A SEM study of the reindeer sinus worm ( Linguatula arctica )

Pentastomids are a group of peculiar parasitic arthropods, often referred to as tongue worms due to the resemblance of some species to a tongue. Linguatula arctica is the sinus worm of the reindeer (Rangifer tarandus), being the only pentastomid to have a direct life cycle and an ungulate as a definite host. Here, the surface structures and internal anatomy of adult L. arctica are described as seen by scanning electron microscope (SEM). Sinus worms were collected in the winter 1991-92 in Finnish Lapland. Paranasal cavities of about 80 reindeer were examined and 30 sinus worms were found. The sinus worms had typical Linguatula sp. morphology, being paddle-shaped, transparent, pale yellow, dorsoventrally flattened and pseudosegmented with a long tapering end. Present at the anteroventral part of the cephalothorax was an oral opening with a large, conspicuous, head-like papillar structure. Bilaterally, on both sides of this opening, was a pair of strong curved hooks. The cephalothorax and abdomen had a segmented appearance, as they showed distinct annulation. There was a small cup-shaped sensory organ present at the lateral margin on each annula. The posterior edge of each annula was roughened by tiny spines projecting backwards. Throughout the cuticular surface, small, circular depressions that represented the apical portion of chloride cells. The genital opening of the male was located medioventrally between the tips of the posterior pair of hooks, and that of the female posteroventrally and subterminally. In both sexes, the genital opening was bilaterally flanked by papillar (in males) or leaf-like (in females) structures. One copulating couple was present, with the male attached to the posteroventral part of the female with its anteroventral hooks and papillae. Several structures typical of arthropods and other pentastomids were identified. Because SEM allows only surfaces to be studied, the morphology and especially the sense organs of L. arctica remain obscure. Transmission electron microscopy should be employed to gain more information about this fascinating creature and its origin.


Introduction
Pentastomids are a group of peculiar parasitic invertebrates, often referred to as tongue worms due to the resemblance of some species to a tongue.There are about 10 species of pentastomids.The majority are obligatory parasites of the respiratory passages of reptiles in tropical regions.However, the subclass also includes some parasites of the upper respiratory tract of birds and mammals (Riley, 1986).Despite their flat and worm-like appearance, pentastomids belong to the phylum Arthropoda, finding their current placement in the phylum quite recently.The fossils from the Upper Cambrian and Lower Ordovicium identified as pentastomids suggest that they had branched very early from other arthropods, and their parasitic lifestyle was established long before the terrestrialization of the ancestors of their present hosts (Walossek & Muller,199).It is perhaps because of this early branching that pentastomids still have morphological similarities to other groups of invertebrates, thus resulting in their phylogeny being controversial.The first attempt to classify pentastomids as crustaceans was by Wingstrand (1972), who showed that the structure of the spermatozoa of pentastomids is iden-raises some doubts about the justification for the current classification, and many textbooks continue to regard pentastomids as a phylum of its own.
Pentastomids are divided into two orders: Cephalobaenida and Porocephalida.In the order Porocephalida, the genus Linguatula (Frölich 1789) presently includes six species, five of which as adults parasitize the respiratory tract of carnivorous mammals, with mammals serving as intermediate hosts (Swetman, 1971), and the sixth, Linguatula arctica, being the sinus worm of the reindeer (Rangifer tarandus) (Haugerud & Nilssen, 1986).Reindeer sinus worm differs from other linguatulids not only by having an ungulate as a definite host but also by possessing a direct life cycle (Haugerud & Nilssen, 1985;1990), although the possibility of an invertebrate intermediated host cannot be totally excluded (Riley et al., 1987).The best known member of the Linguatula family is L. serrata, a cosmopolitan parasite living in the sinuses of canids.Thus, unsurprisingly, all of the early reports of the sinus worm (L.arctica) in reindeer were recorded as L. serrata.Murie (1926) described L. serrata in caribou in Alaska in 1926, andVoblikova (1961) reported a high prevalence (1%, n=10) of L. serrata in reindeer in Taimyr, Russia.According to Skjenneberg (1965), L. serrata was common in Norwegian reindeer, confirmed by extensive population studies of L. arctica in semi-domesticated and wild reindeer in Norway (Haugerud, 1986;1988).In Swedish reindeer, L. serrata was first reported by Christensson et al. (197) and later by Rehbinder and Nordkvist (1982), who autopsied reindeer, 2% of which were positive for L. serrata.In Finland the earliest record of L. serrata in reindeer is from 196 (Collections of University of Oulu, Itämies, pers. comm. 2006).Nikander & Rahko (1989), who studied sinusitis in reindeer in Finnish Lapland, found 5 out of 25 randomly sampled animals to be infected by L. arctica.
The morphology and life cycle of the reindeer sinus worm were described by Riley et al. (1987), and their morphological description was based on findings with light microscopy (LM).Scanning electron microscopy (SEM) is a valuable tool when supplementary information to morphological observations with LM is needed.In the present study, the surface structures and internal anatomy of adult L. arctica are described as seen by SEM.The functional morphology of these structures is also discussed.tical to the corresponding structures of another group of living organisms, namely the branchiuran fish lice, belonging to parasitic crustaceans.There are also similarities to the reproductive behaviour of the fish louse, as sperm are usually transferred to and stored in the spermatheca of the female (Fryer, 1982).Recently, techniques based on molecular biology have been employed to elucidate the taxonomic position of pentastomids.Lavrov et al. (200) determined the complete sequence of the mitochondrial DNA of a pentastomid (Armillifer armillatus) and compared it with complete or nearly complete mitochondrial DNA sequences of four different groups of Crustacea.Their findings indicated "unambiquously that pentastomids are a group of modified crustaceans."To summarize, pentastomids are currently classified as arthropods, more precisely to the class Maxillopoda within the subphylum Crustacea (see e.g.Fauna Europaea at www.faunaeur.org).However, the dissimilar morphology of pentastomids to other maxillopods

Material and methods
Sinus worms were collected in a three months period from December 1991 to February 1992 during the slaughter in Savukoski in the eastern part of Finnish Lapland.The muzzles of reindeer were opened by sawing them transversally behind the back teeth, and the paranasal cavities were carefully examined.About 80 reindeer, mainly calves, were inspected and 0 sinus worms were found.Worms were immediately fixed in a mixture of formaldehyde (10%), paraformaldehyde (2%) and glutaraldehyde (0.1%).The dehydration of the samples was carried out through a series of increasing concentrations of ethanol (50%, 60%, 70%, 80%, 90%, 96% and 100%).The alcohol series was followed by drying using a critical point dryer, mounting on brass stubs and coating with gold (JOEL JFCsputter).The internal organs were brought out by breaking some dried worms before mounting and coating with gold.The specimens were examined under a scanning electron microscope (JEOL JMS-820) operating at -6 kV at the Electron Microscopy Unit of the Institute of Biotechnology, University of Helsinki.

Results
In infected reindeer, one or two sinus worms were usually present unilaterally in the palatinal sinuses.The sinus worms had typical Linguatula sp.morphology, being paddle-shaped, transparent, pale yellow, dorsoventrally flattened and pseudosegmented with a long tapering end.
The biggest females were about 10 cm long.Their body consisted of a 2-cm-wide cephalothorax and a thin abdomen which occupied over half of the length of the worm.The uterus could be seen as an orange coloured elongated mass of eggs (shown dark in the figure) in the middle of the cephalothorax (Fig. 1).Present at the anteroventral part of the cephalothorax was an oral opening with large, conspicuous, head-like papillar structure (Fig. 2).Around this opening, the cuticle formed a well-defined circular ridge.Bilaterally, on both sides of the oral opening, was a pair of strong curved hooks with sharp tips (Figs. 2,3,8).The proximal third of the dorsal side of the hooks was covered by a calotte-like chitinous layer with fine triangular spines (Figs. 3,4).The cephalothorax and abdomen had a segmented appearance, as they showed distinct annulation.There was a small cup-shaped cavity present at the lateral margin on each annula (Fig. 5).Furthermore, at the bottom of the cavity, a globular projection was observed (Fig. 6).Throughout the cuticular surface were scattered, small, circular depressions with a porous, grooved or granular surface (Figs. 5,7,14).Subterminally, bilaterally and ventrolaterally, both the males (Fig. 8) and the females had a cluster of receptors and glands consisting of an opening, a row of or setae and conical papillar projections (Fig. 9).The genital opening of the male was located medioventrally between the tips of the posterior pair of hooks (Fig. 10).The genital opening was bilaterally flanked by broad-based papillary structures (Figs. 10,12).The posterior margin of the oral opening was bumpy, consisting of multiple, papillar elevations that created a surface resembling cobble-stones (Fig. 13).
Clot of blood cells were occasionally observed in the oral opening (Fig. 11).Each thoracic annula overlapped slightly with the adjacent annula, and the posterior edge was roughened by tiny spines projecting backwards (Fig. 14).2).The conspicuous hole is seemingly an opening of the frontal gland.The three delicate setae and conical papillae were considered as sensory organs.Scale bar = 300 μm.Fig. 10.The genital opening (arrow) of the male L. arctica is located between the posterior pair of hooks.This opening is bilaterally flanked with papillar structures (rectangle).An enlargement of the area in the rectangle is depicted in Fig. 12. Scale bar = 500 μm.The reproductive and digestive systems were the dominant structures seen in SEM from dissected females.The ovary resembled a cluster of grapes (Fig. 15), and the paired seminal receptacles were packed with long slender spermatozoa (Fig. 16).The uterus in the adult female was a long, winding tube filled with spherical eggs (Fig. 17) and ending in a posteriorly, subterminally located gonopore.The genital opening was bilaterally flanked by leaf-like extensions (vaginal lips) protruding from the gonopore (Fig. 20).The intestine was a straight tube (Fig. 18) extending from the mouth to the anus (Figs. 19,20).Among the sinus worms studied, one copulating couple was present, with the male attached to the posteroventral end of a small-sized female with its anteroventral hooks and papillae.

Discussion
Since the SEM technique is performed on dried specimens, shrinkage should always be taken into account when these findings are interpreted.The measurements and scale bars presented in this paper should therefore be considered merely indicative.
The difficulties in classifying pentastomids as arthropods are obvious, as they lack the most characteristic morphological feature of arthropods, namely, jointed appendages.Nevertheless, they possess several morphological structures and biological features that are typical of arthropods.These features have been reviewed by, for instance, Riley (1986) and Haugerud (1989).The most important features are chitinous cuticle, muscle striation and its attachment to cuticle, sensory sensillae, lack of free cilia, shape and structure of spermatozoa, dorsal organ and blastoderm cuticle of the egg, paired embryonic appendages, certain parts of the reproductive system and the developmental ecdysis.
The scanning electron microscope is an excellent tool for investigating surface structures.The entire body surface of L. arctica is covered by a chitinous sheath with annuli and spines, resembling insect maggots.Present at the anterior end of sinus worms are five distinct protuberances, inspiring the Latin name Pentastomida ("five mouths").In reality, only one of the protuberances is connected to the mouth, the remaining four being heavily sclerotized holdfast hooks.The size and shape of the hooks have been used to identify pentasto-mids (Riley 1986).However, these measurements must be interpreted cautiously, as hooks increase in size at each molt (Fain, 1964).The hooks have been shown to be of the arthropodal type.They are considered as remnants of limbs that have during evolution been reduced to hooks more useful to the current niche (Riley, 1986;Walossek & Muller, 199).As evidence for such an evolution, in the Cephalobaenida pentastomids, the hooks are located at the tip of leg-like protuberances (Bush et al., 2001).The hooks seen in Porocephala pentastomids arise from the socket provided by the body.What is left of the ancient limbs is two podomeres and a joint membrane between them (Walossek & Muller, 199).In L. arctica, a calotte-like structure dorsally covers the proximal part of all hooks.The surface of this thickened portion has cuticle-like configuration with spines and is regarded as a remnant of the joint membrane.
The mouth was situated between the proximal parts of the inner hooks on the anteroventral side of the sinus worm.The posterior margin of the oral opening was one of the sites where the generally smooth cuticle of L. arctica was uneven.The lumpy, cobble-stone-like cuticular surface probably forms a functional counterpart for oral papilla during feeding.Relatively little is known about the feeding habits of L. arctica, but pentastomids typi- Note that at the time of mating the female sinus worm is about the same size as the male, beingcm in length, while the adult female typically exceeds 10 cm.Scale bar = 1 mm.cally feed on blood (Riley, 1986).With the help of an oral papilla, the sinus worm produces an ulcer on the epithelium and feeds on the blood pooling on the ulcer.A conspicuous ulcerative lesion was a constant finding at the attachment site in the sinusoid mucosa of reindeer.Occasionally, clotted red blood cells could be seen in the oral opening of sinus worms in SEM.The digestive physiology seems to be a unifying feature of all pentastomids.They have a pharyngeal pump, which by rhythmic expansion and contraction enables ingestion (Riley,197).
Bilaterally, in the direction of ten and two o'clock from the head papilla there was a conspicuous hole present.SEM revealed several other structures in the vicinity of these openings.Near the anterior edge of the opening was a row consisting of or delicate setae, and on the opposite side of the opening, were two groups of setae with two stout conical papillae.These structures are apparently sensory organs.The hole is probably an opening of the frontal gland, which, in many pentastomids, has been shown to secrete a lamellate material resembling the pulmonary surfactant produced by type II alveolar epithelial cells of the host (Riley, 1992).The substance produced by the glands covers the entire pentastomid, likely to help the parasite to evade its host's immune system (Riley, 1992).
Small cup-shaped cavities seen as a chain running through the lateral margin of annuli were considered to be sensory structures (lateral sense organs) (Riley, 1986).Storch & Böckeler (1979) described the ultrastructure of sensory cells of Reighardia sternae and showed that they consist of one apical cilium, which is surrounded by two sheat cells, forming a complex with a close resemblance to the sensillae of other arthropods.Because SEM allows only surface structures to be investigated, the sense organs of L. arctica have been poorly studied, and at least transmission electron microscopy should be employed to gain more information about these structures.
The strainer-like, round, abundant, cuticular depressions scattered throughout the body are in fact the visible, apical part of cells, earlier referred to as epidermal glands (Hett,192) and currently as chloride cells, class I cells or ionocytes (Riley, 1986;Bush et al., 2001).They were described in detail by Banaja et al. (1977) and, according to their descrip-tion, they consist of one or more pyriform cells.These cells are probably responsible for maintaining the hydromineral balance for the haemolymph of the sinus worm.Since pentastomids feed on host blood or lymph, both of which are rich in sodium and chloride, chloride cells have been proposed to secrete excess ions (Banaja et al., 1977).One of the typical features of pentastomids is their lack of excretory systems.Interestingly, a group of Aphasmida nematodes, such as Trichuris (Sheffield, 1963;Wright, 1968) and Trichinella (Bruce, 1970), shares this feature.As a substitute for the excretory system, they possess bacillary cells comparable to chloride cells.A question awaiting an answer is whether this is an example of convergent evolution taking place independently in two groups of parasites or whether these groups share related ancestor.
Copulation in Porocephala pentastomids is a complex process.Although being long-lived, females evidently copulate only once in their lifetime, but males are probably able to copulate several times (Riley,198).As mating occurs only once, it has seldom been observed in sinus worms (R. Haugerud, 2006, pers. comm.).The male grasps the tip of the female's abdomen with its hooks such that the genital pores are in juxtaposition.Possibly the calottelike extension of the hook with its spines, functions as a stopper during the attachment to prevent infliction of injuries to the female.Once the initial attachment has taken place, the male can insert its spicules into the vagina.The papillar structures flanking the genital pore of the male correspond in dimension to similar structures in the female, thus these structures are evidently morphological counterparts involved in copulation.The copulation is temporally determined since only in young (small) females are the vagina and the receptacula seminis close enough together for a sperm injection by the cirri into the spermatheca.Thus, the mating happens before oocytes are fully developed, and the spermatozoa are retained by the female in the spermatheca, remaining inactive during the storage period (Riley, 1986).This kind of reproductive behaviour is common in pentastomids and in the forementioned fish lice (Fryer, 1982).This peculiarity of pentastomids explains the small size of the female engaged in copulation in our material.In adult females, stored sperm could be seen in tightly packed bundles and whorls.The infective eggs of L. arctica are spread out on the pasture (Haugerud & Nilssen,1986).We have twice observed in August the narrow tail of the female sinus worm is protruding through the sphenopalatinal foramen of the reindeer into its nasal cavity, suggesting that fertilized eggs are expelled from the palatinal sinus with the aid of the long tail of the female.

Fig. 1 .
Fig. 1.Female Linguatula arctica, the sinus worm of the reindeer.An enlargement of the area in the rectangle is depicted in Fig. 2. The elongated mass of eggs in uterus is seen as the dark median region.Scale bar = 1 cm.Fig. 2. Anteroventral view of a female L. arctica showing the hooks and oral opening with distinct papillae.One pair of papillae (asterisks) is present in the vicinity of the posterior pair of hooks.Note the opening of the frontal gland (arrow).Scale bar = 500 μm.Fig. 3. Close-up of the curved, sharp, retractable hooks of the female L. arctica.An enlargement of the area in the rectangle is depicted in Fig. 4. Scale bar = 200 μm.Fig. 4. Magnification of the calotte-like structure covering the proximal part of the hook with fine triangular cuticular spines.Scale bar = 20 μm.Fig. 5. Lateral view of the cephalothoracic annuli of the female L. arctica, showing cup-shaped lateral sense organs (one in rectangle) and the apical parts of chloride cells seen as round, scattered hollows (arrow).An enlargement of the area in the rectangle is depicted in Fig. 6.Scale bar = 200 μm.Fig. 6.Close-up of the cup-shaped lateral sense organ.Scale bar = 5 μm.Fig. 7. High magnification of the apical part of the chloride cell.Scale bar = 5 μm.

Fig. 8 .
Fig.8.Anteroventral view of the hooks and oral papillae of the male L. arctica.Scale bar = 500 μm.Fig.9.Magnification of the structures located subterminally and anteroventrally (the area is indicated with an arrow in Fig.2).The conspicuous hole is seemingly an opening of the frontal gland.The three delicate setae and conical papillae were considered as sensory organs.Scale bar = 300 μm.Fig.10.The genital opening (arrow) of the male L. arctica is located between the posterior pair of hooks.This opening is bilaterally flanked with papillar structures (rectangle).An enlargement of the area in the rectangle is depicted in Fig.12.Scale bar = 500 μm.Fig. 11.A clot consisting of blood cells and fibrin is seen in the oral opening of the sinus worm.Scale bar = 500 μm.Fig. 12. Magnification of the lateral papilla situated in the vicinity of the genital opening of the male sinus worm.Scale bar = 50 μm.Fig. 13.Posterior margin of the oral opening of L. arctica showing cobble-stone-like cuticular ornamentation.Scale bar = 10 µm.Fig. 14.Posterior edge of one cephalothoracic annula showing cuticular spines projecting backwards.Two chloride cells can also be seen.Scale bar = 100 μm.

Fig. 11 .
Fig.8.Anteroventral view of the hooks and oral papillae of the male L. arctica.Scale bar = 500 μm.Fig.9.Magnification of the structures located subterminally and anteroventrally (the area is indicated with an arrow in Fig.2).The conspicuous hole is seemingly an opening of the frontal gland.The three delicate setae and conical papillae were considered as sensory organs.Scale bar = 300 μm.Fig.10.The genital opening (arrow) of the male L. arctica is located between the posterior pair of hooks.This opening is bilaterally flanked with papillar structures (rectangle).An enlargement of the area in the rectangle is depicted in Fig.12.Scale bar = 500 μm.Fig. 11.A clot consisting of blood cells and fibrin is seen in the oral opening of the sinus worm.Scale bar = 500 μm.Fig. 12. Magnification of the lateral papilla situated in the vicinity of the genital opening of the male sinus worm.Scale bar = 50 μm.Fig. 13.Posterior margin of the oral opening of L. arctica showing cobble-stone-like cuticular ornamentation.Scale bar = 10 µm.Fig. 14.Posterior edge of one cephalothoracic annula showing cuticular spines projecting backwards.Two chloride cells can also be seen.Scale bar = 100 μm.

Fig. 15 .
Fig. 15.The ovary resembles a cluster of grapes.Scale bar = 500 μm.Fig. 16.Tightly packed bundles of spermatozoa stored in the seminal receptacle of the female sinus worm.Scale bar = 10 μm.Fig. 17.Dissected uterus showing the spherical eggs of the female L. arctica.Scale bar = 100 μm.Fig. 18.Cross-section of the intestine of the female L. arctica.Scale bar = 50 μm.Fig. 19.Posterior end of the male L. arctica with a vertical anal opening.Scale bar = 500 μm.Fig. 20.Posterior end of the female L. arctica with a subterminal, slightly dorsally located anus (arrow) and a ventrally located gonopore (asterisk).Note asterisk).Note ).Note the leaf-like protrusions of the genital opening.Scale bar = 500 μm.Fig. 21.Copulating couple of Linguatula arctica.Note that at the time of mating the female sinus worm is about the same size as the male, beingcm in length, while the adult female typically exceeds 10 cm.Scale bar = 1 mm.
. Pentastomids, including L. arctica, are fascinating examples of the the richness and biodiversity of nature.