Journal of Helminthology (1979) 53, 51-63
Strigeoid trematodes of Malaysia with descriptions of a new genus and three new species JAMES R. PALMIERI, M. KRISHNASAMY and JOHN T. SULLIVAN University of California ICMR, and Division of Medical Ecology, Institute for Medical Research, Kuala Lumpur, Malaysia* ABSTRACT Six species of strigeoid trematodes are reported from Malaysia. One new genus and 3 new species are described: Apatemon (Apatemon) jamesi sp. n. (Strigeidae); cercaria Cotylurus sullivani sp. n. (Strigeidae); Neodiplostomum (Neodiplostomum) sp. (Diplostomatidae); Fibricola ramachandrani (Diplostomatidae); Pseudoscolopacitrema otteri gen. n. et sp. n. (Diplostomatidae); and cercaria Cyathocotyle malayi sp. n. (Cyathocotylidae). The life cycles of A. jamesi and C. malayi have also been investigated.
The species of strigeoid trematodes were found during examination of 17,452 vertebrate hosts and several hundred snails collected from January 1964 through June 1977. All three families of the order Strigeatida were found. Vertebrate hosts were live-trapped when possible and returned to the laboratory for examination. Specimens were dissected from the hosts and observed alive, then fixed in AFA. Additional specimens were obtained from collections of worms that had been fixed and transferred to 70 % ethanol for storage. All specimens were stained with Mayer's paracarmine, counter-stained with fast green, dehydrated in ethanol, cleared in methyl salicylate and mounted in Permount. All measurements are in micrometers unless otherwise indicated. Measurements are expressed as an average followed by the range in parentheses. Drawings were made with the aid of a Bausch-and-Lomb microprojector, drawing tube and enlarged photographs. Since forebody width varies with the state of muscular contraction, only hindbody width is reported. Classification of all worms is based on the system presented by Dubois (1968 and 1970). Type specimens have been deposited in the USNM Helminthological Collection and the Division of Medical Ecology, Institute for Medical Research, Kuala Lumpur, Malaysia. Apatemon (Apatemon) jamesi sp. n.
(Strigeidae, Strigeinae, Cotylurini) (Figs. 1-2) Diagnosis: (62 specimens; 15 gravid adults measured). Length 1.34 (1.12-1.52) mm, body curved dorsally. Forebody hemispherical, truncated anteriorly, 0.25 (0.18-0.40) mm long; hindbody elongate, roughly cylindrical, curved dorsally, forms right angle with forebody when contracted, 0.87 (0.80-1.04) mm long. Oral sucker subterminal 83 (78-87) long by 85 (78-97). Prepharynx absent; pharynx continuous with oral sucker, 43 (37—51) long by 44 (37-51) wide. Oesophagus 30 (23-37) long. Gut caeca reaching posterior extremity; acetabulum larger than oral sucker, 99 (74-147) long by 110 (92-129) wide. Proteolytic glands at junction of forebody and hindbody, 50 (41-55) long by 69 (46-83) wide. Tribocytic or adhesive organ two-lobed, lying within the forebody, lobes when withdrawn 158 (129193) long by 180 (124-276) wide. Vitellaria abundant in hindbody, following dorsal curvature of body extending 90-95 % of hindbody length. Ovary pretesticular, round to subcircular, 95 (87-110) long by 84 (60-101) wide. Uterus extending from ovary posteriorly to junction of ejaculatory duct where it becomes the hermaphroditic duct. Mehlis' gland lateral to posterior-lateral to ovary. Laurer's canal present, opening on body surface, ventral and posterior to ovary. Testes tandem in posterior half of body, lobed, square to butterfly-shaped; anterior testis 169 (129-221) long by 156 (115-212) wide; posterior *Reprint address: Editor, G. W. Hooper Foundation, HSW 1699, University of California, San Francisco, California 94143, U.S.A. 51
J. R. PALMIERI, M. KRISHNASAMY and J. T. SULLIVAN
testis 152 (120-239) long by 147 (83-193) wide. Vitelline reservoir between anterior and posterior testis, 66 (51-83) long by 58 (37-69) wide. Seminal vesicle posterior to posterior testis, 91 (69-115) long by 91 (69-110) wide. Eggs usually less than 10 in number, 89 (8292) long by 53 (46-60) wide. Genital cone not delimited from surrounding parenchyma, opening into terminal bursa, not strongly muscular. This species is named in honour of Dr. Hugo A. James. Natural first intermediate host: Lymnaea rubiginosa Michelin. Natural second intermediate hosts: Hirudinaria manillensis Lesson, Dinobdella ferox Blanchard. Natural definitive host: Anas platyrhynchos Linn. Location: Upper to middle small intestine. Type Locality: Kuala Pilah, Negri Sembilan, West Malaysia. Type Specimens: USNM Helm. Collection. Holotype: No. 73048. Paratypes: No. 73049. Remarks
A survey of the parasitic fauna of domestic ducks {Anas platyrhynchos) from Kampung Panglang, Sri Menanti Town, Kuala Pilah, Negri Sembilan, revealed 29 out of 37 ducks positive for Apatemon jamesi. Ducks, bred in the rice fields surrounding the kampung, only return to the kampung at night for shelter. Water buffalo (Bos bubalus Linn), also inhabit the fields. Since the snail and leech commonly serve as the first and second intermediate host, respectively, for Apatemon (McDonald, 1969; Palmieri, 1973) a search for these hosts in this area was conducted. Of the 748 Lymnaea rubiginosa examined, 21 were positive for sporocysts and cercariae of Apatemon (Palmieri et ah, 1977). Buffalo leeches, identified as Hirudinaria manillensis Lesson and Dinobdella ferox Blanchard (Sharma and Fernando, 1961), collected from water buffaloes and from pools which contained L. rubiginosa, were found positive for metacercariae similar to the Apatemon type reported by Palmieri and James (1976b). Leeches naturally infected with metacercariae and fed to laboratory-reared ducks produced trematodes morphologically similar to Apatemon jamesi. Eggs, collected from living gravid worms, were incubated at 26°C for 18 days until hatching occurred. Laboratory-reared L. rubiginosa were exposed to miracidia and were placed in aquaria until they shed furcocercous cercariae approximately 4 weeks postexposure. Snails were allowed to shed cercariae for 10 days in an aquarium containing laboratory-reared leeches (Dinobdella ferox). Cercariae were observed penetrating the leeches and eliciting a typical behavioural reaction as reported by Palmieri and James (1976a). At 14 days postexposure leeches were fed to laboratory-reared ducks. At 3 days postfeeding, ducks were examined and trematodes which morphologically resembled Apatemon jamesi were recovered from the small intestine. We have chosen to use Dubois' (1968) taxonomic system that divides the genus Apatemon Szidat, 1928 into two subgenera (Apatemon) Szidat, 1928 and (Australapatemon) Sudarikov, 1959. Apatemon jamesi is most closely aligned with subgenus (Apatemon). We consider Apatemon jamesi to be a new species with a life cycle typical for the genus. Cercaria Cotylurus sullivani sp. n.
(Strigeidae, Strigeinae, Cotylurini) (Fig. 3) Diagnosis: (20 specimens measured) Furcocercous, pharyngeate, distomous, longifurcate cercariae developing in nature in Melanoides tuberculata Miiller, 1774. Body 170 (156-183) long by 58 (54-66) wide. Tail stem 200 (186-225) long by 38 (36-45) wide; tail furca (right) 138 (114-156), (left) 138 (111-150). 52
o
FIG. 1. Drawing of a wholemount of the type specimen of Apatemon (Apatemon) jamesi sp. n. (Trematoda: Strigeidae) recovered from the intestine of the duck, Anas platyrhynchos. FIG. 2. Drawing of a lateral section of Apatemon (Apatemon) jamesi showing internal morphology. FIG. 3. Drawing of cercaria Cotylurus sulliyani sp. n. (Trematoda: Strigeidae) recovered from the shedding snail, Melanoides tuberculata. FIG. 4. Drawing of a wholemount of Neodiplostomum (Neodiplostomum) sp. (Trematoda: Diplostomatidae) recovered from the owl Ketupa ketupu. 53
J. R. PALMIERI, M. KRISHNASAMY and J. T. SULLIVAN
Two pairs of penetration glands, first pair at level of acetabulum, second pair postacetabular; ducts opening into oral cavity. Mouth terminal. Anterior organ 35 (33-42) long by 28 (27-30) wide surrounded by 26-30 fine spines. Acetabulum muscular 21.5 (21-24) long by 25 (24-27) wide. Mouth leading to a well-developed pharynx 12-15 in diameter. Oesophagus long 37 (32^0); caecal bifurcation halfway between pharynx and acetabulum. Gut caeca long, narrow and regular in outline extending to excretory bladder. Five pairs of flame cells in body and 3 pairs in tail stem, total 4 pairs per side or 16 in all. Posterior bladder distinctly bipartite. Fourteen pairs of lateral sensory hairs on tail stem, 2 pairs per side at the anterior tail stem and 5 pairs per side on the lower 1/3 of tail stem. Genital primordium conspicuous, between gut caeca and excretory bladder. Furca 85% body length. Excretory duct passing through tail stem, bifurcating at caudal fork, opening on posterior edge of each furca less than halfway down its length. Natural host: Melanoides tuberculata Miiller, 1774 Type locality: Klang Gates, Malaysia Remarks
During spring, 1975, James J. Sullivan of the University of California, International Centre for Medical Research, Kuala Lumpur, collected a strigeid cercaria from the operculate snail Melanoides tuberculata Miiller, 1774 at Klang Gates, Malaysia. From examination of data on organ morphology and detailed drawings compiled by him, we have tentatively identified this cercaria as being closely related to those belonging to the genus Cotylurus Szidat, 1928 and have tentatively named it cercaria Cotylurus sullivani sp. n. after Dr. James J. Sullivan. The description reported here is given with his permission. Neodiplostomum (Neodiplostomum) sp.
(Diplostomatidae, Diplostomatinae, Diplostomatini) (Fig. 4) Diagnosis: (1 specimen recovered and measured). Body 2.15 mm long; forebody 1.10 mm long; hindbody 1.05 mm long by 0.32 mm wide. Forebody lightly spinose, spoonshaped ; hindbody cylindrical and aspinose; forebody approximately same length as hindbody. Oral sucker subterminal, 83 long by 78 wide. Prepharynx 9 long. Pharynx well developed, 55 long by 46 wide. Oesophagus 23 long. Acetabulum muscular, 78 long by 97 wide. Holdfast organ oval, 290 long by 179 wide, with a median ventral slit. Ovary large, round to triangular on right-hand side below junction of hindbody and just anterior to anterior testis, 170 long by 611 wide. Uterus runs anteriorly from ovary and turns back upon itself at the forebody-hindbody junction where it then proceeds posteriorly to the genital pore. Vitelline reservoir intertesticular, 37 in diameter. Anterior testis dumbell- or butterfly-shaped with deep posterior cleft, 244 long by 282 wide. Seminal vesicle not seen. Vitelline glands in forebody extend midway between the caecal bifurcation and acetabulum in 4 longitudinal bands with heavy concentrations between the acetabulum and holdfast organ; in the hindbody the vitelline glands are large with 2 dense lateral fields from the posterior edge of the holdfast to the posterior edge of the ovary and also from the posterior edge of the posterior testis to the genital pore. No eggs were found. Natural host: Ketupa ketupu Glenister 1974 Location: Mid-small intestine Type locality: Perak, Malaysia Type specimen: USNM Helm. Coll. Holotype: No. 73050. 54
Strigeoid trematodes in Malaysia Remarks
This single specimen was recovered from the intestine of the fish owl {Ketupa ketupu) commonly found feeding on fish near rice fields along the banks of streams in inland areas (Glenister 1971). The description of Neodiplostomum {Neodiplostomum) sp. is based on a single specimen, and hence we do not feel justified in giving it a species name. Morphologically it resembles several reported species of Neodiplostomum yet its characteristics separate it from any single reported species. Although Ketupa ketupu has never been reported as host for trematodes of the superfamily Strigeoidea Railliet, 1919, Dubois (1970) lists Strigiformes (owls) as hosts for trematodes of the genus Neodiplostomum. N. {Neodiplostomum) sp. morphologically resembles N. {Neodiplostomum) americanum (Chandler et Rausch, 1947) Dubois et Rausch, 1948, the latter also being found in several genera of Strigiformes hosts. TV*. {Neodiplostomum) americanum has been reported from 7 hosts, 4 of which belong to the Strigiformes. However, N. {Neodiplostomum) sp. differs from A". {Neodiplostomum) americanus in having a forebody approximately the same size as its hindbody. N. {Neodiplostomum) reflexum Chandler et Rausch, 1947 also morphologically resembles N. {Neodiplostomum) sp. in having similar body and organ sizes as well as having 4 genera of Strigiformes as hosts (Dubois, 1970) but differs in having the vitellaria extend anteriorly only as far as the level of the acetabulum. Because body size, testis size and shape, vitellaria extent and attachment, and organ size and shapes can vary under the influences of host-induced morphological variation (Palmieri 1976, 1977 a-c), we find it difficult to place N. {Neodiplostomum) sp. into a specific category and therefore leave it without specific name until either more specimens are found or life cycle data compiled. Fibricola ramachandrani
(Betterton, 1976) Palmieri, Krishnasamy and Sullivan (Diplostomatidae, Alariinae, Alariini) Palmieri et al. (in press) reported trematodes resembling Neodiplostomum {Conodiplostomum) ramachandrani Betterton, 1976 from four rodent hosts Echinosorex gymnurus Raffles, Rattus whiteheadi Thos, R. muelleri Jentink, and Callosciurus notatus (Boddaert). After a review of the taxonomy of Neodiplostomum and a careful examination of type and paratype specimens of N. {Conodiplostomum) ramachandrani, Palmieri et al. (in press) renamed this trematode Fibricola ramachandrani (Betterton, 1976) Palmieri, Krishnasamy and Sullivan. F. ramachandrani is highly variable morphologically, undergoes host-induced morphological variation, and is host-nonspecific. We refer the reader to Palmieri et al. (in press) for details on Fibricola ramachandrani. Pseudoscolopacitrema gen. n. Diagnosis: Diplostomatidae, Diplostomini, Crassiphialini, with pseudosuckers. Body clearly divided into two regions, arched dorsad. Forebody shorter than hindbody. Tribocytic organ with two fan- or wing-like lobes, occupying major portion of concavity of forebody. Testes large, lobed, tandem asymmetrical, seminal vesicle intratesticular. Ovary pretesticular, round to oval. Uterus reaching from genital atrium and proceeding anterior to ovary. Muscular sphincter at terminal of body joining uterus. Type species: Pseudoscolopacitrema otteri sp. n. Pseudoscolopacitrema otteri gen. n. et. sp. n. (Figs. 5-7) Diagnosis: (5 specimens; 4 gravid adults measured). Length 4.34 (3.78-5.09) mm, body divided into two regions. Strongly recurved forebody 0.99 (0.87-1.20) mm 55
J. R. PALMIER1, M. KRISHNASAMY and J. T. SULLIVAN
FIG. 5. Drawing of a wholemount of the lateral view of Pseudoscolopacitrema otteri gen. n. et sp. n. recovered from the otter Amblonyx cinerea. FIG. 6. Drawing of the anterior region of Pseudoscolopacitrema otteri gen. n. et sp. n. showing the well developed pseudosuckers. FIG. 7. Drawing of the muscular sphincter at the terminal of the genital atrium of Pseudoscolopacitrema otteri gen. n. et. sp. n. 56
Strigeoid trematodes in Malaysia
long. Hindbody conical, widening posteriorly, 3.35 (2.73-4.14) mm long by 0.54 (0.440.59) mm wide. Tegument aspinose. Oral sucker terminal, 100 (92-104) long by 114 (106120) wide. Pseudosuckers muscular and well developed, lateral and posterior to pharynx, 151 (138-184) long by 93 (87-101) wide. Tribocytic or adhesive organ with two large fanor wing-like lobes 481 (423-580) long by 400 (304-589) wide, lobes never observed in retracted state. Vitellaria abundant in forebody and hindbody from base of oral sucker to level of ovary. Ovary pretesticular, round to oval, 154 (143-166) long by 180 (170-189) wide. Uterus extending from 184-322 anterior to ovary to posterior genital atrium with a muscular sphincter 92 (86-101) long by 110 (92-119) wide. Mehlis' gland lateral to ovary. Eggs usually under 40 in number, operculate, well embryonated, 107 (92-129) long by 59 (51-74) wide. Two testes lobed, asymetrical and tandem; anterior testis 373 (332-409) long by 405 (378-459) wide; posterior testis 290 (240-329) long by 368 (342-396) wide. Seminal vesicle large, elongate, intertesticular. Natural definitive host: Amblonyx cinerea (Illiger) Location: Small intestine Type locality: Zoo Negara, Kuala Lumpur, Malaysia Type specimen: USNM Helm. Coll. Holotype: No. 73047 Remarks Of the 17,452 hosts examined only one individual small-clawed otter {Amblonyx cinerea) was collected. Examination of its intestinal contents revealed four gravid and one mature trematodes of the family Diplostomatidae. Because of the large fan- or wing-like tribocytic organ, recurved forebody and long slightly conical hindbody, these specimens superficially resemble specimens of the genus Crassiphiala van Haitsma, 1925 and Scolopacitrema Sudarikov and Rykovskii, 1959. Careful examination of Pseudoscolopacitrema otteri shows the presence of 2 well developed pseudosuckers, a well developed acetabulum, vitellaria extending into the forebody and the presence of a large muscular sphincter at the posterior junction of the uterus. Neither Crassiphiala nor Scolopacitrema have 2 pseudosuckers; the former also lacking a well developed muscular acetabulum as well as vitellaria extending into the forebody. Scolopacitrema does have vitellaria extending into the tribocytic organ and a sucker-like genital atrium which is similar to Pseudoscolopacitrema but lacks its well developed muscular pseudosuckers. Based on the above we consider these worms to be of a new genus Pseudoscolopacitrema, the name based upon its similarity to Scolopacitrema, the species name otteri being descriptive of the type host. Cercaria Cyathocotyle malayi sp. n. (Cyathocotylidae) (Figs. 8-13) Over 300 Bellamya sumatrensis Dunker, collected from the Kelang River drainage, Kelang Gates, Malaysia, were heavily infected with an unreported cercaria of the family Cyathocotylidae that encysted as tetracotyle-type metacercariae on the fins and in the musculature of freshwater fish. Field-collected Bellamya sumatrensis with advanced infections of the cyathocotylid cercariae were placed in aquaria with 10 red-eyed red swordtail fish (Xiphophorus hellerii) and allowed to shed. After 48 hours, the cyathocotylid-exposed fish were separated and maintained 3-4 weeks to ensure complete development of encysted tetracotyle larvae within the musculature and fins. 57
J. R. PALMIERI, M. KRISHNASAMY and J. T. SULLIVAN
EP
FIG. 8. Drawing of the daughter sporocyst of cercaria Cyathocotyle malayi sp. n. (Trematoda: Cyathocotylidae) recovered from the snail Bellamya sumatrensis. FIG. 9. Drawing of cercaria Cyathocotyle malayi sp. n. (Trematoda: Cyathocotylidae) recovered from the shedding snail Bellamya sumatrensis.
Five species of fish were found naturally infected with metacercariae of Cyathocotyle malayi while Barbus binotatus and Xiphophorus hellerii were experimentally infected with metacercariae (Table 1). Naturally and experimentally infected fish were cut into 1 cm2 pieces and held in saline solution for feeding experiments. Six species of reptiles, 2 birds and 3 mammals were experimentally fed metacercariae but no adult cyathocotylid trematodes were ever recovered (Table 2). Daughter sporocyst: (Fig. 8) Elongate opaque vermiform sacs from 0.6-0.8 mm long and 0.03-0.04 mm wide. Birth pore near anterior end. Interior filled with germ balls and developing daughter sporocysts. Body wall lightly muscular, lined by tegument, and 2-2.5 thick. 58
Strigeoid trematodes in Malaysia
Mother sporocysts developed in the digestive gland of the snail and appeared to move with a twisting motion when dissected from snail. In heavy infections the mother sporocysts extended along the oesophagus and intestine of the snail. TABLE 1 Fish infected with tetracotyles of Cyathocotyle malayi
Host Barbus binotatus B. fasciatus B. lateristriga B. partipentazona Trichogaster trichopterus Xiphophorus hellerii
Experimental infection
Natural infection
X
X X X X X
X
Cercaria: (Fig. 9) Furcocercous, nonoculate, pharyngeate and monostomate; without acetabulum or rudimentary acetabulum. Body 276 (252-312) long by 122 (108-150) wide; tail stem 370 (336-432) by 44 (36-54); furcae 278 (201-336); oral sucker 70 (57-84) by 44 (36-54); pharynx 18.3 (15-21) by 21.6 (18-27). Tegument without thornlike spines; body and tail stem without sensory hairs. One pair of cephalic glands consisting of 9 cells; their ducts opening lateral to the mouth; oral sucker oval; mouth leading to a small tubular prepharynx; pharynx wider than long; oesophagus 3 times the length of the pharynx; intestine with sinuous walls, reaching posterior of body. Genital primordium present intercaecally. One pair of excretory ducts lateral to the intestine; median longitudinal commissure present; excretory duct extends from tail stem through furcae to a terminal excretory pore; excretory vesicle in anterior tail stem; flame cell formula 2 [ l + 3 + 3 (3+3)]=26. Tail without finfold, muscular and bent. Behaviour of the cercaria: Furcocercous cercariae appeared 32 days postinfection when snails were maintained at 27-29° C. Cercariae were shed in large numbers from early morning to midaftemoon. At rest, cercariae slowly sank toward the bottom with furcae upward for 7-13 seconds. When swimming, cercariae spun in an upward clockwise spiral direction around a central axis. Cercaria either made a 1-coil spin taking 3-5 seconds or a 3-coil spin taking 7-13 seconds for a distance of 1.5-2 cm (Fig. 10). Cercariae were never observed crawling or attached to either the bottom or sides of the container. Cercariae showed no phototropic preference and swam toward light or dark. When observed from above, the furcae could be clearly seen and obstructed the view of the tail stem and body (Fig. l i b and c). When viewed from the side, the cercaria had a characteristic bend in the tail stem of approximately 140° (Fig. l l a ) . One hundred cercariae were placed in a 30 cm 500-ml graduated cylinder to observe swimming and resting positions. After 30 min most cercariae were below the 100-ml mark (6 cm) (Fig. 12). Tetracotyle (Fig. 13): Encysted, fully formed, round tetracotyles found either on the scales or in the fins or musculature of fish. Cyst delicate, clear, and about 2 thick leaving no trace after routine paraffin sectioning except for a layer of host cells that covered cysts in muscle tissue. Excysted tetracotyles oval to pear-shaped usually 416-442 long by 214242 wide although varying somewhat in size depending on crowding and age. No body spines present. Distinct forebody and weakly developed hindbody. Genital anlage recognizable just below the holdfast organ. Oral sucker muscular and well developed measuring 60 (53-62) long by 72 (62-80) wide. Muscular pharynx well 59
J. R. PALMIERI, M. KRISHNASAMY AND J. T. SULLIVAN
10 B
1 2.0 in.
. 500 m I.
.400
300
200
.100
FIG. 10. Swimming behaviour of cercaria Cyathocotyle malayi sp. n. a. A one-coil upward spin taking from 3 to 5 seconds to complete. b. A three-coil upward spin taking 7 to 13 seconds to complete. FIG. 11. Different views of a suspended cercaria Cyathocotyle malayi sp. n. a. Side view, b-c. Top view looking down on posterior region. FIG. 12. One hundred cercaria Cyathocotyle malayi sp. n. suspended in 30 cm. 500-ml graduate cylinder after 30 minutes, note: most cercariae are below the 100-ml mark. 60
Strigeoid trematodes in Malaysia
developed. Virtually no oesophagus present. Caeca reaching to midgenital anlage. Holdfast organ measuring 40-43 in diameter. Unicellular cephalic glands present on either side of the pharynx and oral sucker. Infection of 2nd intermediate host: Cercariae actively penetrated and developed into tetracotyles in a variety of fish (Table 1). At Kelang Gates, where about 1 % of all B. sumatrensis were infected with C. malayi, all fish sampled were infected with at least 60 tetracotyles of C. malayi.
13
FIG. 13. Drawing of a wholemount of the excysted tetracotyle of cercaria Cyathocotyle malayi sp. n. recovered from the musculature of a fish.
If a suceptible fish was placed in a beaker of water containing active cercariae, penetration began immediately. Cercariae initially became trapped on the body mucus where they crawled under scales or into the fins and encysted. In most cases cercariae penetrated the musculature. In cases of heavy infection (over 200 tetracotyles/fish (6-10 cm)) tetracotyles were commonly found grouped. In light infections, tetracotyles were found evenly spaced apart. Dead tetracotyles were opaque and yellow. 61
J. R. PALMIERI, M. KRISHNASAMY and J. T. SULLIVAN TABLE 2 Hosts exposed to tetracotyles of Cyathocotyle malayi Number exposed
Host Reptilia Calotes sp. Cuora amboinensis Geomyda bispinose Mabola multifasciata Trionyx hurum Varanus salvator
1 4 4 8 3 1
Aves Anas platyrhynchos Gallus domesticus
17 10
Mammalia Canis familiaris Felis catus
6 1
Macaca nemestrina
1
Remarks Members of the Cyathocotylidae are found in reptilian, avian and mammalian hosts (Yamaguti, 1971). Within the 6 subfamilies of the Cyathocotylidae both cyathocotylinae and prohemistominae have reptilian, avian and mammalian definitive hosts; szidatinae are found in both reptilian and avian hosts; prosostephaninae in both reptilian and mammalian hosts; pseudohemistominae only in avian and muehlingininae only in mammalian hosts. Subfamilies prohemistominae, muehlinginiane and prosostephaninae all contain members which lack an acetabulum, a character held in common with Cyathocotyle malayi. Because a natural or experimental definitive host was never found and since the cercarial stage of C. malayi does not morphologically or behaviorally resemble reported species of Cyathocotylid cercariae, we find it difficult to place this cercaria within a subfamily of cyathocotylidae until further information is available concerning the adult stage in the life cycle. ACKNOWLEDGEMENT This study was supported in part by grant AI 10051 (UC ICMR) to the Department of Epidemiology and International Health, School of Medicine, University of California, San Francisco, from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, U.S. Public Health Service. REFERENCES BETTERTON, C. (1976) Neodiplostomum (Conodiplostomum) ramachandrani sp. n. from Mueller's rat Rattus muelleri in Malaysia. Journal of Helminthology, 50, 157-161. DUBOIS, G. (1968) Synopsis des Strigeidae et des Diplostomatidae (Trematoda). Societe Neuchateloise des Sciences Naturelles, pp. 1-258. DUBOIS, G. (1970) Synopsis des Strigeidae et des Diplostomatidae (Trematoda). Sociite Neuchateloise des Sciences Naturelles, pp. 259-727. GLENISTER, A. G. (1974) The birds of the Malay Peninsula, Singapore and Penang. Oxford University Press, Ely House, London, 291 pp. McDONALD, M. E. (1969) Catalogue of helminths of waterfowl (Anatidae). Bureau of sportfisheriesand wildlife. Special Scientific Report—Wildlife No. 126. Washington D.C. PALMIERI, J. R. (1973) Additional natural and experimental hosts and intraspecific variation in Posthodiplostomum minimum (Trematoda: Diplostomatidae). Journal of Parasitology, 59, 744-746. 62
Strigeoid trematodes in Malaysia PALMIERI, J. R. (1977) Host-induced morphological variations in the strigeoid trematode Posthodiplostomum minimum (Trematoda: Diplostomatidae). II. Body measurements and tegument modifications. Great Basin Naturalist, 37: 129-137. PALMIERI, J. R. (1977) Host-induced morphological variations in the strigeoid trematode Posthodiplostomum minimum (Trematoda: Diplostomatidae). III. Organs of attachment. Great Basin Naturalist, 37, 375-382. PALMIERI, J. R. (1977) Host-induced morphological variations in the strigeoid trematode Posthodiplostomum minimum (Trematoda: Diplostomatidae). IV. Organs of reproduction (ovary and testes), vitelline gland, and egg, Great Basin Naturalist, 37, 481-488. PALMIERI, J. R. and JAMES, H. A. (1976a) The effects of leech behaviour on penetration and localization of Apatemon gracihs (Trematoda: Strigeidae) cercariae and metacercariae. Great Basin Naturalist, 36, 97-100. PALMIERI, J. R. and JAMES, H. A. (1976b) Metacercarial composition and development in Apatemon gracilis (Trematoda: Strigeidae). Iowa State Journal of Research, 50, 409^417. PALMIERI, J. R., KRISHNASAMY, M. and SULLIVAN, J. T. (In Press). Fibricola ramachandrani (Betterton, 1976) Palmieri, Krishnasamy and Sullivan comb. nov. from Malaysian rodent hosts with special note on intraspecific morphological variation. Journal of Helminthology. PALMIERI, J. R., SULLIVAN, J. T. and OW-YANG, C. K. (1977) A survey of snail hosts and larval trematodes collected from Peninsular Malaysia and Singapore during 1972 to 1977. The Southeast Asian Journal of Tropical Medicine and Public Health, 8, 275-277. SHARMA, R. E. and FERNANDO, C. H. (1961) Leeches and their ways. The Malayan Nature Journal, 15, 152-159. YAMAGUTI, S. (1971) Synopsis ofdigenetic trematodes of vertebrates. Vol. I and II. Keigaku Publishing: Tokyo. 1074 pp. Received 20 October, 1977.
63
Strigeoid trematodes of Malaysia with descriptions of a new genus and three new species JAMES R. PALMIERI, M. KRISHNASAMY and JOHN T. SULLIVAN University of California ICMR, and Division of Medical Ecology, Institute for Medical Research, Kuala Lumpur, Malaysia* ABSTRACT Six species of strigeoid trematodes are reported from Malaysia. One new genus and 3 new species are described: Apatemon (Apatemon) jamesi sp. n. (Strigeidae); cercaria Cotylurus sullivani sp. n. (Strigeidae); Neodiplostomum (Neodiplostomum) sp. (Diplostomatidae); Fibricola ramachandrani (Diplostomatidae); Pseudoscolopacitrema otteri gen. n. et sp. n. (Diplostomatidae); and cercaria Cyathocotyle malayi sp. n. (Cyathocotylidae). The life cycles of A. jamesi and C. malayi have also been investigated.
The species of strigeoid trematodes were found during examination of 17,452 vertebrate hosts and several hundred snails collected from January 1964 through June 1977. All three families of the order Strigeatida were found. Vertebrate hosts were live-trapped when possible and returned to the laboratory for examination. Specimens were dissected from the hosts and observed alive, then fixed in AFA. Additional specimens were obtained from collections of worms that had been fixed and transferred to 70 % ethanol for storage. All specimens were stained with Mayer's paracarmine, counter-stained with fast green, dehydrated in ethanol, cleared in methyl salicylate and mounted in Permount. All measurements are in micrometers unless otherwise indicated. Measurements are expressed as an average followed by the range in parentheses. Drawings were made with the aid of a Bausch-and-Lomb microprojector, drawing tube and enlarged photographs. Since forebody width varies with the state of muscular contraction, only hindbody width is reported. Classification of all worms is based on the system presented by Dubois (1968 and 1970). Type specimens have been deposited in the USNM Helminthological Collection and the Division of Medical Ecology, Institute for Medical Research, Kuala Lumpur, Malaysia. Apatemon (Apatemon) jamesi sp. n.
(Strigeidae, Strigeinae, Cotylurini) (Figs. 1-2) Diagnosis: (62 specimens; 15 gravid adults measured). Length 1.34 (1.12-1.52) mm, body curved dorsally. Forebody hemispherical, truncated anteriorly, 0.25 (0.18-0.40) mm long; hindbody elongate, roughly cylindrical, curved dorsally, forms right angle with forebody when contracted, 0.87 (0.80-1.04) mm long. Oral sucker subterminal 83 (78-87) long by 85 (78-97). Prepharynx absent; pharynx continuous with oral sucker, 43 (37—51) long by 44 (37-51) wide. Oesophagus 30 (23-37) long. Gut caeca reaching posterior extremity; acetabulum larger than oral sucker, 99 (74-147) long by 110 (92-129) wide. Proteolytic glands at junction of forebody and hindbody, 50 (41-55) long by 69 (46-83) wide. Tribocytic or adhesive organ two-lobed, lying within the forebody, lobes when withdrawn 158 (129193) long by 180 (124-276) wide. Vitellaria abundant in hindbody, following dorsal curvature of body extending 90-95 % of hindbody length. Ovary pretesticular, round to subcircular, 95 (87-110) long by 84 (60-101) wide. Uterus extending from ovary posteriorly to junction of ejaculatory duct where it becomes the hermaphroditic duct. Mehlis' gland lateral to posterior-lateral to ovary. Laurer's canal present, opening on body surface, ventral and posterior to ovary. Testes tandem in posterior half of body, lobed, square to butterfly-shaped; anterior testis 169 (129-221) long by 156 (115-212) wide; posterior *Reprint address: Editor, G. W. Hooper Foundation, HSW 1699, University of California, San Francisco, California 94143, U.S.A. 51
J. R. PALMIERI, M. KRISHNASAMY and J. T. SULLIVAN
testis 152 (120-239) long by 147 (83-193) wide. Vitelline reservoir between anterior and posterior testis, 66 (51-83) long by 58 (37-69) wide. Seminal vesicle posterior to posterior testis, 91 (69-115) long by 91 (69-110) wide. Eggs usually less than 10 in number, 89 (8292) long by 53 (46-60) wide. Genital cone not delimited from surrounding parenchyma, opening into terminal bursa, not strongly muscular. This species is named in honour of Dr. Hugo A. James. Natural first intermediate host: Lymnaea rubiginosa Michelin. Natural second intermediate hosts: Hirudinaria manillensis Lesson, Dinobdella ferox Blanchard. Natural definitive host: Anas platyrhynchos Linn. Location: Upper to middle small intestine. Type Locality: Kuala Pilah, Negri Sembilan, West Malaysia. Type Specimens: USNM Helm. Collection. Holotype: No. 73048. Paratypes: No. 73049. Remarks
A survey of the parasitic fauna of domestic ducks {Anas platyrhynchos) from Kampung Panglang, Sri Menanti Town, Kuala Pilah, Negri Sembilan, revealed 29 out of 37 ducks positive for Apatemon jamesi. Ducks, bred in the rice fields surrounding the kampung, only return to the kampung at night for shelter. Water buffalo (Bos bubalus Linn), also inhabit the fields. Since the snail and leech commonly serve as the first and second intermediate host, respectively, for Apatemon (McDonald, 1969; Palmieri, 1973) a search for these hosts in this area was conducted. Of the 748 Lymnaea rubiginosa examined, 21 were positive for sporocysts and cercariae of Apatemon (Palmieri et ah, 1977). Buffalo leeches, identified as Hirudinaria manillensis Lesson and Dinobdella ferox Blanchard (Sharma and Fernando, 1961), collected from water buffaloes and from pools which contained L. rubiginosa, were found positive for metacercariae similar to the Apatemon type reported by Palmieri and James (1976b). Leeches naturally infected with metacercariae and fed to laboratory-reared ducks produced trematodes morphologically similar to Apatemon jamesi. Eggs, collected from living gravid worms, were incubated at 26°C for 18 days until hatching occurred. Laboratory-reared L. rubiginosa were exposed to miracidia and were placed in aquaria until they shed furcocercous cercariae approximately 4 weeks postexposure. Snails were allowed to shed cercariae for 10 days in an aquarium containing laboratory-reared leeches (Dinobdella ferox). Cercariae were observed penetrating the leeches and eliciting a typical behavioural reaction as reported by Palmieri and James (1976a). At 14 days postexposure leeches were fed to laboratory-reared ducks. At 3 days postfeeding, ducks were examined and trematodes which morphologically resembled Apatemon jamesi were recovered from the small intestine. We have chosen to use Dubois' (1968) taxonomic system that divides the genus Apatemon Szidat, 1928 into two subgenera (Apatemon) Szidat, 1928 and (Australapatemon) Sudarikov, 1959. Apatemon jamesi is most closely aligned with subgenus (Apatemon). We consider Apatemon jamesi to be a new species with a life cycle typical for the genus. Cercaria Cotylurus sullivani sp. n.
(Strigeidae, Strigeinae, Cotylurini) (Fig. 3) Diagnosis: (20 specimens measured) Furcocercous, pharyngeate, distomous, longifurcate cercariae developing in nature in Melanoides tuberculata Miiller, 1774. Body 170 (156-183) long by 58 (54-66) wide. Tail stem 200 (186-225) long by 38 (36-45) wide; tail furca (right) 138 (114-156), (left) 138 (111-150). 52
o
FIG. 1. Drawing of a wholemount of the type specimen of Apatemon (Apatemon) jamesi sp. n. (Trematoda: Strigeidae) recovered from the intestine of the duck, Anas platyrhynchos. FIG. 2. Drawing of a lateral section of Apatemon (Apatemon) jamesi showing internal morphology. FIG. 3. Drawing of cercaria Cotylurus sulliyani sp. n. (Trematoda: Strigeidae) recovered from the shedding snail, Melanoides tuberculata. FIG. 4. Drawing of a wholemount of Neodiplostomum (Neodiplostomum) sp. (Trematoda: Diplostomatidae) recovered from the owl Ketupa ketupu. 53
J. R. PALMIERI, M. KRISHNASAMY and J. T. SULLIVAN
Two pairs of penetration glands, first pair at level of acetabulum, second pair postacetabular; ducts opening into oral cavity. Mouth terminal. Anterior organ 35 (33-42) long by 28 (27-30) wide surrounded by 26-30 fine spines. Acetabulum muscular 21.5 (21-24) long by 25 (24-27) wide. Mouth leading to a well-developed pharynx 12-15 in diameter. Oesophagus long 37 (32^0); caecal bifurcation halfway between pharynx and acetabulum. Gut caeca long, narrow and regular in outline extending to excretory bladder. Five pairs of flame cells in body and 3 pairs in tail stem, total 4 pairs per side or 16 in all. Posterior bladder distinctly bipartite. Fourteen pairs of lateral sensory hairs on tail stem, 2 pairs per side at the anterior tail stem and 5 pairs per side on the lower 1/3 of tail stem. Genital primordium conspicuous, between gut caeca and excretory bladder. Furca 85% body length. Excretory duct passing through tail stem, bifurcating at caudal fork, opening on posterior edge of each furca less than halfway down its length. Natural host: Melanoides tuberculata Miiller, 1774 Type locality: Klang Gates, Malaysia Remarks
During spring, 1975, James J. Sullivan of the University of California, International Centre for Medical Research, Kuala Lumpur, collected a strigeid cercaria from the operculate snail Melanoides tuberculata Miiller, 1774 at Klang Gates, Malaysia. From examination of data on organ morphology and detailed drawings compiled by him, we have tentatively identified this cercaria as being closely related to those belonging to the genus Cotylurus Szidat, 1928 and have tentatively named it cercaria Cotylurus sullivani sp. n. after Dr. James J. Sullivan. The description reported here is given with his permission. Neodiplostomum (Neodiplostomum) sp.
(Diplostomatidae, Diplostomatinae, Diplostomatini) (Fig. 4) Diagnosis: (1 specimen recovered and measured). Body 2.15 mm long; forebody 1.10 mm long; hindbody 1.05 mm long by 0.32 mm wide. Forebody lightly spinose, spoonshaped ; hindbody cylindrical and aspinose; forebody approximately same length as hindbody. Oral sucker subterminal, 83 long by 78 wide. Prepharynx 9 long. Pharynx well developed, 55 long by 46 wide. Oesophagus 23 long. Acetabulum muscular, 78 long by 97 wide. Holdfast organ oval, 290 long by 179 wide, with a median ventral slit. Ovary large, round to triangular on right-hand side below junction of hindbody and just anterior to anterior testis, 170 long by 611 wide. Uterus runs anteriorly from ovary and turns back upon itself at the forebody-hindbody junction where it then proceeds posteriorly to the genital pore. Vitelline reservoir intertesticular, 37 in diameter. Anterior testis dumbell- or butterfly-shaped with deep posterior cleft, 244 long by 282 wide. Seminal vesicle not seen. Vitelline glands in forebody extend midway between the caecal bifurcation and acetabulum in 4 longitudinal bands with heavy concentrations between the acetabulum and holdfast organ; in the hindbody the vitelline glands are large with 2 dense lateral fields from the posterior edge of the holdfast to the posterior edge of the ovary and also from the posterior edge of the posterior testis to the genital pore. No eggs were found. Natural host: Ketupa ketupu Glenister 1974 Location: Mid-small intestine Type locality: Perak, Malaysia Type specimen: USNM Helm. Coll. Holotype: No. 73050. 54
Strigeoid trematodes in Malaysia Remarks
This single specimen was recovered from the intestine of the fish owl {Ketupa ketupu) commonly found feeding on fish near rice fields along the banks of streams in inland areas (Glenister 1971). The description of Neodiplostomum {Neodiplostomum) sp. is based on a single specimen, and hence we do not feel justified in giving it a species name. Morphologically it resembles several reported species of Neodiplostomum yet its characteristics separate it from any single reported species. Although Ketupa ketupu has never been reported as host for trematodes of the superfamily Strigeoidea Railliet, 1919, Dubois (1970) lists Strigiformes (owls) as hosts for trematodes of the genus Neodiplostomum. N. {Neodiplostomum) sp. morphologically resembles N. {Neodiplostomum) americanum (Chandler et Rausch, 1947) Dubois et Rausch, 1948, the latter also being found in several genera of Strigiformes hosts. TV*. {Neodiplostomum) americanum has been reported from 7 hosts, 4 of which belong to the Strigiformes. However, N. {Neodiplostomum) sp. differs from A". {Neodiplostomum) americanus in having a forebody approximately the same size as its hindbody. N. {Neodiplostomum) reflexum Chandler et Rausch, 1947 also morphologically resembles N. {Neodiplostomum) sp. in having similar body and organ sizes as well as having 4 genera of Strigiformes as hosts (Dubois, 1970) but differs in having the vitellaria extend anteriorly only as far as the level of the acetabulum. Because body size, testis size and shape, vitellaria extent and attachment, and organ size and shapes can vary under the influences of host-induced morphological variation (Palmieri 1976, 1977 a-c), we find it difficult to place N. {Neodiplostomum) sp. into a specific category and therefore leave it without specific name until either more specimens are found or life cycle data compiled. Fibricola ramachandrani
(Betterton, 1976) Palmieri, Krishnasamy and Sullivan (Diplostomatidae, Alariinae, Alariini) Palmieri et al. (in press) reported trematodes resembling Neodiplostomum {Conodiplostomum) ramachandrani Betterton, 1976 from four rodent hosts Echinosorex gymnurus Raffles, Rattus whiteheadi Thos, R. muelleri Jentink, and Callosciurus notatus (Boddaert). After a review of the taxonomy of Neodiplostomum and a careful examination of type and paratype specimens of N. {Conodiplostomum) ramachandrani, Palmieri et al. (in press) renamed this trematode Fibricola ramachandrani (Betterton, 1976) Palmieri, Krishnasamy and Sullivan. F. ramachandrani is highly variable morphologically, undergoes host-induced morphological variation, and is host-nonspecific. We refer the reader to Palmieri et al. (in press) for details on Fibricola ramachandrani. Pseudoscolopacitrema gen. n. Diagnosis: Diplostomatidae, Diplostomini, Crassiphialini, with pseudosuckers. Body clearly divided into two regions, arched dorsad. Forebody shorter than hindbody. Tribocytic organ with two fan- or wing-like lobes, occupying major portion of concavity of forebody. Testes large, lobed, tandem asymmetrical, seminal vesicle intratesticular. Ovary pretesticular, round to oval. Uterus reaching from genital atrium and proceeding anterior to ovary. Muscular sphincter at terminal of body joining uterus. Type species: Pseudoscolopacitrema otteri sp. n. Pseudoscolopacitrema otteri gen. n. et. sp. n. (Figs. 5-7) Diagnosis: (5 specimens; 4 gravid adults measured). Length 4.34 (3.78-5.09) mm, body divided into two regions. Strongly recurved forebody 0.99 (0.87-1.20) mm 55
J. R. PALMIER1, M. KRISHNASAMY and J. T. SULLIVAN
FIG. 5. Drawing of a wholemount of the lateral view of Pseudoscolopacitrema otteri gen. n. et sp. n. recovered from the otter Amblonyx cinerea. FIG. 6. Drawing of the anterior region of Pseudoscolopacitrema otteri gen. n. et sp. n. showing the well developed pseudosuckers. FIG. 7. Drawing of the muscular sphincter at the terminal of the genital atrium of Pseudoscolopacitrema otteri gen. n. et. sp. n. 56
Strigeoid trematodes in Malaysia
long. Hindbody conical, widening posteriorly, 3.35 (2.73-4.14) mm long by 0.54 (0.440.59) mm wide. Tegument aspinose. Oral sucker terminal, 100 (92-104) long by 114 (106120) wide. Pseudosuckers muscular and well developed, lateral and posterior to pharynx, 151 (138-184) long by 93 (87-101) wide. Tribocytic or adhesive organ with two large fanor wing-like lobes 481 (423-580) long by 400 (304-589) wide, lobes never observed in retracted state. Vitellaria abundant in forebody and hindbody from base of oral sucker to level of ovary. Ovary pretesticular, round to oval, 154 (143-166) long by 180 (170-189) wide. Uterus extending from 184-322 anterior to ovary to posterior genital atrium with a muscular sphincter 92 (86-101) long by 110 (92-119) wide. Mehlis' gland lateral to ovary. Eggs usually under 40 in number, operculate, well embryonated, 107 (92-129) long by 59 (51-74) wide. Two testes lobed, asymetrical and tandem; anterior testis 373 (332-409) long by 405 (378-459) wide; posterior testis 290 (240-329) long by 368 (342-396) wide. Seminal vesicle large, elongate, intertesticular. Natural definitive host: Amblonyx cinerea (Illiger) Location: Small intestine Type locality: Zoo Negara, Kuala Lumpur, Malaysia Type specimen: USNM Helm. Coll. Holotype: No. 73047 Remarks Of the 17,452 hosts examined only one individual small-clawed otter {Amblonyx cinerea) was collected. Examination of its intestinal contents revealed four gravid and one mature trematodes of the family Diplostomatidae. Because of the large fan- or wing-like tribocytic organ, recurved forebody and long slightly conical hindbody, these specimens superficially resemble specimens of the genus Crassiphiala van Haitsma, 1925 and Scolopacitrema Sudarikov and Rykovskii, 1959. Careful examination of Pseudoscolopacitrema otteri shows the presence of 2 well developed pseudosuckers, a well developed acetabulum, vitellaria extending into the forebody and the presence of a large muscular sphincter at the posterior junction of the uterus. Neither Crassiphiala nor Scolopacitrema have 2 pseudosuckers; the former also lacking a well developed muscular acetabulum as well as vitellaria extending into the forebody. Scolopacitrema does have vitellaria extending into the tribocytic organ and a sucker-like genital atrium which is similar to Pseudoscolopacitrema but lacks its well developed muscular pseudosuckers. Based on the above we consider these worms to be of a new genus Pseudoscolopacitrema, the name based upon its similarity to Scolopacitrema, the species name otteri being descriptive of the type host. Cercaria Cyathocotyle malayi sp. n. (Cyathocotylidae) (Figs. 8-13) Over 300 Bellamya sumatrensis Dunker, collected from the Kelang River drainage, Kelang Gates, Malaysia, were heavily infected with an unreported cercaria of the family Cyathocotylidae that encysted as tetracotyle-type metacercariae on the fins and in the musculature of freshwater fish. Field-collected Bellamya sumatrensis with advanced infections of the cyathocotylid cercariae were placed in aquaria with 10 red-eyed red swordtail fish (Xiphophorus hellerii) and allowed to shed. After 48 hours, the cyathocotylid-exposed fish were separated and maintained 3-4 weeks to ensure complete development of encysted tetracotyle larvae within the musculature and fins. 57
J. R. PALMIERI, M. KRISHNASAMY and J. T. SULLIVAN
EP
FIG. 8. Drawing of the daughter sporocyst of cercaria Cyathocotyle malayi sp. n. (Trematoda: Cyathocotylidae) recovered from the snail Bellamya sumatrensis. FIG. 9. Drawing of cercaria Cyathocotyle malayi sp. n. (Trematoda: Cyathocotylidae) recovered from the shedding snail Bellamya sumatrensis.
Five species of fish were found naturally infected with metacercariae of Cyathocotyle malayi while Barbus binotatus and Xiphophorus hellerii were experimentally infected with metacercariae (Table 1). Naturally and experimentally infected fish were cut into 1 cm2 pieces and held in saline solution for feeding experiments. Six species of reptiles, 2 birds and 3 mammals were experimentally fed metacercariae but no adult cyathocotylid trematodes were ever recovered (Table 2). Daughter sporocyst: (Fig. 8) Elongate opaque vermiform sacs from 0.6-0.8 mm long and 0.03-0.04 mm wide. Birth pore near anterior end. Interior filled with germ balls and developing daughter sporocysts. Body wall lightly muscular, lined by tegument, and 2-2.5 thick. 58
Strigeoid trematodes in Malaysia
Mother sporocysts developed in the digestive gland of the snail and appeared to move with a twisting motion when dissected from snail. In heavy infections the mother sporocysts extended along the oesophagus and intestine of the snail. TABLE 1 Fish infected with tetracotyles of Cyathocotyle malayi
Host Barbus binotatus B. fasciatus B. lateristriga B. partipentazona Trichogaster trichopterus Xiphophorus hellerii
Experimental infection
Natural infection
X
X X X X X
X
Cercaria: (Fig. 9) Furcocercous, nonoculate, pharyngeate and monostomate; without acetabulum or rudimentary acetabulum. Body 276 (252-312) long by 122 (108-150) wide; tail stem 370 (336-432) by 44 (36-54); furcae 278 (201-336); oral sucker 70 (57-84) by 44 (36-54); pharynx 18.3 (15-21) by 21.6 (18-27). Tegument without thornlike spines; body and tail stem without sensory hairs. One pair of cephalic glands consisting of 9 cells; their ducts opening lateral to the mouth; oral sucker oval; mouth leading to a small tubular prepharynx; pharynx wider than long; oesophagus 3 times the length of the pharynx; intestine with sinuous walls, reaching posterior of body. Genital primordium present intercaecally. One pair of excretory ducts lateral to the intestine; median longitudinal commissure present; excretory duct extends from tail stem through furcae to a terminal excretory pore; excretory vesicle in anterior tail stem; flame cell formula 2 [ l + 3 + 3 (3+3)]=26. Tail without finfold, muscular and bent. Behaviour of the cercaria: Furcocercous cercariae appeared 32 days postinfection when snails were maintained at 27-29° C. Cercariae were shed in large numbers from early morning to midaftemoon. At rest, cercariae slowly sank toward the bottom with furcae upward for 7-13 seconds. When swimming, cercariae spun in an upward clockwise spiral direction around a central axis. Cercaria either made a 1-coil spin taking 3-5 seconds or a 3-coil spin taking 7-13 seconds for a distance of 1.5-2 cm (Fig. 10). Cercariae were never observed crawling or attached to either the bottom or sides of the container. Cercariae showed no phototropic preference and swam toward light or dark. When observed from above, the furcae could be clearly seen and obstructed the view of the tail stem and body (Fig. l i b and c). When viewed from the side, the cercaria had a characteristic bend in the tail stem of approximately 140° (Fig. l l a ) . One hundred cercariae were placed in a 30 cm 500-ml graduated cylinder to observe swimming and resting positions. After 30 min most cercariae were below the 100-ml mark (6 cm) (Fig. 12). Tetracotyle (Fig. 13): Encysted, fully formed, round tetracotyles found either on the scales or in the fins or musculature of fish. Cyst delicate, clear, and about 2 thick leaving no trace after routine paraffin sectioning except for a layer of host cells that covered cysts in muscle tissue. Excysted tetracotyles oval to pear-shaped usually 416-442 long by 214242 wide although varying somewhat in size depending on crowding and age. No body spines present. Distinct forebody and weakly developed hindbody. Genital anlage recognizable just below the holdfast organ. Oral sucker muscular and well developed measuring 60 (53-62) long by 72 (62-80) wide. Muscular pharynx well 59
J. R. PALMIERI, M. KRISHNASAMY AND J. T. SULLIVAN
10 B
1 2.0 in.
. 500 m I.
.400
300
200
.100
FIG. 10. Swimming behaviour of cercaria Cyathocotyle malayi sp. n. a. A one-coil upward spin taking from 3 to 5 seconds to complete. b. A three-coil upward spin taking 7 to 13 seconds to complete. FIG. 11. Different views of a suspended cercaria Cyathocotyle malayi sp. n. a. Side view, b-c. Top view looking down on posterior region. FIG. 12. One hundred cercaria Cyathocotyle malayi sp. n. suspended in 30 cm. 500-ml graduate cylinder after 30 minutes, note: most cercariae are below the 100-ml mark. 60
Strigeoid trematodes in Malaysia
developed. Virtually no oesophagus present. Caeca reaching to midgenital anlage. Holdfast organ measuring 40-43 in diameter. Unicellular cephalic glands present on either side of the pharynx and oral sucker. Infection of 2nd intermediate host: Cercariae actively penetrated and developed into tetracotyles in a variety of fish (Table 1). At Kelang Gates, where about 1 % of all B. sumatrensis were infected with C. malayi, all fish sampled were infected with at least 60 tetracotyles of C. malayi.
13
FIG. 13. Drawing of a wholemount of the excysted tetracotyle of cercaria Cyathocotyle malayi sp. n. recovered from the musculature of a fish.
If a suceptible fish was placed in a beaker of water containing active cercariae, penetration began immediately. Cercariae initially became trapped on the body mucus where they crawled under scales or into the fins and encysted. In most cases cercariae penetrated the musculature. In cases of heavy infection (over 200 tetracotyles/fish (6-10 cm)) tetracotyles were commonly found grouped. In light infections, tetracotyles were found evenly spaced apart. Dead tetracotyles were opaque and yellow. 61
J. R. PALMIERI, M. KRISHNASAMY and J. T. SULLIVAN TABLE 2 Hosts exposed to tetracotyles of Cyathocotyle malayi Number exposed
Host Reptilia Calotes sp. Cuora amboinensis Geomyda bispinose Mabola multifasciata Trionyx hurum Varanus salvator
1 4 4 8 3 1
Aves Anas platyrhynchos Gallus domesticus
17 10
Mammalia Canis familiaris Felis catus
6 1
Macaca nemestrina
1
Remarks Members of the Cyathocotylidae are found in reptilian, avian and mammalian hosts (Yamaguti, 1971). Within the 6 subfamilies of the Cyathocotylidae both cyathocotylinae and prohemistominae have reptilian, avian and mammalian definitive hosts; szidatinae are found in both reptilian and avian hosts; prosostephaninae in both reptilian and mammalian hosts; pseudohemistominae only in avian and muehlingininae only in mammalian hosts. Subfamilies prohemistominae, muehlinginiane and prosostephaninae all contain members which lack an acetabulum, a character held in common with Cyathocotyle malayi. Because a natural or experimental definitive host was never found and since the cercarial stage of C. malayi does not morphologically or behaviorally resemble reported species of Cyathocotylid cercariae, we find it difficult to place this cercaria within a subfamily of cyathocotylidae until further information is available concerning the adult stage in the life cycle. ACKNOWLEDGEMENT This study was supported in part by grant AI 10051 (UC ICMR) to the Department of Epidemiology and International Health, School of Medicine, University of California, San Francisco, from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, U.S. Public Health Service. REFERENCES BETTERTON, C. (1976) Neodiplostomum (Conodiplostomum) ramachandrani sp. n. from Mueller's rat Rattus muelleri in Malaysia. Journal of Helminthology, 50, 157-161. DUBOIS, G. (1968) Synopsis des Strigeidae et des Diplostomatidae (Trematoda). Societe Neuchateloise des Sciences Naturelles, pp. 1-258. DUBOIS, G. (1970) Synopsis des Strigeidae et des Diplostomatidae (Trematoda). Sociite Neuchateloise des Sciences Naturelles, pp. 259-727. GLENISTER, A. G. (1974) The birds of the Malay Peninsula, Singapore and Penang. Oxford University Press, Ely House, London, 291 pp. McDONALD, M. E. (1969) Catalogue of helminths of waterfowl (Anatidae). Bureau of sportfisheriesand wildlife. Special Scientific Report—Wildlife No. 126. Washington D.C. PALMIERI, J. R. (1973) Additional natural and experimental hosts and intraspecific variation in Posthodiplostomum minimum (Trematoda: Diplostomatidae). Journal of Parasitology, 59, 744-746. 62
Strigeoid trematodes in Malaysia PALMIERI, J. R. (1977) Host-induced morphological variations in the strigeoid trematode Posthodiplostomum minimum (Trematoda: Diplostomatidae). II. Body measurements and tegument modifications. Great Basin Naturalist, 37: 129-137. PALMIERI, J. R. (1977) Host-induced morphological variations in the strigeoid trematode Posthodiplostomum minimum (Trematoda: Diplostomatidae). III. Organs of attachment. Great Basin Naturalist, 37, 375-382. PALMIERI, J. R. (1977) Host-induced morphological variations in the strigeoid trematode Posthodiplostomum minimum (Trematoda: Diplostomatidae). IV. Organs of reproduction (ovary and testes), vitelline gland, and egg, Great Basin Naturalist, 37, 481-488. PALMIERI, J. R. and JAMES, H. A. (1976a) The effects of leech behaviour on penetration and localization of Apatemon gracihs (Trematoda: Strigeidae) cercariae and metacercariae. Great Basin Naturalist, 36, 97-100. PALMIERI, J. R. and JAMES, H. A. (1976b) Metacercarial composition and development in Apatemon gracilis (Trematoda: Strigeidae). Iowa State Journal of Research, 50, 409^417. PALMIERI, J. R., KRISHNASAMY, M. and SULLIVAN, J. T. (In Press). Fibricola ramachandrani (Betterton, 1976) Palmieri, Krishnasamy and Sullivan comb. nov. from Malaysian rodent hosts with special note on intraspecific morphological variation. Journal of Helminthology. PALMIERI, J. R., SULLIVAN, J. T. and OW-YANG, C. K. (1977) A survey of snail hosts and larval trematodes collected from Peninsular Malaysia and Singapore during 1972 to 1977. The Southeast Asian Journal of Tropical Medicine and Public Health, 8, 275-277. SHARMA, R. E. and FERNANDO, C. H. (1961) Leeches and their ways. The Malayan Nature Journal, 15, 152-159. YAMAGUTI, S. (1971) Synopsis ofdigenetic trematodes of vertebrates. Vol. I and II. Keigaku Publishing: Tokyo. 1074 pp. Received 20 October, 1977.
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