JOURNAL OF INVERTEBRATE PATHOLOGY
26, 81-90 (1975)
Echinocephalus crassostreai sp, nov., A larval Nematode
from the Oyster Crassostrea gigas in the Orient I THOMAS C. CHENG
lnstitutefor Pathobiology, Center for Health Sciences, Lehigh University. Bethlehem. Pennsylvania 18015 Received Octoher 24, 1974 A new gnathostomatid nematode, Echinocephalus crassostreai sp. nov., is described from the Japanese oyster, Crassostrea gigas. from Hong Kong and the People's Republic of China. The erection of the new species is based on conspicuous differences between the third-stage larva of the nematode and the other valid species of the genus. A description of the secondstage larva is also provided. Echinocephalus erassostreai third-stage larvae are found primarily in the gonoducts of the molluscan host. There are no histopathological changes of the cells lining the gonoduct associated with these parasites. There is, however, a tunic of reaction elements surrounding the gonoduct. Occasional larvae may invade the gonad and cause some damage to adjacent molluscan gametes. Based on preliminary findings by others, E. crassostreai third-stage larvae may be of public health importance.
INTRODUCTION
dors who informed me that they were collected that morning from the oyster beds in Hau Hoi Wan (Deep Bay). This body of water extends from the coast of the People's Republic of China to that of the New Territories, Hong Kong (Fig. 1). Preparation for light microscopy. Fresh as well as fixed and cleared specimens of nematodes that had been removed from dissected oysters were studied in Hong Kong. In addition, specimens from oysters shipped to this Institute were isolated by digesting the molluscs for 2 hr at 3T C in a 1% pepsin solution acidified with 1% HC!. Subsequently, the worms were collected by transferring the digestion mixture into sedimentation cones. Selected specimens of both second- and third-stage larvae were fixed and cleared in hot glycerol alcohol (80% ethanol plus 10% glycerol) and prepared either as permanent or temporary mounts for microscopical examination. Nomarski interference optics was employed to examine
The occurrence of a species of nematodes belonging to the family Gnathostomatodae in the Japanese oyster, Crassostrea gigas. off the shores of the British Crown Colony of Hong Kong was brought to my attention over 2 yr ago by Dr. Brian Morton and Miss Patsy S. Wong of the Department of Zoology, University of Hong Kong. Subsequently, during July and August 1974, I traveled to Hong Kong to study this nematode and to ascertain the ecological conditions existing in the waters from which the infected oysters were collected. As a result of a detailed analysis of specimens of this nematode, it has been concluded that the larval worms in C. gigas represent a new species of Echinocephalus Molin, 1858. Descriptions of the second- and third-stage larvae of the new parasite are presented below, along with an account of aspects of the ecology of the natural habitat of the molluscan host and histopathological changes in parasitized oysters.
'This research was supported by Contract 223-742092 from the Food and Drug Administration, Public Health Service, U.S. Department of Health, Education, and Welfare.
MATERIALS AND METHODS Origin of oysters. Specimens of C. gigas were purchased from local Hong Kong ven81 Copyright © 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
82
THOMAS C. CHENG PEOPLE'S REPUBLIC OFCHINA Kwanat_ Province
FIG. 1. Map showing site at which the oysters, Crassostrea gigas, were collected. The shaded areas represent the oyster beds.
the specimens and all of the measurements were made with a calibrated ocular micrometer. Additional oysters were fixed in 10% seawater-formalin after their valves had been removed. These were subsequently embedded in Tissuemat, sectioned at 7 JLm, and selected sections were stained with Delafield's hematoxylin and eosin. In all, a total of 50 oysters were prepared as histological sections and an additional 150 were either dissected or digested to isolate the nematodes. Scanning electron microscopy (SEM). In preparation for SEM, whole specimens of the nematode removed from infected oysters by digestion were fixed in 2.5% glutaraldehyde in Millipore-filtered seawater for 4 hr and postfixed in 2% OS04 for 2 hr. They were subsequently dehydrated, dried by the critical point method of Ruffolo (1974), coated with a film of gold in vacuo, and examined with an ETEC SEM operated at 10 and 20 kV. OBSERVATIONS
Ecological observations. The waters from which the oysters were obtained have a salinity ranging from 10 to 30 0/00' depending on the tide. The oysters grow on the sand
and mud bottom of the intertidal zone, which extends from 7;1 to over Y2 mile from the high tide line. The distance between the high and low tide lines is approximately 9 ft. The temperatures on the days that I visited the oyster grounds during July and August 1974, ranged from 29.5 to 34°C and the relative humidity from 75 to 90%. During the winter the temperature can drop to as low as 1-3°e. Spawning of oysters. Although spawning of C. gigas at the sites mentioned is thought to occur from mid-April to the beginning of May (P. S. Wong, pers. commun.), examination of sections of 50 oysters revealed that 36 were in the female and 13 were in the male phase. The remaining specimen was in the transitional phase. Specimens of both sexes included gonads filled with ova or sperm and did not give the appearance of having spawned. However, judging by previous experience, it would not be surprising if the oysters commenced to spawn during the third week of August. The parasite. Both second- and thirdstage larvae of the gnathostomatid nematode were found in C. gigas. Of the 150 oysters dissected or digested, 22 harbored this parasite. One oyster contained both second- and third-stage larvae. Of the 50
83
Echinocephalus crassostreai SP . NOV.
oysters examined as histological sections, five included the parasite. Thus, out of 200 oysters examined, 27, or 13.5%, were infected with the nematode. Examination of whole worms led to the conclusion that they represent a new species
of the genus Echinocephalus, described below as E. crassostreai sp. nov. Echinocephalus erassostreai sp. nov.
Third-stage Echinocephalus erassostreai sp. nov. larva (Figs . 2-5). Description (based
CG
ES
CG
I.'
IN'
1h'j "1® .J e i !
FIGS . 2-8. AN, anus; CG, cervical gland ; CGO, duct of cervical gland ; CH, cephalic hook; ES, esophagus; EV, esophageal valve; GP, genital primordium; INT, intestine; L, lip; MO, mouth ; NR, nerve ring. FIG . 2. Anterior portion of third-stage larva of Echinocephalus crassostreai sp. nov. FIG. 3. Cephalic hook from eighth row of third- stage larva of Echino cephalus crassos treai sp. nov. FIG . 4. Third-stage larva of Echinocephalus crassos treai sp. nov . FIG . 5. En face view of third-st age larva of Echino cephalus crassostreai sp. nov. showing first two rows of cephalic hooks only. FIG . 6. Second-stage larva of Echin ocephalus crassos treai sp, nov. FIG. 7. Anterior portion of second-stage larva of Echinocephalus crassostreai sp. nov . FIG .8. Drawing showing esophageal valve in larval Echino cephalus crassostreai sp, nov.
84
THOMAS C. CHENG
on 12 specimens): Gnathostomatidae. Body 10.93 mm (8.16-13.59 mm) long and 0.33 mm (0.25-0.39 mm) wide at widest level; head bulb 0.15 mm (0.13-0.16 mm) long and 0.25 mm (0.24-0.26 mm) wide; head armed with eight circular rows of hooks (Figs. 2-5, 9, 10) projecting from the cuticle and with those comprising the posterior six rows being prominent; each hook of the eighth (most posterior) row averages 0.0025 mm long, each of the seventh row averages 0.0025 mm long, each of the sixth row averages 0.0023 mm long, each of the fifth row averages 0.0020 mm long, each of the fourth row averages 0.0018 mm long, and each of the third row averages 0.0016 mm long; hooks of first and second rows inconspicuous, with each of the second row averaging 0.0014 mm long and each of the first row averaging 0.0007 mm long; number of hooks in third through eighth rows varies,
with the numbers in rows 1-8 being 48, 56, 46-48,44-45,44-52,44-56,48-51, and 4648, respectively (Figs. 2, 9); terminal mouth guarded by two smooth-surfaced, bulbous lips (Figs. 2, 5, 9); four cervical glands, with ducts leading to anterior terminal, lying alongside and terminating at approximate midlength of esophagus (Fig. 4); esophagus, 1.63 mm (1.54-1.70 mm) long, with slightly bulbous posterior terminal which averages 0.20 mm (0.19-0.21 mm) wide; esophageal valve present (Fig. 8); intestine 6.71 mm (6.68-6.73 mm) long byO.ll mm (0. Jl-O.12 mm) wide, terminating at midventral anus located approximately 0.03 mm from posterior tip of body (Figs. 4, I 1); genital primordium, 0.01 by 0.01 mm, at approximate midlength of body, comprised of 10-25 cells (Fig. 4). No papillae present in pre- or postanal regions (Fig. II); phasmids, nerve ring, and excretory system not observed.
FIG . 9. Scanning electron micrograph of cephalic end of third-stage larva of Echinocephalus crassostreai sp. nov. L,lip. 400x .
Echinocephalus crassostreai
SP . NOV .
85
FIG . 10. Scanning electron micrograph of some cephalic hooks of third-stage larva of Ech inocephalus crasso s treai sp. nov. 1300x .
Since these were immature worms, bursae, spicules, and fully developed reproductive organs were not observed . Host: Crassostrea gigas, Japanese oyster. Location: Primarily in gonoduct. Locality: Off the coasts of the New Territories, Hong Kong, and the People's Republic of China in Hau Hoi Wan (Deep Bay). Holotype specimen: U.S. National Museum Helminthological Collection No. 73739. Second-stage larva (Figs. 6, 7). Description (based on two specimens): Gnathostomatidae. Body 4.98 mm long and 0.14 mm wide at widest level; head bulb 0.07 mm long and 0.09 mm wide; head armed with eight circular rows of minute hooks with largest averaging 0.008 mm long; two prominent smooth-surfaced lips present around terminal mouth (Figs. 6, 7); esophagus 0.95
mm long by 0.06 mm wide; four cervical glands situated alongside esophagus for most of its length (Fig. 6); intestine terminating posteriorly at midventral subterminal anus (Fig. 6); circumesophageal nerve ring present. No portions of reproductive system present. Justification for new species. Scanning electron microscopy has contributed to the elucidation of the morphological characteristics of E. crasso streai which, as pointed out below, distinguish the new species from those already described. Eight species of Echinicephalus have been named previously: E. uncinatus Molin, \858; E. striatus Monticelli, 1889; E. gracilis Stossich, 1906; E. spinosissimus (von Linstow, 1905); E. southwelli Baylis and Lane, 1920; E. multidentatus Baylis and Lane, 1929; E. aetobati MacCallum, 1921 ; and E. pseudouncinatus Millemann, 1951. However, according to Yamaguti (1961),
86
THOMAS C. CHENG
FIG. II. Scanning electron micrograph of posterior portion of third-stage larva of Echinocephalus crassostreai sp. nov. A, anus. 320x.
both E. gracilis and E. aetobati are synonymous with the generotype, E. uncinatus. Furthermore, Baylis and Lane (1920) have pointed out that there is no way of identifying Monticelli's (1889) E. striatus from the stomach of Scyllium sp. from Payta, Peru, and hence it must be considered a nomen nudum. In addition, Millemann (1963) has reduced von Linstow's (in Shipley and Hornell, 1904-1905) E. spinosissimus into synonymy with E. uncinatus. Consequently, only four species of Echinocephalus are considered to be valid prior to this report. Echinocephalus crassostreai differs from E. pseudounicinatus and E. multidentatus in that its lips do not bear teeth. There are two teeth on each of the lips of E. pseudouncinatus and eight to ten teeth on the lips of E. multidentatus. The new species can be distinguished from E. southwelli and E. uncinatus by the number of cephalic hooks. In the case of E. uncinatus, Baylis and Lane
(1920) have reported that the comparable larval stage in the marine bivalve Pinna sp. has six rows of 40~50 hooks and in the case of E. southwelli, Baylis and Lane (1920) have reported the presence of 15-18 rows of hooks with more than 100 hooks per row. Thus, it is apparent that E. crassostreai is distinct from the other known species. Millemann (1963), as a result of studying changes in the morphology of E. pseudouncinatus during growth, has noted that the definitive number of rows of cephalic hooks is attained by this group of worms at the third-stage molt, i.e., during the third larval stage, hence it would appear that the lesser number of rows of hooks, specifically eight, in E. crassostreai is a valid criterion for distinguishing it from E. southwelli, even if the only available description of the latter is of the adult. Also, studies by Millemann (1951, 1963) have revealed that dentitions on the lips persist from the larval stages through
Echinocephalus crassostreai
SP. NOV .
87
FIG . 12. C ross section through gonoduct of Crassostrea gigas showing third-stage larva of Echinocephalus ~rassos treai sp. nov. in lumen . Note tunic of reaction elements in perigonoductal region. GO, lumen of gonoduct; RT, reaction tunic. 130x. FIG. 13. Section showing third-stage larva of Echinocephalus crassostreai sp. nov. in ovary of Crasso strea gigas. Note abnorm al-appearing ova in proximity of parasite. DO, degener ate ova. 130x .
88
THOMAS C. CHENG
the adult form. Hence, the absence of teeth on the lips of E. crassostreai must be considered a valid criterion for distinguishing it from E. pseudouncinatus and E. multidentatus. Histopathology. Almost all the nematodes observed in histological preparations were found within the lumen of the gonoduct, especially in the proximal portion where the wall includes digitiform folds (Galtsoff, 1964) (Fig. 12). Echinocephalus crassostreai does not appear to cause appreciable histopathological changes associated with the ciliated epithelium lining the host's gonoduct. However, there is a conspicuous tunic consisting of connective tissue fibers, hemolymph cells (primarily granulocytes), myofibers, and tightly packed Leydig cells. These structural elements are embedded in an eosinophic ground substance (Fig. 12). This tunic, which averages 0.15 mm in thickness, does not exist in nonparasitized oysters. Only in one parasitized oyster were E. crassostreai larvae found in the gonad. The parasites were third-stage larvae and they were in the ovary (Fig. 13). The presence of nematodes appeared to cause the displacement of ova and many of the host's gametes in the adjacent zones were shrunken, ruptured, or compressed (Fig. 13). DISCUSSION By comparing the morphology of thirdstage larvae of an Echinocephalus from Crassostrea gigas collected in Hong Kong and the People's Republic of China with the descriptions of the other valid species of Echinocephalus, it is evident that the collected specimens represent a new species, E. crassostreai. The adult form is not yet known. It is noted, however, that Ko et al. (1974), who were the first to record the existence of what I have formally designated as E. crassostreai larvae, have stated that the ray, Aetabatus flagellum, is most likely the natural definitive host. Their speculation is based on the finding of third- and fourthstage larvae as well as adults of a species of
Echinocephalus in 11 of 15 rays caught near the oyster beds from which the oysters that contained E. crassostreai were obtained. However, direct proof that the Echinocephalus found by Ko et al. is the adult form of E. crassostreai is still wanting. Ko et al. (1974) also reported experimental infections of a cat and a rhesus monkey, Macaca mulatta, with E. crassostreai third-stage larvae isolated from C. gigas. The larvae penetrated the stomach and the small and large intestines of these experimental hosts. Because their report was in the form of an abstract, no details were presented. I have attempted to infect laboratory rats with third-stage larvae of E. crassostreai isolated from oysters by employing gastric tube feeding, but these attempts were unsuccessful. However, these were merely preliminary experiments involving feeding four larvae to each of four rats. Ko et al. fed 45 larvae to the cat and 200 larvae to the monkey and examined these hosts at 17 and 19 hr postfeeding, respectively. It should also be noted that P. S. Wong (pers. commun.) informed me that subsequent attempts to infect both kittens and monkeys have not been successful. Nevertheless, the preliminary report by Ko et al. suggests that the third-stage larva of E. crassostreai in C. gigas could be of potential public health importance. The occurrence of Echinocephalus larvae in marine molluscs has also been noted by Shipley and Hornell (1904-1905) and by Stossich (in Shipley and Hornell, 1906). These investigators have reported an undetermined species, perhaps E. uncinatus (= E. gracilis), encysted in the adductor muscle of the pearl oyster, Margaritifera vulgaris, in Ceylon. Also, Baylis and Lane (1920) have found E. uncinatus larvae encysted in Pinna sp. in Ceylon and Millemann (1951) has reported E. pseudouncinatus larvae encysted in the foot of the abalone, Haliotis corrugata, in southern California. In addition, Johnston and Mawson (1945) have recorded an unknown species in the gastropods Policines conica and Katylesia scalarina in
Echinocephalus crassos treai SP.
Australia. Of these earlier reports, only Millemann (1951) has made observations on the pathology caused by E. pseudouncinatus larvae in the molluscan host. He has reported: "Only old abalones were found to harbor the worms; these encyst in the ventral portion of the foot producing a blister-like effect and the burrowing of the larvae through the foot prior to encystment, apparently weakens the muscle and decreases the efficacy of this structure as a hold-fast organ." In the case of E. crassostreai in C. gigas, as stated, the parasite is not encapsulated. Furthermore, there are no external signs of infection, although histological examination of parasitized oysters has revealed a reaction complex surrounding the genital ducts in which the worms are located. It is noted, however, that of the constituents of the reaction complex, i.e., connective tissue fibers, hemolymph cells, tightly packed Leydig cells, and myofibers, the last mentioned are believed to be normally present surrounding gonoducts. Roughley (1933) has reported their presence surrounding gonoducts in Crassostrea commercialis, but Galtsoff (1938) has reported that they are missing at this location in C. virginica. Examination of sections of nonparasitized C. gigas has revealed that myofibers normally occur in the perigonoductal zone and hence their presence in this area of parasitized molluscs does not constitute a reaction. Nevertheless, the development of a reaction tunic around gonoducts enclosing E. crassostreai larvae is of interest. The lack of visible damage to the lining cells but the occurrence of the described reaction surrounding such a duct suggests possibly that a substance secreted or excreted by the parasite elicits the host response. It is noted that although some damage to adjacent ova has been observed in the oyster that harbored E. crassostreai in its gonad, the pathology was too slight to be regarded as parasitic castration. Finally, although Leydig cells occur in the perigonoductal zone of nonparasitized
NOV.
89
oysters, they become compressed in parasitized ones. Cytochemical studies are currently under way to ascertain whether there are any chemical alterations associated with this compression. ACKNOWLEDGMENTS I am grateful to Professor B. Lofts, Department of Zoology, University of Hong Kong, for making laboratory facilities available to me during my sojourn in Hong Kong; Dr. Ronald Ko of the same institution for assisting me in many ways; and Messers. John T. Sullivan and Thomas A. Garrabrant and Mrs. Diane H. Raymond, all of this Institute, for technical assistance.
REFERENCES BAYLIS, H. A. AND LANE, C. 1920. A revision of the nematode family Gnathostomatidae. Proc. Zool. Soc. London, 245-310. GALTSOFF, P. S. 1938. Physiology of reproduction of Ostrea virginica. Spawning reactions of the female and male. Bioi. Bull.. 74, 461-486. GALTSOFF, P. S. 1964. The American oyster Crassostrea virginica Gmelin. Fisher. Bull. Fish WildlifeServ., 64,1-480. JOHNSTON, T. H. AND MAWSON, P. M. 1945. Parasitic nematodes. Rep. Brit. Austral. New Zeal. Antarct. Res. Exped. 1929-/931,5,8(2),141. xo, R. C., MORTON, 8., AND WONG, P. S. 1974. Echinocephalus sp. Molin, 1858 (Spiruroidea: Gnathostornatidae), an unusual nematode from the oyster, Crassostrea gigas Thunberg, 1793. Proc. 3rd Inti. Congr. Parasitol., Munich, Germany, 3, 17311732. MILLEMANN, R. E. 1951. Echinocephalus pseudouncinatus n. sp., a nematode parasite of the abalone. J. Parasitol., 37,435- 439. MILLEMANN, R. E. 1963. Studies on the taxonomy and life history of echinocephalid worms (Nematoda: Spiruroidea) with a complete description of Echinocephalus pseudouncinatus Millemann, 1951. J. Parasitol., 49, 754--764. MONTICELLI, S. 1889. Elenco degli elminti raccolte da1 Capitano G. Chierchia durante 11 viaggio di circumnaviganzione della R. Corvetta "Vetter Pisano." Bull. Soc. Nat. Napoli, 3, G7-71. ROUGHLEY, T. C. 1933. The life history of the Australian oyster (Ostrea commercialis). Proc. Linn. Soc. New S. Wales, 58,279-333. RUFFOLO, J. J., JR. 1974. Critical point drying of protozoan cells and other biological specimens for scanning electron microscopy: apparatus and methods of specimen preparation. Trans. A mer. Microsc. Soc., 93, 124-131. SHIPLEY, A. E. AND HORNELL, J. 1904-1905. The parasites of the pearl oyster. In "Report to the
90
THOMAS C. CHENG
Government of Ceylon on Pearl Oyster Fisheries in the Gulf of Manaar" (W. A. Herdman, 00.), Pt. 2, pp. 77-106; Pt . 3,pp.49-56. London . SHIPLEY, A. E. AND HORNELL, J. 1906. Report on the cestode and nematode parasites from the marine fishes of Ceylon . In "Report to the Government of
Ceylon on Pearl Oyster Fisheries of the Gulf of Manaar" (W. A. Herdman, 00.), Pt. 5, pp. 43-96 . London . YAMAGUTI, S. 1961. "Systerna Helminthum," Vol. Ill. The Nematodes of Vertebrates, Pts. I and II . Interscience Publ., New York.
26, 81-90 (1975)
Echinocephalus crassostreai sp, nov., A larval Nematode
from the Oyster Crassostrea gigas in the Orient I THOMAS C. CHENG
lnstitutefor Pathobiology, Center for Health Sciences, Lehigh University. Bethlehem. Pennsylvania 18015 Received Octoher 24, 1974 A new gnathostomatid nematode, Echinocephalus crassostreai sp. nov., is described from the Japanese oyster, Crassostrea gigas. from Hong Kong and the People's Republic of China. The erection of the new species is based on conspicuous differences between the third-stage larva of the nematode and the other valid species of the genus. A description of the secondstage larva is also provided. Echinocephalus erassostreai third-stage larvae are found primarily in the gonoducts of the molluscan host. There are no histopathological changes of the cells lining the gonoduct associated with these parasites. There is, however, a tunic of reaction elements surrounding the gonoduct. Occasional larvae may invade the gonad and cause some damage to adjacent molluscan gametes. Based on preliminary findings by others, E. crassostreai third-stage larvae may be of public health importance.
INTRODUCTION
dors who informed me that they were collected that morning from the oyster beds in Hau Hoi Wan (Deep Bay). This body of water extends from the coast of the People's Republic of China to that of the New Territories, Hong Kong (Fig. 1). Preparation for light microscopy. Fresh as well as fixed and cleared specimens of nematodes that had been removed from dissected oysters were studied in Hong Kong. In addition, specimens from oysters shipped to this Institute were isolated by digesting the molluscs for 2 hr at 3T C in a 1% pepsin solution acidified with 1% HC!. Subsequently, the worms were collected by transferring the digestion mixture into sedimentation cones. Selected specimens of both second- and third-stage larvae were fixed and cleared in hot glycerol alcohol (80% ethanol plus 10% glycerol) and prepared either as permanent or temporary mounts for microscopical examination. Nomarski interference optics was employed to examine
The occurrence of a species of nematodes belonging to the family Gnathostomatodae in the Japanese oyster, Crassostrea gigas. off the shores of the British Crown Colony of Hong Kong was brought to my attention over 2 yr ago by Dr. Brian Morton and Miss Patsy S. Wong of the Department of Zoology, University of Hong Kong. Subsequently, during July and August 1974, I traveled to Hong Kong to study this nematode and to ascertain the ecological conditions existing in the waters from which the infected oysters were collected. As a result of a detailed analysis of specimens of this nematode, it has been concluded that the larval worms in C. gigas represent a new species of Echinocephalus Molin, 1858. Descriptions of the second- and third-stage larvae of the new parasite are presented below, along with an account of aspects of the ecology of the natural habitat of the molluscan host and histopathological changes in parasitized oysters.
'This research was supported by Contract 223-742092 from the Food and Drug Administration, Public Health Service, U.S. Department of Health, Education, and Welfare.
MATERIALS AND METHODS Origin of oysters. Specimens of C. gigas were purchased from local Hong Kong ven81 Copyright © 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
82
THOMAS C. CHENG PEOPLE'S REPUBLIC OFCHINA Kwanat_ Province
FIG. 1. Map showing site at which the oysters, Crassostrea gigas, were collected. The shaded areas represent the oyster beds.
the specimens and all of the measurements were made with a calibrated ocular micrometer. Additional oysters were fixed in 10% seawater-formalin after their valves had been removed. These were subsequently embedded in Tissuemat, sectioned at 7 JLm, and selected sections were stained with Delafield's hematoxylin and eosin. In all, a total of 50 oysters were prepared as histological sections and an additional 150 were either dissected or digested to isolate the nematodes. Scanning electron microscopy (SEM). In preparation for SEM, whole specimens of the nematode removed from infected oysters by digestion were fixed in 2.5% glutaraldehyde in Millipore-filtered seawater for 4 hr and postfixed in 2% OS04 for 2 hr. They were subsequently dehydrated, dried by the critical point method of Ruffolo (1974), coated with a film of gold in vacuo, and examined with an ETEC SEM operated at 10 and 20 kV. OBSERVATIONS
Ecological observations. The waters from which the oysters were obtained have a salinity ranging from 10 to 30 0/00' depending on the tide. The oysters grow on the sand
and mud bottom of the intertidal zone, which extends from 7;1 to over Y2 mile from the high tide line. The distance between the high and low tide lines is approximately 9 ft. The temperatures on the days that I visited the oyster grounds during July and August 1974, ranged from 29.5 to 34°C and the relative humidity from 75 to 90%. During the winter the temperature can drop to as low as 1-3°e. Spawning of oysters. Although spawning of C. gigas at the sites mentioned is thought to occur from mid-April to the beginning of May (P. S. Wong, pers. commun.), examination of sections of 50 oysters revealed that 36 were in the female and 13 were in the male phase. The remaining specimen was in the transitional phase. Specimens of both sexes included gonads filled with ova or sperm and did not give the appearance of having spawned. However, judging by previous experience, it would not be surprising if the oysters commenced to spawn during the third week of August. The parasite. Both second- and thirdstage larvae of the gnathostomatid nematode were found in C. gigas. Of the 150 oysters dissected or digested, 22 harbored this parasite. One oyster contained both second- and third-stage larvae. Of the 50
83
Echinocephalus crassostreai SP . NOV.
oysters examined as histological sections, five included the parasite. Thus, out of 200 oysters examined, 27, or 13.5%, were infected with the nematode. Examination of whole worms led to the conclusion that they represent a new species
of the genus Echinocephalus, described below as E. crassostreai sp. nov. Echinocephalus erassostreai sp. nov.
Third-stage Echinocephalus erassostreai sp. nov. larva (Figs . 2-5). Description (based
CG
ES
CG
I.'
IN'
1h'j "1® .J e i !
FIGS . 2-8. AN, anus; CG, cervical gland ; CGO, duct of cervical gland ; CH, cephalic hook; ES, esophagus; EV, esophageal valve; GP, genital primordium; INT, intestine; L, lip; MO, mouth ; NR, nerve ring. FIG . 2. Anterior portion of third-stage larva of Echinocephalus crassostreai sp. nov. FIG. 3. Cephalic hook from eighth row of third- stage larva of Echino cephalus crassos treai sp. nov. FIG . 4. Third-stage larva of Echinocephalus crassos treai sp. nov . FIG . 5. En face view of third-st age larva of Echino cephalus crassostreai sp. nov. showing first two rows of cephalic hooks only. FIG . 6. Second-stage larva of Echin ocephalus crassos treai sp, nov. FIG. 7. Anterior portion of second-stage larva of Echinocephalus crassostreai sp. nov . FIG .8. Drawing showing esophageal valve in larval Echino cephalus crassostreai sp, nov.
84
THOMAS C. CHENG
on 12 specimens): Gnathostomatidae. Body 10.93 mm (8.16-13.59 mm) long and 0.33 mm (0.25-0.39 mm) wide at widest level; head bulb 0.15 mm (0.13-0.16 mm) long and 0.25 mm (0.24-0.26 mm) wide; head armed with eight circular rows of hooks (Figs. 2-5, 9, 10) projecting from the cuticle and with those comprising the posterior six rows being prominent; each hook of the eighth (most posterior) row averages 0.0025 mm long, each of the seventh row averages 0.0025 mm long, each of the sixth row averages 0.0023 mm long, each of the fifth row averages 0.0020 mm long, each of the fourth row averages 0.0018 mm long, and each of the third row averages 0.0016 mm long; hooks of first and second rows inconspicuous, with each of the second row averaging 0.0014 mm long and each of the first row averaging 0.0007 mm long; number of hooks in third through eighth rows varies,
with the numbers in rows 1-8 being 48, 56, 46-48,44-45,44-52,44-56,48-51, and 4648, respectively (Figs. 2, 9); terminal mouth guarded by two smooth-surfaced, bulbous lips (Figs. 2, 5, 9); four cervical glands, with ducts leading to anterior terminal, lying alongside and terminating at approximate midlength of esophagus (Fig. 4); esophagus, 1.63 mm (1.54-1.70 mm) long, with slightly bulbous posterior terminal which averages 0.20 mm (0.19-0.21 mm) wide; esophageal valve present (Fig. 8); intestine 6.71 mm (6.68-6.73 mm) long byO.ll mm (0. Jl-O.12 mm) wide, terminating at midventral anus located approximately 0.03 mm from posterior tip of body (Figs. 4, I 1); genital primordium, 0.01 by 0.01 mm, at approximate midlength of body, comprised of 10-25 cells (Fig. 4). No papillae present in pre- or postanal regions (Fig. II); phasmids, nerve ring, and excretory system not observed.
FIG . 9. Scanning electron micrograph of cephalic end of third-stage larva of Echinocephalus crassostreai sp. nov. L,lip. 400x .
Echinocephalus crassostreai
SP . NOV .
85
FIG . 10. Scanning electron micrograph of some cephalic hooks of third-stage larva of Ech inocephalus crasso s treai sp. nov. 1300x .
Since these were immature worms, bursae, spicules, and fully developed reproductive organs were not observed . Host: Crassostrea gigas, Japanese oyster. Location: Primarily in gonoduct. Locality: Off the coasts of the New Territories, Hong Kong, and the People's Republic of China in Hau Hoi Wan (Deep Bay). Holotype specimen: U.S. National Museum Helminthological Collection No. 73739. Second-stage larva (Figs. 6, 7). Description (based on two specimens): Gnathostomatidae. Body 4.98 mm long and 0.14 mm wide at widest level; head bulb 0.07 mm long and 0.09 mm wide; head armed with eight circular rows of minute hooks with largest averaging 0.008 mm long; two prominent smooth-surfaced lips present around terminal mouth (Figs. 6, 7); esophagus 0.95
mm long by 0.06 mm wide; four cervical glands situated alongside esophagus for most of its length (Fig. 6); intestine terminating posteriorly at midventral subterminal anus (Fig. 6); circumesophageal nerve ring present. No portions of reproductive system present. Justification for new species. Scanning electron microscopy has contributed to the elucidation of the morphological characteristics of E. crasso streai which, as pointed out below, distinguish the new species from those already described. Eight species of Echinicephalus have been named previously: E. uncinatus Molin, \858; E. striatus Monticelli, 1889; E. gracilis Stossich, 1906; E. spinosissimus (von Linstow, 1905); E. southwelli Baylis and Lane, 1920; E. multidentatus Baylis and Lane, 1929; E. aetobati MacCallum, 1921 ; and E. pseudouncinatus Millemann, 1951. However, according to Yamaguti (1961),
86
THOMAS C. CHENG
FIG. II. Scanning electron micrograph of posterior portion of third-stage larva of Echinocephalus crassostreai sp. nov. A, anus. 320x.
both E. gracilis and E. aetobati are synonymous with the generotype, E. uncinatus. Furthermore, Baylis and Lane (1920) have pointed out that there is no way of identifying Monticelli's (1889) E. striatus from the stomach of Scyllium sp. from Payta, Peru, and hence it must be considered a nomen nudum. In addition, Millemann (1963) has reduced von Linstow's (in Shipley and Hornell, 1904-1905) E. spinosissimus into synonymy with E. uncinatus. Consequently, only four species of Echinocephalus are considered to be valid prior to this report. Echinocephalus crassostreai differs from E. pseudounicinatus and E. multidentatus in that its lips do not bear teeth. There are two teeth on each of the lips of E. pseudouncinatus and eight to ten teeth on the lips of E. multidentatus. The new species can be distinguished from E. southwelli and E. uncinatus by the number of cephalic hooks. In the case of E. uncinatus, Baylis and Lane
(1920) have reported that the comparable larval stage in the marine bivalve Pinna sp. has six rows of 40~50 hooks and in the case of E. southwelli, Baylis and Lane (1920) have reported the presence of 15-18 rows of hooks with more than 100 hooks per row. Thus, it is apparent that E. crassostreai is distinct from the other known species. Millemann (1963), as a result of studying changes in the morphology of E. pseudouncinatus during growth, has noted that the definitive number of rows of cephalic hooks is attained by this group of worms at the third-stage molt, i.e., during the third larval stage, hence it would appear that the lesser number of rows of hooks, specifically eight, in E. crassostreai is a valid criterion for distinguishing it from E. southwelli, even if the only available description of the latter is of the adult. Also, studies by Millemann (1951, 1963) have revealed that dentitions on the lips persist from the larval stages through
Echinocephalus crassostreai
SP. NOV .
87
FIG . 12. C ross section through gonoduct of Crassostrea gigas showing third-stage larva of Echinocephalus ~rassos treai sp. nov. in lumen . Note tunic of reaction elements in perigonoductal region. GO, lumen of gonoduct; RT, reaction tunic. 130x. FIG. 13. Section showing third-stage larva of Echinocephalus crassostreai sp. nov. in ovary of Crasso strea gigas. Note abnorm al-appearing ova in proximity of parasite. DO, degener ate ova. 130x .
88
THOMAS C. CHENG
the adult form. Hence, the absence of teeth on the lips of E. crassostreai must be considered a valid criterion for distinguishing it from E. pseudouncinatus and E. multidentatus. Histopathology. Almost all the nematodes observed in histological preparations were found within the lumen of the gonoduct, especially in the proximal portion where the wall includes digitiform folds (Galtsoff, 1964) (Fig. 12). Echinocephalus crassostreai does not appear to cause appreciable histopathological changes associated with the ciliated epithelium lining the host's gonoduct. However, there is a conspicuous tunic consisting of connective tissue fibers, hemolymph cells (primarily granulocytes), myofibers, and tightly packed Leydig cells. These structural elements are embedded in an eosinophic ground substance (Fig. 12). This tunic, which averages 0.15 mm in thickness, does not exist in nonparasitized oysters. Only in one parasitized oyster were E. crassostreai larvae found in the gonad. The parasites were third-stage larvae and they were in the ovary (Fig. 13). The presence of nematodes appeared to cause the displacement of ova and many of the host's gametes in the adjacent zones were shrunken, ruptured, or compressed (Fig. 13). DISCUSSION By comparing the morphology of thirdstage larvae of an Echinocephalus from Crassostrea gigas collected in Hong Kong and the People's Republic of China with the descriptions of the other valid species of Echinocephalus, it is evident that the collected specimens represent a new species, E. crassostreai. The adult form is not yet known. It is noted, however, that Ko et al. (1974), who were the first to record the existence of what I have formally designated as E. crassostreai larvae, have stated that the ray, Aetabatus flagellum, is most likely the natural definitive host. Their speculation is based on the finding of third- and fourthstage larvae as well as adults of a species of
Echinocephalus in 11 of 15 rays caught near the oyster beds from which the oysters that contained E. crassostreai were obtained. However, direct proof that the Echinocephalus found by Ko et al. is the adult form of E. crassostreai is still wanting. Ko et al. (1974) also reported experimental infections of a cat and a rhesus monkey, Macaca mulatta, with E. crassostreai third-stage larvae isolated from C. gigas. The larvae penetrated the stomach and the small and large intestines of these experimental hosts. Because their report was in the form of an abstract, no details were presented. I have attempted to infect laboratory rats with third-stage larvae of E. crassostreai isolated from oysters by employing gastric tube feeding, but these attempts were unsuccessful. However, these were merely preliminary experiments involving feeding four larvae to each of four rats. Ko et al. fed 45 larvae to the cat and 200 larvae to the monkey and examined these hosts at 17 and 19 hr postfeeding, respectively. It should also be noted that P. S. Wong (pers. commun.) informed me that subsequent attempts to infect both kittens and monkeys have not been successful. Nevertheless, the preliminary report by Ko et al. suggests that the third-stage larva of E. crassostreai in C. gigas could be of potential public health importance. The occurrence of Echinocephalus larvae in marine molluscs has also been noted by Shipley and Hornell (1904-1905) and by Stossich (in Shipley and Hornell, 1906). These investigators have reported an undetermined species, perhaps E. uncinatus (= E. gracilis), encysted in the adductor muscle of the pearl oyster, Margaritifera vulgaris, in Ceylon. Also, Baylis and Lane (1920) have found E. uncinatus larvae encysted in Pinna sp. in Ceylon and Millemann (1951) has reported E. pseudouncinatus larvae encysted in the foot of the abalone, Haliotis corrugata, in southern California. In addition, Johnston and Mawson (1945) have recorded an unknown species in the gastropods Policines conica and Katylesia scalarina in
Echinocephalus crassos treai SP.
Australia. Of these earlier reports, only Millemann (1951) has made observations on the pathology caused by E. pseudouncinatus larvae in the molluscan host. He has reported: "Only old abalones were found to harbor the worms; these encyst in the ventral portion of the foot producing a blister-like effect and the burrowing of the larvae through the foot prior to encystment, apparently weakens the muscle and decreases the efficacy of this structure as a hold-fast organ." In the case of E. crassostreai in C. gigas, as stated, the parasite is not encapsulated. Furthermore, there are no external signs of infection, although histological examination of parasitized oysters has revealed a reaction complex surrounding the genital ducts in which the worms are located. It is noted, however, that of the constituents of the reaction complex, i.e., connective tissue fibers, hemolymph cells, tightly packed Leydig cells, and myofibers, the last mentioned are believed to be normally present surrounding gonoducts. Roughley (1933) has reported their presence surrounding gonoducts in Crassostrea commercialis, but Galtsoff (1938) has reported that they are missing at this location in C. virginica. Examination of sections of nonparasitized C. gigas has revealed that myofibers normally occur in the perigonoductal zone and hence their presence in this area of parasitized molluscs does not constitute a reaction. Nevertheless, the development of a reaction tunic around gonoducts enclosing E. crassostreai larvae is of interest. The lack of visible damage to the lining cells but the occurrence of the described reaction surrounding such a duct suggests possibly that a substance secreted or excreted by the parasite elicits the host response. It is noted that although some damage to adjacent ova has been observed in the oyster that harbored E. crassostreai in its gonad, the pathology was too slight to be regarded as parasitic castration. Finally, although Leydig cells occur in the perigonoductal zone of nonparasitized
NOV.
89
oysters, they become compressed in parasitized ones. Cytochemical studies are currently under way to ascertain whether there are any chemical alterations associated with this compression. ACKNOWLEDGMENTS I am grateful to Professor B. Lofts, Department of Zoology, University of Hong Kong, for making laboratory facilities available to me during my sojourn in Hong Kong; Dr. Ronald Ko of the same institution for assisting me in many ways; and Messers. John T. Sullivan and Thomas A. Garrabrant and Mrs. Diane H. Raymond, all of this Institute, for technical assistance.
REFERENCES BAYLIS, H. A. AND LANE, C. 1920. A revision of the nematode family Gnathostomatidae. Proc. Zool. Soc. London, 245-310. GALTSOFF, P. S. 1938. Physiology of reproduction of Ostrea virginica. Spawning reactions of the female and male. Bioi. Bull.. 74, 461-486. GALTSOFF, P. S. 1964. The American oyster Crassostrea virginica Gmelin. Fisher. Bull. Fish WildlifeServ., 64,1-480. JOHNSTON, T. H. AND MAWSON, P. M. 1945. Parasitic nematodes. Rep. Brit. Austral. New Zeal. Antarct. Res. Exped. 1929-/931,5,8(2),141. xo, R. C., MORTON, 8., AND WONG, P. S. 1974. Echinocephalus sp. Molin, 1858 (Spiruroidea: Gnathostornatidae), an unusual nematode from the oyster, Crassostrea gigas Thunberg, 1793. Proc. 3rd Inti. Congr. Parasitol., Munich, Germany, 3, 17311732. MILLEMANN, R. E. 1951. Echinocephalus pseudouncinatus n. sp., a nematode parasite of the abalone. J. Parasitol., 37,435- 439. MILLEMANN, R. E. 1963. Studies on the taxonomy and life history of echinocephalid worms (Nematoda: Spiruroidea) with a complete description of Echinocephalus pseudouncinatus Millemann, 1951. J. Parasitol., 49, 754--764. MONTICELLI, S. 1889. Elenco degli elminti raccolte da1 Capitano G. Chierchia durante 11 viaggio di circumnaviganzione della R. Corvetta "Vetter Pisano." Bull. Soc. Nat. Napoli, 3, G7-71. ROUGHLEY, T. C. 1933. The life history of the Australian oyster (Ostrea commercialis). Proc. Linn. Soc. New S. Wales, 58,279-333. RUFFOLO, J. J., JR. 1974. Critical point drying of protozoan cells and other biological specimens for scanning electron microscopy: apparatus and methods of specimen preparation. Trans. A mer. Microsc. Soc., 93, 124-131. SHIPLEY, A. E. AND HORNELL, J. 1904-1905. The parasites of the pearl oyster. In "Report to the
90
THOMAS C. CHENG
Government of Ceylon on Pearl Oyster Fisheries in the Gulf of Manaar" (W. A. Herdman, 00.), Pt. 2, pp. 77-106; Pt . 3,pp.49-56. London . SHIPLEY, A. E. AND HORNELL, J. 1906. Report on the cestode and nematode parasites from the marine fishes of Ceylon . In "Report to the Government of
Ceylon on Pearl Oyster Fisheries of the Gulf of Manaar" (W. A. Herdman, 00.), Pt. 5, pp. 43-96 . London . YAMAGUTI, S. 1961. "Systerna Helminthum," Vol. Ill. The Nematodes of Vertebrates, Pts. I and II . Interscience Publ., New York.
