한국가축위생학회지 제39권 제4호 (2016) Korean J Vet Serv, 2016, 39(4), 271-276 Korean Journal of ISSN 1225-6552, eISSN 2287-7630 Veterinary Service https://doi.org/10.7853/kjvs.2016.39.4.271 Available online at http://kjves.org
Abstract
Horsehair or gordian worms (Nematomorpha) were identified with 22 genera (Gordiida) and 5 marine species (Nectonema) until now. During juvenile phase in development, they gain parasitic activity in arthropods. In this study, a gordian worm was detected in the feces of a dog living in Nonsan-si, Chungcheongnam-do, Korea. Using this worm, we evaluated the morphological characteristics by light microscopic analysis. Furthermore, the morphological classification was re-evaluated by scanning and transverse electron microscopes. The worm was determined that it is male adult having a bi-lobed tail and male gonads in cross sections. Based on the morphological characteristics including cross sections of body and areole on the cuticle, the parasite was also identified as Gordius sp. (Nematomorpha: Gordiidae).
Key words : Gordius, Dog, Korea, Nematomorpha
INTRODUCTION the free-living stage (Brivio et al, 2000). Although terrestrial insects act as a host, there are The phylum Nematomorpha include about 300 fresh- some reports on the presence of Nematomorpha in frogs, water species in 22 genera (Gordiida) and 5 marine spe- fishes, birds, mammals including human being (Bolek cies in one marine genus (Nectonema) (Bleidorn et al, and Coggins, 2002; Brivio et al, 2000). Mammalian par- 2002). The class Gordioidea, commonly known as asitism of gordian worms is uncommon in the literature, horsehair or gordian worms, are usually found emerging while many gordian worms have been identified in dif- insects. The juvenile gordian worms are obligate para- ferent parts of the world from specimens recovered from sites of terrestrial insects (e.g., carabid beetles, crickets, the mouth, urethra, and anus (Ali-khan and Ali-khan, or others) and, as adults, are free–living in freshwater 1977; Herter and Nesse, 1989; Saito et al, 1987; Wei sites including lakes, streams, and rivers (Poinar et al, and Yang, 1981). To date, a total of 9 species in 3 gen- 2004; Schmidt-Rhaesa, 1997). After the larval stage, era have been recorded in the gordioidean fauna in adult worms kill and leave the host for the beginning of Korea (Baek and Noh, 1992; Baek, 1993; Lee et al, 2003). Even though the distribution and identification *Corresponding author: Bae-keun Park, Tel. +82-42-821-5849, Fax. +82-42-821-8903, E-mail. [email protected] for gordian worms have been studied, there still re- †First author equivalent.
Copyright © 2016, The Korean Society of Veterinary Service. All Rights Reserved. 271 272 Eui-Ju Hong, Na-Ri Ha, Si-Yun Ryu, Joon-Seok Chae, Hyeon-Cheol Kim, Jinho Park, Kyoung-Seong Choi, Do-Hyeon Yu, Bae-Keun Park mained a lot of the undefined gordian worms. lene oxide, and embedded in epon mixture. The ultra- In this study, we examined the Gordius sp. (Nemato- thin sections were stained with ura3nyl acetate and lead morpha: Gordiida) through mammalian parasitism. To citrate and examined in Tecnai G2 transmission electron identify the gordian worm in a canine feces, it was microscope at 120 kV (FEI, Oregon, USA). studied by light, scanning and transverse electron In gross finding, the body is 19.5 mm in length 1 microscopes. mm in diameter (Fig. 1A), and its color is light brown. In more details, the anterior end (calotte) is rounded with dark color and the mouth is presented at the cen- CASE DESCRIPTION ter, but the white tips were not found on the cuticle. The posterior end is bi-lobed with blunt lobes, each In July 2014, a gordian worm was passed in canine lobe about twice as long as wide. The cuticle is smooth (5 month old, male) feces, and was alive and wiggling and slippery. vigorously at that time (Fig. 1). The worm was placed In SEM finding of transverse section, the worm is in the lacto-phenol solution (glycerin 20 mL, lactic acid composed of epicuticle, cuticle, epidermis, muscle, mes- 10 mL, phenol 10 mL, D.W. 10 mL) for 24 hrs and enchyme, pseudocoel, nerve cord, gut and testis tubules tentatively identified under the light microscope. (Figs. 2A, 2C). While the ventral nerve cord is pre- For the scanning electron microscope (SEM), the sented in sub-epidermal under the muscle layer, the di- pieces of parasite were washed 5 times with 0.2 M ca- ameter of intestine would appear small (Fig. 2B). codylate buffer (pH 7.3) and fixed in 2.5% gluta- Gonads, filled with sperm, occupy much of the pseudo- ldehyde, post fixed in 1% osmium tetroxide at 4oC. The coel within the body (Figs. 2A, 2C), the male re- specimens were then dehydrated in a graded ethyl alco- productive system is made up of two testis tubes that hol series, dried by CO2 critical point, coated with os- fuse at their posterior end by a vertical duct and lead mium and examined by SEM (S-4800, Hitachi) at 15 via a single duct into the intestine (Figs. 2A, 2C). The kV. epidermis is thin (Fig. 3A) and has a connection with a For the transmission electron microscopy (TEM), the ventral nerve cord through the ventral neural lamella, as middle part’s pieces of worm were fixed with 2.5% glu- shown in Figs. 2A, 2B. The parenchyma fills the rest of talaldehyde solution in 0.1 M phosphate buffer (pH 7.4) the body and surrounds the visceral organs such as in- for 4 hours. After washing with buffer, the specimens testine, ventral nerve cord (Figs. 2A∼D). were post-fixed with 1% osmium tetroxide at 4oC for 4 In TEM finding of transverse section, the body wall hours. Afterward, the specimens were dehydrated in a is composed with three components which are cuticle, graded ethyl alcohol series and two changes of propy- epidermis, muscle layers. The cuticle is composed of 21∼22 layers of fibers, followed by a thin epidermis and longitudinal musculature (Figs. 3A∼3C). The epi- cuticle is thin, and it is secreted by epidermis appears as thick and being composed of radial fibrillar layers (Figs. 3A, 3B). According to our expectation, the nuclei were rarely detected in the epidermis (Fig. 3D). The muscle plates are anchored on all sides to the matrix by a little weakly developed hemidesmosomes which appear in transverse view as irregular densities beneath the plasma membranes of the muscle plates (Fig. 3F). The tonofila- ments inserted in muscle layers were weakly developed (Figs. 3E, 3F). Also, glycogen granules are scattered in Fig. 1. The gross finding of Gordius sp. Note rounded calotte. the muscle plate (Fig. 3F). The collagenous matrix of
Korean J Vet Serv, 2016, Vol. 39, No. 4 Morphological study of a horsehair worm, Gordius sp. (Nematomorpha: Gordiida), passed in canine feces 273
Fig. 2. SEM findings from the transverse sectioned soma of Gordius sp. (A) The posterior part of worm. Note the testis tubes are fused to one. (B) The magnified square in Fig. A. Note the nerve cord and intestine. The nerve cord is connected to the epidermis by a neural lamella (arrow). (C) The anterior part of worm. Note the two testis tubes. (D) Parenchyme. I: Intestine, NC: nerve cord, P: parenchyme, PIC: peri-intestinal cavity, PS: pseu- docole, TT: testis tubes. parenchyma does not fuse to basal lamina of mesen- (Schmidt-Rhaesa, 2005). On the other hand, the family chymal cells (Figs. 3F, 3H), and tests tubes are occu- Gordiidae have a smooth cuticle, even though the are- pied by aflagellate spermatozoa (Figs. 3G, 3I). oles in some species of Gordiidae do also occur. They have characteristic two post-anal projections at the pos- terior end in male. In our study, simple areoles are DISCUSSION found on the anterior and posterior end, but rarely found on the mid-body. The tail is a bifulcated posterior Members of class Gordiacea belong to 2 recognized end. families, Chordodidae and Gordiidae. In Chordodidae, The class Gordiacea includes 4 families (Gordiidae, the worms are characterized by the rough cuticle with Chordodidae, Lanochordodidae, Spinochordodidae): Gor- true areoles. They have a bifulcated or grooved posterior diidae is easily distinguished by postcloacal crescent end without post-anal crescent in male (Son et al, 2009). (Gordius and Acutogordius). Spinochordodidae is includ- These subfamily groups include 18 genera (Chordodes, ing the genus Spinochordodes, Lanochordodidae includ- Paragordius, Parachordodes, Neochordodes, Pseudogordius ing the monotypic genus Lanochordodes, and Chordodi- and others). There are also other genera with 2 types of dae is including all remaining genera (Bleidorn et al, areoles, but the characteristic for Parachordodes is that 2002). The useful diagnostic features of nematomorphs 1 type, superareole, is very large and has a pore on top include the shape of the posterior end and cuticular
Korean J Vet Serv, 2016, Vol. 39, No. 4 274 Eui-Ju Hong, Na-Ri Ha, Si-Yun Ryu, Joon-Seok Chae, Hyeon-Cheol Kim, Jinho Park, Kyoung-Seong Choi, Do-Hyeon Yu, Bae-Keun Park
Fig. 3. TEM finding from the transverse sections of Gordius sp. (A) The cutcle consist of three layers, epicuticle, germinal layer of cuticle, fibrillar layer. (B) Subepidermal position. (C) Fibrillar layer. (D) Epidermis. (E) Muscle layer. (F) Magnified muscle layer. Tonofilament (arrows). Hemidesmosomes (circles) (G) Parenchyme and gonad. (H) Collagenous matrix of parenchyme. (I) Early stage of the gonad anlage. Aflagellate spermatozoa (arrows). C: cuticle, CM: collagenous matrix, EC: epicuticle, ED: epidermis, FL: fibrillar layer, G: glycogen granules, GL: germinal layer of cuticle, N: nucleus, P: parenchyme, TF: tonofilament. structures. These structures are most reliably docu- rounded tail lobes. mented with SEM, which is the primary tool in gordiid Gordius species often lack diagnostic characters be- taxonomy (Schmidt-Rhaesa et al, 2003). A semicircular cause of postcloacal crescent. In more details, the male or parabolic crescent is located on the posterior of the worm usually lacks bristles or spines in posterior end, cloacal opening in males, the postcloacal crescent, char- and have a smooth cuticle without areoles. For some acterizes the genus Gordius. But, this structure is also species, a rhomboidal pattern has been reported, which present in the genus Acutogordius (South American and is due to the underlying cuticular fibers arranged in Southeast-Asian), which is easy to distinguish from crossed layers where fibers of adjacent layers form an Gordius by the possession of pointed tail lobes in the angle. White spots on the cuticle are also a common males. In our study, the postcloacal crescent was located characteristic, the latter is not a reliable diagnostic fea- on the posterior of cloacal opening and possessed ture (Schmidt-Rhaesa, 2002). In our case, the areoles are
Korean J Vet Serv, 2016, Vol. 39, No. 4 Morphological study of a horsehair worm, Gordius sp. (Nematomorpha: Gordiida), passed in canine feces 275 shown on the anterior and posterior portion, rarely on especially at the upper surface of the cell, that they are mid-body, but the white spots are not found. almost a continuous belt (Eakin and Brandenburger, The ultrastructural cuticles of gordian worm have 1974). In our sample, the tonofilaments terminate at the been studied by many researchers. But, there is no cell boundaries in hemidesmosomes, but hemidesmosomes agreement which has been reached on naming the dif- are irregularly located at the upper surface of the cell. ferent strata. The cuticle has two structurally different The worms have been frequently reported in humans, layers, a proximal layer containing fibers and a distal but hardly ever in domestic animals. In most cases of one. The fibers of the proximal layer were arranged in mammalian gordiosis, the worms were passed through one layer parallely aligned. The number of fiber sheets anus or vomited out. A human case of a Gordius worm varies widely according to species and researchers. Also, found in the vomitus and another case of a Parachordodes the cuticle has two fundamentally different functions: worm found in the urinary system have also been re- One is for protection during the free-living phase and ported in Korea (Lee et al, 2003; Schmidt-Rhaesa et al, the other is for absorbing nutrients through the cuticle 2003). In animal cases, Saito et al (1987) reported a (Schmidt-Rhaesa, 2005; Skaling and MacKinnon, 1988). hairworm vomited by a domestic cat. Although the The cuticle of larval stage is replaced towards the end worm seems to be similar to Gordius ogatai, it could of the parasitic phase by the adult cuticle. In the para- not be closely identified in G. ogatai (Saito et al, 1987). sitic phase, it is thin, homogeneous and flexible (Sch- A male worm of Chododes koreensis also was reported midt-Rhaesa, 2005; Skaling and MacKinnon, 1988) re- in the canine vomitus in Korea (Son et al, 2009). ported that the cuticle of Chordodes koreensis appears In earlier study analyzing Gordius species in Korea, thick and is composed of radial fibrillar layers. The fi- Baek and Noh (1992) reported 2 species, Gordius ro- bers of proximal layer are comparatively large, and the bustus and Gordionus lineatus. G. lineatus is easily dis- number of fiber sheets is counted as 17 layers. On other tinguished by characteristics of dorsal and ventral line. hand, Swanson (1971) described the two components of This worm resembles Gordius aquaticus because it was the cuticle (homogeneous and fibrous layers) in his not possessed a row of hairs around the male cloacal work on Paragordius varius and G. robustus. Zapotosky aperture, but differ from G. robustus and G. aquaticus (1971) described three main cuticular layers (the cortex, which had wick areoles on the surface of cuticles. the areolar and the basal, which are in turn subdivided Moreover, this worm is similar to G. robustus because into sublayers) in his work on P. varius. Bresciani it has short areole over the whole body cuticle, espe- (1991, 1970), when referring to G. aquaticus, determined cially anterior and posterior portion. the presence of two cuticular layers, the protective and In our study, the worm was similar to G. robustus the fibrillar components. Eakin and Brandenburger (1974), and G. aquaticus because it has short areoles over the working on an unnamed Gordius sp., also found two whole body cuticle, especially anterior and posterior compartments including epicuticle and the fibrillar cuticle. portion, and the post-cloacal crescent was posteriorly lo- In our study, the cuticle consists of three layers, epi- cated at the cloaca and the inverted V-shaped ridge con- cuticle, germinal layer of cuticle, fibrillar layer. And, taining bristle anterior. Like G. lineatus and G. aqua- the cuticle is composed of 21∼22 layers of fibers, fol- ticus, our sample was not possessed a row of hairs lowed by a thin epidermis and longitudinal musculature. around the male cloacal aperture except the dorsal and The epicuticle is thin, and it is secreted by epidermis ventral lines of G. lineatus. The cuticle consists of three appears as thick and being composed of radial fibrillar layers, epicuticle, germinal layer of cuticle, fibrillar layers. layer. This resembles G. robustus which is described by The epidermis of C. koreensis has many clear va- Zapotosky (1971). Gonads, filled with sperm, occupy cuoles, tonofilaments, and numerous hemidesmosome (Son much of the pseudocoel within the body. Therefore, the et al, 2009). The tonofilaments terminate at the cell worm is adult male which is progressed the larval stage. boundaries in hemidesmosomes, which are so numerous, We found that the worm was an unknown species.
Korean J Vet Serv, 2016, Vol. 39, No. 4 276 Eui-Ju Hong, Na-Ri Ha, Si-Yun Ryu, Joon-Seok Chae, Hyeon-Cheol Kim, Jinho Park, Kyoung-Seong Choi, Do-Hyeon Yu, Bae-Keun Park
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