicular to the cuticular layers, numerous pore canals cross the cuticle (TEM); H: The cuticle of the pharynx furrow is covered by a distinct mucous layer. Apically, a small sclerotised cuticular layer is developed, interrupted by small, unsclerotised sections (TEM); I: Several epidermal glands are found between the foregut epithelium around the pharynx apparatus (TEM). ac, aberrant cuticle; c: cuticle; dm: dilatator muscle; eg: epidermal gland; me: ‘multi-layered’
epithelium; ml: mucous layer; p: pistil; pf: pharynx furrow; po: pore canals; vi: ventral invagination.
The whole gut is surrounded by circular muscles (Fig. 3G). Seifert (1967) described several muscles (ventral and dorsoventral dilators) that are attached to the cuticle of the foregut. The attachment of one of the dilator muscles is shown in Fig. 3F. The epithelium of the foregut is single-layered (Fig. 3E). Scattered epidermal glands are found between the cells of its epithelium (Fig. 2I). Only in the epithelium around the furrow and the insertion area of the pistil the epithelium appears ‘multi-layered’. The nuclei in the region of the furrow are situated laterally (Fig. 2A). The ‘multi-layered’ epithelium is formed by stretched longitudinal arranged cells; their projections are ventrally directed and equipped with several mitochondria (Fig. 2D–E). In cross sections of the median area of the furrow, the cellular projections appear more or less roundish (Fig. 2D). It seems that the projections of these cells run obliquely around the furrow, as seen in a more laterally situated area (Fig. 2E).
The pharynx furrow is formed by two cuticular flaps, linked ventromedially by a small differentiated cuticle area (Fig. 2A–C). At its connection, a ventral invagination is developed (Fig 2B–D). In LM, the cuticle of the pharynx furrow appears strongly sclerotised (Fig. 2A).
However, in TEM, the specialised cuticle of the furrow is not homogeneously structured and sclerotised. Laterally, the lamellated endocuticle of the pharynx is displaced by the cuticle of the furrow. Only in its basal part, endocuticular lamellae are densely packed, whereas in the apical part, the distances between the lamellae are conspicuously widened (Fig. 2C, D). Only apically is a small sclerotised cuticular layer developed, interrupted by small but not sclerotised sections (Fig. 2H). Perpendicular to these cuticular layers, hundreds of pore canals cross the cuticle (Fig. 2C–D). These canals originate from the underlying epithelium and seem to open at the surface of the furrow (Fig. 2G–H). Also in SEM-images it is observable that the whole cuticle of the inner side of the furrow is pierced by numerous pores (Fig. 1B–C). The cuticle of the pharynx furrow is covered by a distinct mucous layer (Fig. 2H). Often bacteria are attached to this layer (Fig. 3G), that is probably secreted by epidermal glands, located in the epithelium of the foregut (Fig. 2I). In the region where the flaps are ventrally connected, the cuticle is conspicuously structured. Endocuticular lamellae and pore canals in this region are not observable (Fig. 2C, F). Apically, a broad and sclerotised area is observable (Fig.
2B–D). Horizontally arranged sclerotised stripes draw into the underlaying cuticle (Fig. 2F).
The pistil consists of an elongated shaft and a spherical end piece (Figs 1E, 3A, B). The cuticle of the pistil is mainly sclerotised in its marginal parts; a small sclerotised band is observable in the middle part of the shaft (Fig. 3A, B). The remaining cuticle of the foregut is folded and underlain by a subcuticle (Fig. 3C, F). The broad lamellated endocuticle is covered by a small sclerotised exocuticle (Fig. 3F). Scattered epidermal glands are arranged between the epithelium of the foregut (Fig. 2I).
A
B
C
D
E
F
G
2.5 µm 2.5 µm
2.5 µm
2.5 µm
2.5 µm
2 µm
p
me
se c
cm pc
c
dm c
c b me
sc
sc c
me
Fig. 3 A–G: The pharynx apparatus of Scutigera coleoptrata (TEM). – A-B: Pistil and the underlaying ‘multi-layered’ epithelium. The pistil consists of an elongated shaft and a spherical end piece; C: Transition zone between the ‘multilayered’ epithelium and the connected single-layered epithelium in the dorsolateral region of the foregut. A distinct subcuticle is observable beside a lamellated endocuticle; D: Transition zone between the
‘multilayered’ epithelium and the regular single-layered epithelium; E: Insertion of one of the ventrolateral dilatator muscles; F: Circular muscle, surrounding the cuticle of the foregut; F: Bacteria, attached to the wall of the foregut. b: bacteria; c: cuticle; cm: circular muscle; dm: dilatator muscle; me: ‘multi-layered’ epithelium; p: pistil; sc: subcuticle; se:
single-layered epithelium.
4. Discussion
The digestive tract of Chilopoda is divided into a foregut, a midgut, and the hindgut. In the foregut of different Chilopoda conspicuous cuticular structures are developed. In Lithobiomorpha the anterior part of the pharynx is armed with backward directed spines (Gibson-Carmichael 1885) and in representatives of the Scolopendromorpha, the cuticle of the anterior part of gizzard is characterised by cuticular plates with ridges and backward directed spines (Balbiani 1890, Jangi 1966, Koch et al. 2009). The pharynx apparatus in the foregut of notostigmophoran Chilopoda was overlooked by Haase (1884) and Verhoeff (1902–25); it was first recognised by Seifert (1967).
Why does the Notostigmophora develop such a complicated structure in the foregut? Seifert (1967) suggests some functional aspects: The pistil seems to fit into the pharynx furrow like a milling device. This study shows that the pistil is quite a delicate structure that is not as strongly sclerotised as it seems to be in LM images. Thus, its function as a tool for trituration remains uncertain. Additionally, the furrow is not suitable to act as a friction surface because of the relatively less sclerotised cuticle. It seems to be more likely that the pharynx apparatus may serve primarily as a structure to separate large from small food particles and to grind the food particles. A gliding of the pistil in the pharynx furrow may be supported by the mucous layer that acts as a lubricant. The mucus might originate from epidermal glands, located in the foregut epithelium, and from salivary glands in the head region.
Seifert (1967) stated that the pistil can be shifted into or extracted out of the pharynx furrow only in its posterior end section. According to our study, the two flaps of pharynx furrow seem to be moveable. Due to the basal invagination and the specialised cuticle of the connecting area between the flaps it is likely that they can be pulled apart by ventral dilatator muscles.
These muscles were already described in detail by Seifert (1967). The connecting cuticle between the two flaps seems to have a high elasticity. Thus, we assume that the pistil can be inserted and extracted over the whole range of the pharynx furrow, when the furrow opens dorsally. A main function of the pharynx apparatus as a mill structure appears questionable, and a main function as a sorting structure seems to be more plausible.
The investigation of the fine structure of the pharynx furrow shows that the wall of the furrow is pierced by hundreds of pore canals. The function of these canals is not clear yet.
However, the thick mucous layer that covers the cuticle of the furrow apically is probably formed by the underlying specialised epithelium, although this epithelium shows no glandular character on its own. Within the lateral wall of the foregut, we documented several epidermal glands releasing their secretion into the foregut. However, it is not clear, whether these glands produce digestive enzymes already in this part of the gut.
The described ‘multi-layered’ epithelium in the ventral and dorsal part of the pharynx apparatus is interpreted as a network of obliquely directed elongated cells. It seems that this epithelium follow the extensions of the foregut, depending of the filling degree of the foregut.
Similar epithelia in Chilopoda are described as ‘pseudo-multilayered’ in the maxillary organ of Scutigera coleoptrata(Hilken & Rosenberg 2006). Actually, even this kind of epithelia is most likely single-layered.
5. Acknowledgements
We are grateful to Dr Markus Koch (Institute of Biology, Freie Universität Berlin) for his helpful comments on the manuscript.
6. References
Balbiani, E.-G. (1890): Étude anatomique et histologique sur le tube digestif des Cryptops. – Archives de zoologie expérimentale et générale 2. Sér. 8: 1–82.
Edgecombe, G. D. & M. Koch (2008): Phylogeny of scolopendromorph centipedes (Chilopoda):
morphological analysis featuring characters from the peristomatic area. – Cladistics 24: 872–901.
Gibson-Carmichael, T. D. (1885): Notes on the anatomy of the Myriapoda. – Proceedings of the Royal Physical Society of Edinburgh 8: 377–381.
Haase, E. (1884): Schlundgerüst und Maxillarorgan von Scutigera. – Zoologische Beiträge 1: 97–108.
Hilken, G. & J. Rosenberg (2006): Ultrastructure of the maxillary organ of Scutigera coleoptrata (Chilopoda, Notostigmophora): Description of a multifunctional head organ. – Journal of Morphology 267: 152–165.
Jangi, B. S. (1966): Scolopendra(The Indian Centipede). – The Zoological Society of India, Calcutta, 109 pp.
Kaufman, Z. S. (1960): The structure of the digestive tract in Geophilus proximusKoch (Chilopoda) (title translated) [in Russian]. – Doklady Akademii Nauk SSSR 135: 1274–1277.
Kaufman, Z. S. (1961a): Postembryonic development and structure of the alimentary tract in Lithobius forficatusL. (Chilopoda) [in Russian with English summary]. – Entomologiceskoe Obozrenie 40:
109–119.
Kaufman, Z. S. (1961b): Digestive tract in Scutigera coleoptrataL. (title translated) [in Russian]. - Doklady Akademii Nauk SSSR 139: 1483–1486.
Kaufman, Z. S. (1962): The structure of digestive tract in Scolopendra cingulataLatr. (Chilopoda) [in Russian with English summary]. – Zoologicheskij zurnal 41: 859–869.
Koch, M. & G. D. Edgecombe (2006): Peristomatic structures in Scutigeromorpha (Chilopoda): a comparative study, with new characters for higher-level systematics. – Zoomorphology 125: 187–207.
Koch, M. & G. D. Edgecombe (2008): The peristomatic structures of Lithobiomorpha (Myriapoda, Chilopoda): comparative morphology and phylogenetic significance. – Journal of Morphology 266:
153–174.
Koch, M., S. Pärschke & G. D. Edgecombe (2009): Phylogenetic implications of gizzard morphology in scolopendromorph centipedes (Chilopoda). – Zoologica Scripta 38(3): 269–288.
Lewis, J. G. E. (1981): The biology of centipedes. – Cambridge University Press, Cambridge: 476 pp.
Manton, S. M. (1965): The evolution of arthropod locomotory mechanisms. Part 8. Functional requirements and body design in Chilopoda, together with a comparative account of their skeleto- muscular systems and an appendix on a comparison between burrowing forces of annelids and chilopods and its bearing upon the evolution of the arthropodan haemocoel (Figs 1–99; plates 1–7). – Journal of the Linnean Society of London / Zoology 46: 251–483.
Minelli, A. (1993): Chilopoda. – In: Harrison, F. W. & M. E Rice (eds): Microscopic anatomy of invertebrates, Vol. 12, ‘Onychophora, Chilopoda, and lesser Protostomata’. – Wiley-Liss, New York:
114 pp.
Plateau, F. (1878): Recherches sur les phénomènes de la digestion et sur la structure de l’appareil digestif chez les Myriapodes de Belgique. – Mémoires de l’Académie Royale des Sciences, des Lettres et des Beaux-Arts de Belgique 42: 1–94.
Rilling, G. (1968): Lithobius forficatus. Grosses Zoologisches Praktikum 13b. – Fischer, Stuttgart:
136 pp.
Rosenberg, J. (2009): Die Hundertfüßer (Chilopoda). – Die Neue Brehm-Bücherei Bd. 285. Westarp Wissenschaften-Verlagsgesellschaft mbH, Hohenwarsleben: 534 pp.
Seifert, G. (1967): Der Pharynxapparat von Scutigera coleoptrata L. – Zeitschrift Morphologie und Ökologie der Tiere 58: 347–354.
Sograff, N. (1880): Anatomie Lithobius forficatus Lin. (title translated) [in Russian]. – Izvestija Imperatorskago Obscestva Ljubitelej Estestvoznanija, Antropologii i Etnografii – Moskovskij Universitet 32(2): 1–34.
Takakuwa, Y. (1955): Morphology and classification of the Scutigera, with a memory of the late Dr.
Asajiro Oka [in Japanese]. – Gakufu-Shoin Publisher, Tokyo: 59 pp.
Trump, B.-F., E. A. Schmuckler & E. P. Bendit (1961): A method for staining exopxy sections for light microscopy. – Journal of ultrastructure research 5: 343–348.
Verhoeff, K. W. (1902–25): Fünfter Band. II. Abteilung Gliederfüssler: Arthropoda Klasse Chilopoda (Tafeln 1–30). – In: Bronn, H. G. (ed): H. G. Bronn’s Klassen und Ordnungen des Tier-Reichs.
Akademische Verlagsgesellschaft m.b.H., Leipzig: 725 pp.
Willem, V. (1889): Note sur l’existence d’un gésier et sur sa structure dans la famille des Scolopendrides.
– Bulletins de l’Académie Royale des Sciences, des Lettres et des Beaux-Arts de Belgique 3: 532–547.
Accepted 25 September 2009
