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An unidentified protistan parasite in the ova of the blacklipped oyster, Crassostrea echinata, from northern Australia
JOLJRNAL
OP INYERTEBRATE
PATHOLOGY
29, 244-246 (1977)
An Unidentified Protistan Parasite in the Ova of the Blacklipped Oyster, Crassos&ea ec/Gnala, from Northern Australia During a routine histological examination of numerous specimens of blacklipped oysters, Crassostrea echinata , from the Northern Territory, Australia, an unidentified protistan was discovered in the developing and mature ova of almost 57% of the females examined. Parasitization of the gonads of males the same area was not observed. The specimens were obtained from Darwin Harbour for a detailed histological examination and were fixed in Davidson’s fluid, embedded, sectioned at 6-7 pm, and stained Harris’ hematoxylin and eosin (HHE) or Heidenhain’s hematoxylin. After the detection of the protistan in many ova of the fixed specimens (Fig. l), several dozen-iive oysters from the same
area were examined for the purpose of making in vivo observations of the intracellular organism. The oysters were carefully removed from their shells and cut transversely across the gonadal region. To obtain a smear, the cut oyster was rubbed against a glass slide. After about lo- 15 min, the ova, squashed by the weight of the coverslip, became flattened and, in many cases, the invading protistan was ejected and floated freely among the debris (Fig. 2). A similar procedure involving male specimens failed to reveal the presence of the intracellular organism in the testes examined. Several oysters suspected of being parasitized, were placed in a beaker containing sea water to which acridine orange (1:
FIG. 1. Ovum with parasite. HHE (x 1000). P = parasite, N = nucleus of ovum.
244 ISSN @XL?-2011
NOTES
FIG. 2. Ovum of Crassostrea echinata. Live smear/squash preparation showing nucleus of ovum and protista ,n almost extruded from egg. Note septum in cyst. (Interference contrast; x 1000.)
FIG. 3. Two ova of Cramostrea echinata. Smear of material which was stained with acridine orange in vivo under fluorescent light. Note two cysts in one ovum, division septum of cysts, and grr undated nuclei of protistan. (Incident fluorescence; x 1000.)
NOTES 100,000) was added and were left for 24 hr. On opening the shells, the oysters were distinct yellow, particularly the gills. Under fluorescent light, in a smear prepared as described above, observation revealed up to two cysts (Fig. 2) with four parasites adjacent to the egg nucleus (Fig. 3) within some ova. The organism occupies the yolk portion of the ovum adjacent to the nuclear vesicle. The number of cysts per ovum varies from one to two. The individual cyst has a clear membrane, is spherical, and is divided in the center by a transverse, equally transparent membrane. The latter is not readily discernible in fixed histological material (Fig. 1) but can be clearly seen in live preparations under interference contrast optics. Thus the sphere is divided into two chambers, each of which harbors one unicellular organism. The discovery of an intracellular protistan in the ova of oysters certainly is important enough to be recorded, although its identity, even at the subclass level, has hitherto remained unsolved in spite of the efforts of specialists such as Dr. V. Sprague and Dr. Norman D. Levine, to whom photographs have been sent for identification. It is likely, however, that the invading protistan belongs to the subclass Coccidiida (N. D. Levine, pers. comm.) The question whether to consider the invading organism
to be a pathogen or merely one which seeks convenient dispersal cannot be conclusively answered without further detailed research on its full life cycle. However, the suggestion is made that the organism most likely is not pathogenic but, rather, utilizes the ripening ovum as a vector for completion of its life cycle, perhaps involving a final host other than the oyster. In support of this suggestion, the sex ratio and incidence of parasitization are taken into account. The normal sex ratio in oysters is 2.7 females to one male at an age of 2 years. If 57% of all the observed females, or 43% of the total population, would have been infected by a highly damaging organism, a mass mortality of oysters in the area would not have passed unnoticed. However, most of the specimens examined were in excellent condition, with their gonads ripe and running, in spite of the infected ova. It is at present unknown at what stage of development of the ovum the protistan enters. Some very immature young ova have been noticed containing the organism which most likely invades them at a very early stage, PETERH. WOLF New South Wales State Fisheries, Scient$c Section 211 Kent Street Sydney, New South Wales Australia Received June 22, 1976
OP INYERTEBRATE
PATHOLOGY
29, 244-246 (1977)
An Unidentified Protistan Parasite in the Ova of the Blacklipped Oyster, Crassos&ea ec/Gnala, from Northern Australia During a routine histological examination of numerous specimens of blacklipped oysters, Crassostrea echinata , from the Northern Territory, Australia, an unidentified protistan was discovered in the developing and mature ova of almost 57% of the females examined. Parasitization of the gonads of males the same area was not observed. The specimens were obtained from Darwin Harbour for a detailed histological examination and were fixed in Davidson’s fluid, embedded, sectioned at 6-7 pm, and stained Harris’ hematoxylin and eosin (HHE) or Heidenhain’s hematoxylin. After the detection of the protistan in many ova of the fixed specimens (Fig. l), several dozen-iive oysters from the same
area were examined for the purpose of making in vivo observations of the intracellular organism. The oysters were carefully removed from their shells and cut transversely across the gonadal region. To obtain a smear, the cut oyster was rubbed against a glass slide. After about lo- 15 min, the ova, squashed by the weight of the coverslip, became flattened and, in many cases, the invading protistan was ejected and floated freely among the debris (Fig. 2). A similar procedure involving male specimens failed to reveal the presence of the intracellular organism in the testes examined. Several oysters suspected of being parasitized, were placed in a beaker containing sea water to which acridine orange (1:
FIG. 1. Ovum with parasite. HHE (x 1000). P = parasite, N = nucleus of ovum.
244 ISSN @XL?-2011
NOTES
FIG. 2. Ovum of Crassostrea echinata. Live smear/squash preparation showing nucleus of ovum and protista ,n almost extruded from egg. Note septum in cyst. (Interference contrast; x 1000.)
FIG. 3. Two ova of Cramostrea echinata. Smear of material which was stained with acridine orange in vivo under fluorescent light. Note two cysts in one ovum, division septum of cysts, and grr undated nuclei of protistan. (Incident fluorescence; x 1000.)
NOTES 100,000) was added and were left for 24 hr. On opening the shells, the oysters were distinct yellow, particularly the gills. Under fluorescent light, in a smear prepared as described above, observation revealed up to two cysts (Fig. 2) with four parasites adjacent to the egg nucleus (Fig. 3) within some ova. The organism occupies the yolk portion of the ovum adjacent to the nuclear vesicle. The number of cysts per ovum varies from one to two. The individual cyst has a clear membrane, is spherical, and is divided in the center by a transverse, equally transparent membrane. The latter is not readily discernible in fixed histological material (Fig. 1) but can be clearly seen in live preparations under interference contrast optics. Thus the sphere is divided into two chambers, each of which harbors one unicellular organism. The discovery of an intracellular protistan in the ova of oysters certainly is important enough to be recorded, although its identity, even at the subclass level, has hitherto remained unsolved in spite of the efforts of specialists such as Dr. V. Sprague and Dr. Norman D. Levine, to whom photographs have been sent for identification. It is likely, however, that the invading protistan belongs to the subclass Coccidiida (N. D. Levine, pers. comm.) The question whether to consider the invading organism
to be a pathogen or merely one which seeks convenient dispersal cannot be conclusively answered without further detailed research on its full life cycle. However, the suggestion is made that the organism most likely is not pathogenic but, rather, utilizes the ripening ovum as a vector for completion of its life cycle, perhaps involving a final host other than the oyster. In support of this suggestion, the sex ratio and incidence of parasitization are taken into account. The normal sex ratio in oysters is 2.7 females to one male at an age of 2 years. If 57% of all the observed females, or 43% of the total population, would have been infected by a highly damaging organism, a mass mortality of oysters in the area would not have passed unnoticed. However, most of the specimens examined were in excellent condition, with their gonads ripe and running, in spite of the infected ova. It is at present unknown at what stage of development of the ovum the protistan enters. Some very immature young ova have been noticed containing the organism which most likely invades them at a very early stage, PETERH. WOLF New South Wales State Fisheries, Scient$c Section 211 Kent Street Sydney, New South Wales Australia Received June 22, 1976