Rhabdias agkistrodonis (Nematoda: Rhabdiasidae): morphology, distribution and specificity

FOLIA PARASITOLOGICA 46: 59-66, 1999 Rhabdias agkistrodonis (Nematoda: Rhabdiasidae): morphology, distribution and specificity Yuriy I. Kuzmin Department of Parasitology, Shmalhausen Institute of Zoology, National Academy of Sciences of Ukraine, 15 B. Khmelnitskii Street, Kiev - 30, 252601, Ukraine Key words: Rhabdias, free-living generation, parasitic hermaphrodites, Far East, viperid snakes Abstract. Morphology of adult parasitic hermaphrodites, free-living males and females, rhabditoid and infective larvae of Rhabdias agkistrodonis Sharpilo, 1976 is described. Adult parasites of the species differ from corresponding stage of other Rhabdias species from snakes in the presence of short cuticular needle on the tip of the tail. Free-living generation stages of R. agkistrodonis have typical rhabditoid morphology. Homogonic infective larvae differ from heterogonic ones in the shape of stoma and oesophagus. Three new hosts: Halys viper (Agkistrodon halys) (Pallas) from Altaiskii Krai (Russia), Okinawa habu (Trimeresurus flavoviridis) (Hallowell) and T. elegans (Gray) from Okinawa Island (Japan) are added to the host range of R. agkistrodonis known previously exclusively from short-tailed viper (Agkistrodon blomhoffi) from the Russian Far East. The original description of Rhabdias agkistrodonis Sharpilo, 1976 was based on the morphology of five parasitic hermaphrodites from one specimen of Agkistrodon blomhoffi (Boie) from the Russian Far East (Kedrovaya Pad' reserve) (Sharpilo 1976). The description was fairly brief and the differential diagnosis was not given. Since then, no data concerning the findings of the species have been published. The characteristic features of R. agkistrodonis life cycle have been already outlined (Kuzmin 1996) but neither free-living adults nor larval stages have been described. Studying the helminthological collection of the Department of Parasitology of Institute of Zoology of NAS of Ukraine I found several samples of nematodes that appeared to belong to R. agkistrodonis. Most of them were collected in the Russian Far East by Dr. V. Sharpilo. These worms had been obtained from the type host, A. blomhoffi. The species was also found in the material from Agkistrodon halys (Pallas) and from two species of the genus Trimeresurus - T. elegans (Gray) and T. flavoviridis (Hallowell) (the latter has been kindly send by Dr. H. Hasegawa from Okinawa, Japan). The experimental investigation of R. agkistrodonis free-living development in culture was carried out and the morphology of free-living stages was studied. Newly obtained data on the morphology, distribution and specificity of R. agkistrodonis are presented in this paper. MATERIALS AND METHODS A total of 31 specimens of Rhabdias agkistrodonis parasitic hermaphrodites were studied. Fifteen of them were from the type host Agkistrodon blomhoffi collected in Primorskii Krai (Russia), 10 from A. halys collected in Altaiskii Krai (Russia), 2 from Trimeresurus elegans and 4 from T. flavoviridis, all from Okinawa Island (Japan). The material studied is stored in the Department of Parasitology, Institute of Zoology of NAS of Ukraine. Two specimens of Agkistrodon blomhoffi caught in Kedrovaya Pad reserve were used in experimental studies of R. agkistrodonis development. Free-living stages of the parasite were cultured using the method of Chu (1936b) in the faeces of the host at room temperature (18-20 C). The method permits the separation of homogonic infective larvae, which migrated from the container into the surrounding water on the third day of culturing, from heterogonic stages. Adult males and females of free-living generation were observed in cultures on the third to fourth day of culturing. Heterogonic infective larvae appeared in cultures on the sixth to eighth day and started to migrate from the container soon afterward. Observations were made on the free-living development of the species in five separate cultures. By the end of experiments both short-tailed vipers had been examined. One and three adult hermaphrodites of R. agkistrodonis, respectively, were found in their lungs and no other nematodes were observed. All material from the collection was fixed in 4% solution of formaldehyde in saline. The material obtained in experiments was studied alive and then fixed in hot mixture of 1 part of 4% formaldehyde and 2 parts of 70% alcohol, with the addition of glycerol (not over 10% of the mixture). Nematodes were cleared in glycerol and studied under a Zeiss-Axiolab light microscope. RESULTS Parasitic hermaphrodites Fig. 1, Table 1 Maximum body width in vulva region. Anterior end of body bluntly pointed, posterior end tapered. Body cuticle relatively uniform in width, slightly swollen and transversely striated in anterior and posterior parts of Address for correspondence: Yu.I. Kuzmin, Department of Parasitology, Shmalhausen Institute of Zoology, National Academy of Sciences of Ukraine, 15 B. Khmelnitskii Street, Kiev - 30, 252601, Ukraine. Phone: ++380 44 266-3380; E-mail: ussp@inzoo.freenet.kiev.ua 59

Fig. 1. Rhabdias agkistrodonis, parasitic hermaphrodite: A general view; B head end, apical view; C anterior part of the body, lateral view; D tail end, lateral view. Scale bars: A = 500 µm; B = 50 µm; C, D = 200 µm. Table 1. Morphometry of parasitic generation adults of Rhabdias agkistrodonis (31 specimens from various hosts). Distance to nerve ring - from anterior edge of oesophagus. All dimensions in µm, unless otherwise indicated. Characters aver. min. max. SD* CV** Body length, mm 5.239 4.075 6.441 0.662 12.63 Body width 220 133 307 43.3 19.71 Buccal capsule depth 13 10 18 1.8 13.99 Buccal capsule width 20 16 23 1.50 7.48 Distance to nerve ring 136 91 170 17.38 12.76 The same, % to oesophagus length 51.7 44.0 63.6 4.26 8.20 Oesophagus length 266 208 300 21.4 8.04 The same, % to body length 5.1 4.1 6.4 0.63 12.15 Oesophageal bulb width 59 40 72 7.6 12.84 Distance to vulva, mm 2.405 1.955 3.154 0.370 15.39 The same, % to body length 45.6 41.5 49.9 2.15 4.71 Tail length 167 91 224 33.14 19.82 The same, % to body length 3.2 2.0 4.1 0.55 17.10 * SD standard deviation ** CV coefficient of variation body. Outer surface of cuticle finely longitudinally striated. Mouth opening round. Cephalic lips prominent. Each lip having short, pointed papilla. Vestibulum short. Buccal capsule wide, cup-shaped, with sclerotized walls. Oesophagus relatively short, club-shaped, prominently widened in posterior part where oesophageal glands (one dorsal and two subventral) with distinct nuclei are situated. Nerve ring surrounding oesophagus slightly behind its middle. Excretory duct short and curved ventrally, situated near posterior edge 60

Kuzmin: Rhabdias agkistrodonis Table 2. Morphometry of free-living generation females of Rhabdias agkistrodonis (46 spec.). Distance to nerve ring - from anterior edge of oesophagus; stoma length - excluding cheilostoma. All dimensions in µm, unless otherwise indicated. Characters aver. min. max. SD CV Body length, mm 1.144 0.838 1.394 0.122 10.67 Body width 65 50 80 6.9 10.70 Stoma length 11 10 12 1 8.92 Distance to nerve ring 91 78 112 8.6 9.34 The same, % to oesophagus length 65.8 56.9 71.2 3.4 5.2 Oesophagus length 139 116 164 10.44 7.51 The same, % to body length 12.3 10.1 15.1 1.23 10.08 Oesophageal bulb width 24 20 26 1.4 6.09 Distance from anterior ovary loop to vulva 355 224 473 57.94 16.32 Distance from vulva to posterior ovary loop 325 207 456 57.6 17.69 Distance between ovary loops 680 448 904 113.2 16.63 The same, % to body length 59.3 48.3 69.6 5.6 9.46 Distance to vulva 595 464 713 56.2 9.43 The same, % to body length 52.2 49.7 61.7 1.8 3.40 Tail length 84 64 122 13.1 15.46 The same, % to body length 7.4 5.8 9.5 0.8 11.19 Number of eggs in anterior uterus 3.5 0 8 2.0 57.14 Number of eggs in posterior uterus 3.2 0 7 1.92 59.32 Number of eggs in both uteri 6.7 0 15 3.85 57.17 Table 3. Morphometry of free-living generation males of Rhabdias agkistrodonis (10 spec.). Distance to nerve ring - from anterior edge of oesophagus; stoma length - excluding cheilostoma. All dimensions in µm, unless otherwise indicated. Characters aver. min. max. SD CV Body length, mm 0.918 0.763 1.013 0.088 9.55 Body width 48 42 52 3.4 7.08 Stoma length 9 8 10 1.0 10.28 Distance to nerve ring 77 70 86 4.9 6.40 The same, % to oesophagus length 64.6 61.4 68.3 2.2 3.45 Oesophagus length 119 110 136 7.79 6.54 The same, % to body length 13.1 11.3 14.9 1.3 9.93 Oesophageal bulb width 20 18 22 1.333 6.67 Length of testis reflexed part 170 120 196 21.4 12.57 Length of straight part of genital system 648 539 722 53.1 8.20 The same, % to body length 70.8 64.7 77.4 3.1 4.40 Total length of genital system, % to body length 89.8 84.6 94.3 2.75 3.06 Tail length 55 40 62 6.34 11.53 The same, % to body length 6.0 5.2 6.5 0.45 7.48 Length of spicules 40 38 42 1.13 2.82 of nerve ring. Excretory glands long, wider posteriorly. Three large granulated subventral coelomocytes present in anterior part of body, two located near anterior ovary loop and one between anterior ovary loop and anterior uterus. Intestine wide, thick-walled. Rectum funnelshaped, with thick sclerotized walls and narrow lumen. Vulva pre-equatorial, lips distinct. Each lip with minute papilla, observed only in live specimens. Uteri wide, sac-like, filled with eggs, most containing larvae. Ovaries narrow, slightly twisted. Tail short, with awllike end formed by a short cuticular needle on the tip. Free-living females Fig. 2 B-E, Table 2 Maximum body width in region of vulva. Body cuticle thin, smooth, slightly swollen in caudal region. 61

Fig. 2. Free-living generation of Rhabdias agkistrodonis, adult stages: A free-living male, general view; B young free-living female, general view; C female with unfertilised eggs in uteri, general view; D gravid female, general view; E gravid female, anterior part of the body. Scale bars: A-D = 100 µm, E = 50 µm. 62

Kuzmin: Rhabdias agkistrodonis Fig. 3. Free-living larvae of Rhabdias agkistrodonis: A rhabditoid 1-stage larvae of the first generation, general view; B rhabditoid 1-stage larvae of the second generation, general view; C moulting L 1 of the second generation, general view; D rhabditoid 2-stage larvae, anterior part of the body; E heterogonic infective larvae, anterior part of the body; F homogonic infective larvae, anterior part of the body; G homogonic infective larvae, general view. Scale bars: 100 µm. 63

Table 4. Morphometry of Rhabdias agkistrodonis first-stage rhabditoid larvae (upper value - from parasitic generation, 68 spec.; lower value - from free-living generation, 25 spec.). Distance to nerve ring - from anterior edge of oesophagus; stoma length - excluding cheilostoma; distance to genital primordium - from anterior end of body. All dimensions in µm, unless otherwise indicated. Characters aver. min. max. SD CV Body length, mm 0.355 0.300 0.456 0.034 9.50 0.426 0.282 0.573 0.076 17.79 Body width 19 18 24 1.6 8.47 22 18 26 2.0 9.10 Stoma length 8 8 8 0 0 9.7 8 10 0.75 7.73 Distance to nerve ring 56 50 66 2.7 4.80 57 44 72 8.5 14.9 The same, % to oesophagus length 64.2 60.9 69.0 1.83 2.85 63.6 55.0 87.8 6.77 10.64 Oesophagus length 87 80 100 3.5 4.04 89 76 108 8.7 9.78 The same, % to body length 24.7 19.5 28.1 1.86 7.54 21.4 18.2 28.4 2.51 11.73 Oesophageal bulb width 12 10 16 1.3 10.62 14 12 16 1.2 8.83 Distance to genital primordium 187 158 224 15.3 8.17 231 158 307 38.2 16.52 The same, % to body length 52.9 47.3 55.3 1.50 2.84 54.4 52.4 57.1 1.20 2.21 Genital primordium length 28 14 62 8.18 28.99 9.5 8 10 0.87 9.16 Tail length 45 38 56 3.5 7.64 56 42 64 5.2 9.16 The same, % to body length 12.9 10.2 15.4 0.95 7.41 13.5 11.2 15.6 1.33 9.88 Stoma and oesophagus rhabditoid. Swelling of oesophageal corpus wide, with distinct muscular filaments. Oesophageal bulb round and valved. Nerve ring surrounding middle of isthmus. Excretory cells situated subventrally behind bulb of oesophagus. Vulva near middle of body or (in the oldest females) slightly behind it. Genital system amphidelphic, its anterior limb being slightly longer than posterior one. Uteri wide and thinwalled. Eggs arranged in one row, in various stages of development. Some females with unfertilised egg cells in uteri (Fig. 2 C) occurred in the cultures. In old females, seminal receptacles and distal parts of ovaries deformed by enlarged uteri filled with eggs. Anterior ovary situated to the right and posterior one to the left of intestine. Tail conical, elongated and sharply pointed. Free-living males Fig. 2 A, Table 3 Morphology of the anterior part of the body is like that in females. Intestine walls thinner than those in females. Testis relatively long. Anterior part of testis curved backward and situated to right of intestine. Ejaculatory duct with two anterolateral diverticuli. Sperm cells oval-shaped, about 8 µm in length. Spicules short, equal, slightly curved ventrally. Seven pairs of ventrolateral genital papillae (3 pairs preanal and 4 pairs postanal) supporting reduced genital bursa, and 1 unpaired ventral adanal papillae present. Tail short, conical, with tapered end. Rhabditoid larvae Fig. 3 A-D, Table 4 Maximum width in region of oesophageal bulb. Stoma narrow, indistinct in L 1. Oesophagus rhabditoid, bulb oval. Intestine wide, thick-walled, with distinct lumen widened in anterior part. Ten pairs of intestine cell nuclei visible in lateral view. Lens-like genital primordium situated near middle of body close to ventral wall of intestine. Genital primordium differentiated into male and female reproductive systems in heterogonic larvae of amphimictic generation but remaining small and undifferentiated in homogonic larvae and in rhabditoid heterogonic larvae of hermaphrodite generation. Rapid growth of the genital 64

Kuzmin: Rhabdias agkistrodonis Table 5. Morphometry of Rhabdias agkistrodonis infective larvae (upper value - homogonic, 7 spec.; lower value - heterogonic, 12 spec.); distance to nerve ring - from anterior edge of oesophagus; stoma length - excluding cheilostoma; distance to genital primordium - from anterior end of body. All dimensions in µm, unless otherwise indicated. Characters aver. min. max. SD CV Body length, mm 0.854 0.830 0.880 0.017 1.98 0.847 0.788 0.938 0.043 5.12 Body width 33 30 34 1.5 4.64 31 28 34 1.6 5.09 Stoma length 15.7 14 16 0.8 4.81 15.8 14 16 0.6 3.65 Distance to nerve ring 85 80 88 3.207 3.792 84 76 90 5.060 6.052 The same, % to esophagus length 69.5 65.1 73.3 3.20 4.60 50.3 43.3 56.9 3.79 7.55 Esophagus length 122 118 126 2.7 2.21 170 144 190 11.5 6.80 The same, % to body length 14.3 13.7 14.7 0.40 2.78 20.1 15.4 23.4 2.13 10.60 Esophageal bulb width 15 14 16 1.1 7.06 14 14 14 0 0 Distance to genital primordium 453 432 465 11.6 2.56 460 415 531 32.5 7.07 The same, % to body length 53.1 51.9 54.4 0.92 1.73 54.3 51.9 56.6 1.38 2.54 Genital primordium length 34 30 38 3.06 8.98 34 30 40 3.39 9.88 Tail length 78 70 88 6.3 8.00 88 80 92 3. 6 4.06 The same, % to body length 9.2 8.4 10.6 0.80 8.77 10.4 8.5 11.4 0.73 7.03 primordium starting after first moult of rhabditoid larvae; before this time impossible to distinguish homoand heterogonic larvae. Rectum thin-walled, slightly twisted. Tail conical, elongated and tapered. Homogonic infective larvae Fig. 3 E-G, Table 5 Body covered with sheath. Outer surface of sheath smooth, inner surface transversely striated. Stoma narrow, elongated, rhabditoid in shape. Oesophagus with distinct metacorpus and bulb. Nerve ring in middle of isthmus. Intestine wide and straight, without distinct lumen. Rectum straight and thin-walled. Genital primordium situated behind middle of body. Tail end of sheath conical and sharply pointed, tail tip of larva rounded. Heterogonic infective larvae Fig. 3 B,C; Table 5 They differed from homogonic ones in the cylindrical shape of stoma and more elongated oesophagus with indistinct metacorpus and bulb lacking valve. Hosts and localities. Short-tailed viper (Agkistrodon blomhoffi) from Kedrovaya Pad reserve, Ussurijskii reserve and Olga town vicinity (Primorskii Krai, Russia); Halys viper (Agkistrodon halys) from Altaiskii reserve (Altaiskii Krai, Russia); Trimeresurus elegans and Okinawa habu (T. flavoviridis) from Okinawa Island (Japan). All known hosts belong to the subfamily Crotalinae, family Viperidae. DISCUSSION Our studies of available material on 7 Rhabdias species (R. agkistrodonis, R. elaphe, R. fuscovenosa, R. horigutii, R. kurilensis, R. martinoi and R. vibakari) from Palaearctic snakes have shown that only R. agkistrodonis has an awl-like tail tip. R. agkistrodonis may be also distinguished from R. fuscovenosa by the larger number of eggs (about 100) in the uteri of gravid parasites and by predominantly heterogonic development. Samples of R. agkistrodonis specimens were generally similar in various hosts. Specimens from Trimeresurus spp., however, appeared to be smaller than those from Agkistrodon spp. Bogdanov et al. (1969) described Rhabdias species from A. halys from Kazakhstan and Russian Far East as R. annulosa Hsu, 1933 which has been synonymized with R. fuscovenosa by Chu (1936a). The authors stated 65

that their material differed from R. fuscovenosa in the shape of buccal capsule (cup-like, not funnel-shaped), tail length and specificity. We believe that the worms described by Bogdanov et al. (1969) belong to R. agkistrodonis. Acknowledgements. Author wishes to give sincere thanks to Dr. Hideo Hasegawa, who has kindly send the material from his collection, and to Nikolai Barvinskii for his help in experimental work. REFERENCES BOGDANOV O.P., LUTTA A.S., MARKOV G.S., RYZHIKOV K.M. 1969: New data on fauna of parasites of the Pallasian mumushi Ancistrodon halys (Reptilia, Crotalidae). Zool. Zh. 48: 179-183. (In Russian.) CHU T. 1936a: A review of the status of the reptilian nematodes of the genus Rhabdias with a description of Rhabdias fuscovenosa var. catanensis (Rizzo, 1902) new rank. J. Parasitol. 22: 130-139. CHU T. 1936b: Studies on the life history of Rhabdias fuscovenosa var. catanensis (Rizzo, 1902). J. Parasitol. 22: 140-160. KUZMIN Yu. 1996: The free-living development and types of life cycles of rhabdiasid nematodes (Nematoda: Rhabdiasidae) from Palaearctic region. In: Proceedings of the Jubilee Conference of the Ukrainian Scientific Society of Parasitologists Parasitology in the Ukraine. Yesterday, Today and Tomorrow, Kiev, May 16-17, 1995, pp. 133-138. SHARPILO V.P. 1976: Parasitic Worms of Reptiles of the Fauna of the USSR. Naukova dumka, Kiev, 287 pp. (In Russian.) Received 17 April 1998 Accepted 25 August 1998 66