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The role of Uronema marinum (Protozoa) in oyster hatchery production
Aquaculture, 15 (1978) 219-225 o Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
THE ROLE OF URONEMA MARINUM (PROTOZOA) HATCHERY PRODUCTION
LINDA PLUNKET and HERBERT
219
IN OYSTER
HIDU
University of Maine, Ira C. Darling Center, Walpole, Maine 04573 (U.S.A.) Ira C. Darling Center Contribution No. 124. (Received 4 May 1978; revised 7 August 1978)
ABSTRACT Plunket, L. and Hidu, H., 1978. The role of Uronema marinum (Protozoa) hatchery production. Aquaculture, 15: 219-225.
in oyster
In Maine’s hatchery production of oysters, significant mortalities at the early juvenile stage have been associated with ciliate infestations. The predominant ciliate isolated from live infested oysters was identified as Uronema marinum. Feeding experiments, designed to identify the food source of the ciliate in the oyster tank environment, determined that U. marinum is a bacteriophage and not a histophage, thereby clarifying its role in the oyster mortalities.
INTRODUCTION
This work investigated the feeding type of Uronema marinum, a ciliate found invading hatchery reared cultchless oysters. In recent years, the University of Maine has adapted and modified hatchery techniques for commercial application in this subboreal region, A particularly vexing hatchery problem has been consistently high losses of both American oysters Crassostrea virginica Gmelin and European oysters Ostrea edulis Linnaeus during the early cultchless juvenile phase following metamorphosis. The oysters are removed from marble, the setting substrate, approximately 48 h after metamorphosis, with some unavoidable shell damage. During the 5day period following metamorphosis, mortalities of 10 to 80% have occurred and the higher mortalities have coincided with heavy infestations of ciliates. Tubiash et al. (1965) noted the presence of ciliates in moribund larval and juvenile mollusks and felt that the ciliates functioned as scavengers on oysters weakened by bacterial infestation. In reporting on a fungal disease of lamellibranch larvae, Davis et al. (1954) stated that “usually several species of flagellates and ciliates invade the dead or dying larvae to feed upon their tissues leaving . . . empty shells with practically no traces of the fungus”.
220
The tetrahymenid hymenostomes are known to include a wide spectrum of feeding types. Three families in this suborder, Cohnilembidae, Philasteridae, and Ophryoglenidae, display different stages in the evolution from bacteriophages to histophages and parasites (Fenchel, 1968). Species included in the family Cohnilembidae have been known to exhibit histophagic and bacteriophagic behavior simultaneously. Fenchel(1968) suggested that ciliates feeding on the bacteria associated with decaying tissue might have specialized and evolved as histophages. The ecology and feeding type of U. marinum has been speculated on by many workers. Mugard (1949) referred to U. marinum as a histophage. Borror (1963) noted that U. marinum congregates in areas containing high concentrations of bacteria and feeds while practically motionless. Fenchel (1968) observed U. marinum gathering and multiplying around dead and decaying animals in samples of marine sediment and noted how easily the species can be cultured in bacterized peptone solutions. Hamilton and Preslan (1969) observed an oceanic species of Uronema feeding upon bacteria. They reported that Uronemu sp. was able to utilize the following genera of bacteria: Chromobucterium, Serratiu, Vibrio, Pseudomonas, and Micrococcus. Hanna and Lilly (1974) speculated that under natural conditions U. marinum might feed on the bacteria associated with the decomposition of the common sea lettuce, Ulvu luctucu. Thompson and Berger (1965) described a marine ciliate, Puranophrys marina, family Uronematidae, which is found in association with a hydroid, Plumuluriu. This species is also capable of surviving in the absence of this host. Thompson and Moewus (1964) reported on a marine facultative parasite, Miumiensis avidus, associated with the skin tumors of a seahorse. This species was originally placed in the family Uronematidae by Thompson, but Corliss (1974) assigned this species to the family Philasteridae. Thus, as the work on ciliate feeding types progresses it is apparent that a greater number of species are able to utilize a greater variety of food sources than was previously assumed (Fenchel, 1968). The experiments reported here were a necessary prerequisite in determining the importance of controlling the ciliate infestations in our oyster nursery. It was mandatory to know whether the ciliates attacked oyster tissue directly or fed exclusively on the bacterial populations that may have multiplied on moribund oysters which were harmed by other causes. MATERIALS
AND METHODS
Hatchery reared oyster spat were obtained by modifications of techniques derived by Loosanoff and Davis (1963). Cultchless spat were procured by first setting larvae on polished marble (Hidu et al., 1974). Spat were then removed with a blade 24-48 h after attachment at which time a band of early juvenile shell growth was evident. The cultchless spat were rinsed thoroughly and placed in stacks of screens
221
in a continuously circulating, closed water system. The flagellates Isochrysis galbana Parke, Monochrysis lutheri Droop and the diatom Phaeodactylum tri cornutum Bohlin were routinely fed to juveniles commensurate with clearing rates. Isochrysis galbana was, at all times, the dominant food source. The ciliates were isolated by placing a live infested oyster in a sterile boveri dish containing sterile sea water. After 1 to 2 days the empty oyster shell was removed leaving a viable culture of cilia& and a polished rice grain was added as the food source. Transfers were made weekly from cultures which were kept at room temperature in the dark. A single ciliate was isolated from these cultures to initiate a clone in a test tube of sea water containing a polished rice grain under bacteria-free conditions. Many cultures which originated from a single clone were combined to provide the high concentration of ciliates required for acceptable staining. The cultures were centrifuged, the supernatant discarded, and the pellet of ciliates resuspended in additional media containing ciliates. This process was repeated until a sufficient concentration of ciliates was achieved to facilitate staining with the wet silver stain, the Feulgen nuclear reaction, and the stain-fixature nigrosin-HgCl? -formalin. Ciliates were sterilized using four antibiotics; penicillin, streptomycin, polymyxin B sulfate and Fungizone (Lee et al., 1971). The antibiotics were sterilized by dissolving them in appropriate amounts of sterile sea water and passing the liquid through a 0.45 pm filter housed in a sterile Millipore filtration unit. Small aliquots were then aseptically dispensed into test tubes and frozen until needed. Eight successive transfers were performed before axenic culture was achieved using a standard inoculum of 0.1 ml per 3 ml of fresh medium. The length of time between each transfer was dependent on the number of days necessary to repopulate the test tubes, this ranging between 3 days for early and 20 days for later tranfers. The presence or absence of bacteria was monitored using thioglycolate and Difco marine broth. Thioglycolate was found to be unsuitable as the sole monitoring agent since it is specific for fresh water or euryhaline anaerobes. The two diets employed during the sterilization procedures were suggested by Hanna and Lilly (1970,1974). The feeding experiments were designed to ascertain which food sources in the tank environments were utilized by the ciliates. The three food types were procured as follows. The original clone of I. galbana was obtained from Helen Stanley of the Woods Hole Oceanography Institution and the sterility monitored as above. The bacteria were obtained from sea lettuce, Ulua lactuca Linnaeus, harvested from rafts on the Damariscotta River, and allowed to disintegrate in sterile sea water at room temperature. After 72 h a test tube containing sterile marine broth was inoculated but the resulting bacterial flora was not plated or identified. If the food source was bacterial, it seemed unlikely that the ciliates had specialized on a single species of bacteria. The ideal oyster tissue food source would have been aseptic juvenile tissue; however due to difficulties in sterilizing this tissue in
222
unmodified form, adult oyster heart tissue was utilized. To avoid contamination during dissection, it was necessary to relax the oyster in a 0.0015 solution of propylene phenoxytol. Once dissected, the hearts were quickly placed in sterile sea water containing four antibiotics, namely 200 units/ml mycostatin, 300 mg/ml tylocene (anti-PPLO agent), 750 units/ml penicillin G. and 100 mg/ml streptomycin sulfate (B.L. Nicholson, personal communication, 1975). Sterility was monitored as above. The first experiment tested the nutritive value of each food source alone. The six treatments offered to U. marinum included two concentrations of sterile I. gulbana, two concentrations of bacteria, sterile oyster tissue, and sterile sea water as the control. Triplicates of the six treatments were sampled at Days 3, 5 and 7. Initially, 54 test tubes contained 5 ml of sterile sea water and out of these, five groups of nine tubes were inoculated as follows: 0.2 ml of a bacteria-free culture of I. gulbana in log phase growth; 1 ml of the same culture; one drop of a 24-h bacterial culture; two drops from the same culture; and, finally, oyster heart rinsed in sterile sea water. All test tubes received 0.3 ml of a sterile ciliate culture at a density of 10 000 per ml and then were incubated at room temperature in the dark. On Day 3, triplicates of each treatment were killed by adding three drops of 10% (u/u) formalin to each test tube. The ciliates were counted under a compound microscope after staining with Lugol’s solution. On Day 5 and Day 7 the process was repeated with the remaining live cultures. The results were computer analysed using programs from the Statistical Package for the Social Sciences. ANOVA tables were constructed and the Duncan’s multiple range test applied to the results. The second experiment investigated the possibility that bacterized (lysed) oyster tissue might function as a food source. The three treatments offered were sterile oyster tissue, bacteria, and the combination of oyster tissue and bacteria. Growth was designated as the increase in the number of ciliates per unit volume per unit time with experimental cultures compared to the unfed control cultures. Triplicates of each of three treatments were inoculated as follows: four drops of an 18-h bacterial culture; a single oyster heart; and lastly, four drops of an 18-h bacterial culture plus a single oyster heart. All tubes received 0.3 ml of a sterile ciliate culture and were incubated at room temperature in the dark. On Day 6, tests determined that the tissue treatment was indeed sterile. The contents of all test tubes were killed, stained, counted and analyzed as in the first experiment. RESULTS
The ciliate isolated from infested juvenile oysters was identified as U. as redescribed by Thompson (1964). This strain was slightly smaller than the type species, averaging 30 X 15 pm rather than 33 X 17 pm and exhibited an average of ten kineties. The usual number for this species ranges from 13 to 16. marinum
223
Feulgen’s nuclear reaction was applied three times to this strain of U. marinum with poor results. The material was lightly stained never achieving the clarity which normally characterizes this reaction. This strain, however, was unquestionably amicronucleate; and is the first reported amicronucleate strain of U. marinum. Of the five food sources tested in the first experiment only the two bacterial treatments resulted in growth of the ciliates (Fig. 1). There were no
Fig. 1. Numbers of Uronema marinum in response to several food sources as follows: Control 0 l ; Isochrysis galbana (A) l - - - - l ; Isochrysis galbana (B) l Bacterial (A) n - - - - n ; Bacterial (B) 0 -------0 ; Oyster tissue l - - - -4.
l
;
significant differences among treatments on Day 3, but significant differences were apparent in the bacterial treatments on Day 5 and 7. Only bacterial treatment B was significantly different from the control on Day 5, while on Day 7, both bacterial treatments A and B were significantly different from the control as revealed by the Duncan’s multiple range test. These results were confirmed in the second experiment where, again, only the bacterial treatments were associated with ciliate reproduction (Fig. 2). The combination of bacteria plus oyster tissue did not result in additional reproduction of the ciliates; thus it is apparent that the ciliates did not derive benefit from oyster tissue that may have been lysed by bacterial action. DISCUSSION
This investigation defined a specific trophic niche for U. marinum as a bacteriophage, not as a histophage, in our oyster tank environment. Thus, the consistently high mortalities accompanying the early juvenile stage of hatchery oysters cannot be attributed primarily to the ciliate infestation. Rather, these infestations are a symptom of different problems possibly including: shell damage during removal from the cultch and/or high bacterial concentration. Oyster shell damage which occurs on removal is the most plausible explanation. Invariably, as the metamorphosed juveniles are removed from the cultch they are damaged to some extent. The endemic population of bacteria
224
A=Bactsria B=Oystsr
tissue
C=Bacterio
8
Oyster
tissue
IOOO-
0
A TRE&NTC
Fig. 2. Numbers of Uronema marinum in response to bacteria and oyster food sources, alone and in combination, after 6 days of culture.
then, no doubt, responds to the damaged oyster tissue. Bacteriophagic ciliates, which appear to be ubiqitous, respond to the expanding bacterial population and enter an exponential growth phase. These speculations agree with the findings of L. Leibovitz (personal communication, 1975) who experimentally determined that ciliates characteristically appear in larval cul tures as the bacterial concentrations increase. During the Spring of 1974 and 1975 all of the ciliate isolates were identified as U. marinum, but other unidentified species were observed in low numbers in cultures of larval and juvenile oysters. It is not unreasonable to assume that one or more of these species might initiate an infestation. The ciliate infestations occurring in the hatchery are not unique. Other workers have noted Uronema infesting recently killed crustacea and have speculated on the feeding type of the ciliate. Finenko and Zaika (1970) placed dead Arcartiu clausi Giesbrecht in a sea water bath and made observations on the microorganisms which infiltrated the dead individuals. After 3 h, ciliates of the genera Euplotes and Uronemu as well as nematodes were visible in the animals. Although no controlled experiments were performed and no bacterial counts taken, high numbers of Uronema seemed to correspond with low concentrations of bacteria and low numbers of Uronemu seemed to correspond with high concentrations of bacteria. From these observations Finenko and Zaika speculated that Uronemu was not a histophage but a bacteriophage, a conclusion supported by these experiments.
225
ACKNOWLEDGEMENTS
We thank Dr. David Dean and Samuel R. Chapman for significant advice and support. Dr. Arthur Borror of the University of New Hampshire kindly provided much technical information with the Protozoa. The study was supported by NOAA, Office of Sea Grant, Project No. NG40-72.
REFERENCES Borror, A.C., 3.963. Morphology and ecology of the benthic ciliated Protozoa of Alligator Harbor, Florida. Arch. Protistenkd., 106: 465-534. Corliss, J.O., 1974. The changing world of ciliate systematics: historical analysis of past efforts and a newly proposed phylogenetic scheme of classification for the protistan phylum Ciliophora. Syst. Zool., 23: 91-138. Davis, H.C., Loosanoff, V.L., Weston, W.H. and Martin, C., 1954. A fungus disease in clam and oyster larvae. Science, 120: 36-38. Fenchel, T., 1968. The ecology of marine microbenthos II. The food of marine benthic ciliates. Ophelia, 5 : 73-l 21. Finenko, Z.Z. and Zaika, V.W., 1970. Particulate organic matter and its role in the productivity of the sea. In: J.M. Steele (Editor), Marine Food Chains. University of California Press, Berkeley, Calif., pp. 32-44. Hamilton, R.D. and Preslan, J.E., 1969. Cultural characteristics of a pelagic marine hymenostome ciliate, Uronema sp. J. Exp. Mar. Biol. Ecol., 4: 90-99. Hanna, B.A. and Lilly, D.M., 1970. Axenic culture of Uronema marinum. Am. Zoo]., 10: 539-540. Hanna, B.A. and Lilly, D.M., 1974. Growth of Uronema marinum in chemically defined medium. Mar. Biol., 26: 153-160. Hidu, H., Chapman, S. and Soule, P.W., 1974. Cultchless setting of European oysters, Ostrea edulis, using polished marble. Proc. Nat. Shellfish Assoc., 65: 13-14. Lee, J.J., Teitjen, J.H. and Mastropaolo, C., 1971. Axenic culture of the marine hymenostome ciliate Urnonema morinum in chemically defined medium. J. Protozool., 18: (Suppl.), 11. Loosanoff, V.L. and Davis, H.C., 1963. Rearing of bivalve mollusks. In: F.S. Russell (Editor), Advances in Marine Biology. Academic Press, London, pp. l-136. Mugard, H., 1949. Contribution a 1’Btude des infusoires hymenostomes histiophages. Ann Sci. Nat. Zool. Biol. Anim. (Ser. II), 10: 171-268. Thompson Jr., J.C., 1964. A redescription of Uronema marinum and a proposed new family, Uronematidae. Va. J. Sci., 15: 80-87. Thompson Jr., J.C. and Berger, J., 1965. Paranophrys marina n. g., n. sp., a new ciliate associated with a hydroid from the northeast Pacific (Ciliata: Hymenostomatida). J. Protozool., 12: 527-531. Thompson Jr., J.C. and Moewus, L., 1964. Miamiensis auidus n. g., n. ap., a marine facultative parasite in the ciliate order Hymenostomatida. J. Protozool., 11: 378-381. Tubiash, H.S., Chanley, P.E. and Leifson, E., 1965. Bacillary necrosis, a disease of larval and juvenile bivalve mollusks. J. Bacterial., 90: 1036-1044.
THE ROLE OF URONEMA MARINUM (PROTOZOA) HATCHERY PRODUCTION
LINDA PLUNKET and HERBERT
219
IN OYSTER
HIDU
University of Maine, Ira C. Darling Center, Walpole, Maine 04573 (U.S.A.) Ira C. Darling Center Contribution No. 124. (Received 4 May 1978; revised 7 August 1978)
ABSTRACT Plunket, L. and Hidu, H., 1978. The role of Uronema marinum (Protozoa) hatchery production. Aquaculture, 15: 219-225.
in oyster
In Maine’s hatchery production of oysters, significant mortalities at the early juvenile stage have been associated with ciliate infestations. The predominant ciliate isolated from live infested oysters was identified as Uronema marinum. Feeding experiments, designed to identify the food source of the ciliate in the oyster tank environment, determined that U. marinum is a bacteriophage and not a histophage, thereby clarifying its role in the oyster mortalities.
INTRODUCTION
This work investigated the feeding type of Uronema marinum, a ciliate found invading hatchery reared cultchless oysters. In recent years, the University of Maine has adapted and modified hatchery techniques for commercial application in this subboreal region, A particularly vexing hatchery problem has been consistently high losses of both American oysters Crassostrea virginica Gmelin and European oysters Ostrea edulis Linnaeus during the early cultchless juvenile phase following metamorphosis. The oysters are removed from marble, the setting substrate, approximately 48 h after metamorphosis, with some unavoidable shell damage. During the 5day period following metamorphosis, mortalities of 10 to 80% have occurred and the higher mortalities have coincided with heavy infestations of ciliates. Tubiash et al. (1965) noted the presence of ciliates in moribund larval and juvenile mollusks and felt that the ciliates functioned as scavengers on oysters weakened by bacterial infestation. In reporting on a fungal disease of lamellibranch larvae, Davis et al. (1954) stated that “usually several species of flagellates and ciliates invade the dead or dying larvae to feed upon their tissues leaving . . . empty shells with practically no traces of the fungus”.
220
The tetrahymenid hymenostomes are known to include a wide spectrum of feeding types. Three families in this suborder, Cohnilembidae, Philasteridae, and Ophryoglenidae, display different stages in the evolution from bacteriophages to histophages and parasites (Fenchel, 1968). Species included in the family Cohnilembidae have been known to exhibit histophagic and bacteriophagic behavior simultaneously. Fenchel(1968) suggested that ciliates feeding on the bacteria associated with decaying tissue might have specialized and evolved as histophages. The ecology and feeding type of U. marinum has been speculated on by many workers. Mugard (1949) referred to U. marinum as a histophage. Borror (1963) noted that U. marinum congregates in areas containing high concentrations of bacteria and feeds while practically motionless. Fenchel (1968) observed U. marinum gathering and multiplying around dead and decaying animals in samples of marine sediment and noted how easily the species can be cultured in bacterized peptone solutions. Hamilton and Preslan (1969) observed an oceanic species of Uronema feeding upon bacteria. They reported that Uronemu sp. was able to utilize the following genera of bacteria: Chromobucterium, Serratiu, Vibrio, Pseudomonas, and Micrococcus. Hanna and Lilly (1974) speculated that under natural conditions U. marinum might feed on the bacteria associated with the decomposition of the common sea lettuce, Ulvu luctucu. Thompson and Berger (1965) described a marine ciliate, Puranophrys marina, family Uronematidae, which is found in association with a hydroid, Plumuluriu. This species is also capable of surviving in the absence of this host. Thompson and Moewus (1964) reported on a marine facultative parasite, Miumiensis avidus, associated with the skin tumors of a seahorse. This species was originally placed in the family Uronematidae by Thompson, but Corliss (1974) assigned this species to the family Philasteridae. Thus, as the work on ciliate feeding types progresses it is apparent that a greater number of species are able to utilize a greater variety of food sources than was previously assumed (Fenchel, 1968). The experiments reported here were a necessary prerequisite in determining the importance of controlling the ciliate infestations in our oyster nursery. It was mandatory to know whether the ciliates attacked oyster tissue directly or fed exclusively on the bacterial populations that may have multiplied on moribund oysters which were harmed by other causes. MATERIALS
AND METHODS
Hatchery reared oyster spat were obtained by modifications of techniques derived by Loosanoff and Davis (1963). Cultchless spat were procured by first setting larvae on polished marble (Hidu et al., 1974). Spat were then removed with a blade 24-48 h after attachment at which time a band of early juvenile shell growth was evident. The cultchless spat were rinsed thoroughly and placed in stacks of screens
221
in a continuously circulating, closed water system. The flagellates Isochrysis galbana Parke, Monochrysis lutheri Droop and the diatom Phaeodactylum tri cornutum Bohlin were routinely fed to juveniles commensurate with clearing rates. Isochrysis galbana was, at all times, the dominant food source. The ciliates were isolated by placing a live infested oyster in a sterile boveri dish containing sterile sea water. After 1 to 2 days the empty oyster shell was removed leaving a viable culture of cilia& and a polished rice grain was added as the food source. Transfers were made weekly from cultures which were kept at room temperature in the dark. A single ciliate was isolated from these cultures to initiate a clone in a test tube of sea water containing a polished rice grain under bacteria-free conditions. Many cultures which originated from a single clone were combined to provide the high concentration of ciliates required for acceptable staining. The cultures were centrifuged, the supernatant discarded, and the pellet of ciliates resuspended in additional media containing ciliates. This process was repeated until a sufficient concentration of ciliates was achieved to facilitate staining with the wet silver stain, the Feulgen nuclear reaction, and the stain-fixature nigrosin-HgCl? -formalin. Ciliates were sterilized using four antibiotics; penicillin, streptomycin, polymyxin B sulfate and Fungizone (Lee et al., 1971). The antibiotics were sterilized by dissolving them in appropriate amounts of sterile sea water and passing the liquid through a 0.45 pm filter housed in a sterile Millipore filtration unit. Small aliquots were then aseptically dispensed into test tubes and frozen until needed. Eight successive transfers were performed before axenic culture was achieved using a standard inoculum of 0.1 ml per 3 ml of fresh medium. The length of time between each transfer was dependent on the number of days necessary to repopulate the test tubes, this ranging between 3 days for early and 20 days for later tranfers. The presence or absence of bacteria was monitored using thioglycolate and Difco marine broth. Thioglycolate was found to be unsuitable as the sole monitoring agent since it is specific for fresh water or euryhaline anaerobes. The two diets employed during the sterilization procedures were suggested by Hanna and Lilly (1970,1974). The feeding experiments were designed to ascertain which food sources in the tank environments were utilized by the ciliates. The three food types were procured as follows. The original clone of I. galbana was obtained from Helen Stanley of the Woods Hole Oceanography Institution and the sterility monitored as above. The bacteria were obtained from sea lettuce, Ulua lactuca Linnaeus, harvested from rafts on the Damariscotta River, and allowed to disintegrate in sterile sea water at room temperature. After 72 h a test tube containing sterile marine broth was inoculated but the resulting bacterial flora was not plated or identified. If the food source was bacterial, it seemed unlikely that the ciliates had specialized on a single species of bacteria. The ideal oyster tissue food source would have been aseptic juvenile tissue; however due to difficulties in sterilizing this tissue in
222
unmodified form, adult oyster heart tissue was utilized. To avoid contamination during dissection, it was necessary to relax the oyster in a 0.0015 solution of propylene phenoxytol. Once dissected, the hearts were quickly placed in sterile sea water containing four antibiotics, namely 200 units/ml mycostatin, 300 mg/ml tylocene (anti-PPLO agent), 750 units/ml penicillin G. and 100 mg/ml streptomycin sulfate (B.L. Nicholson, personal communication, 1975). Sterility was monitored as above. The first experiment tested the nutritive value of each food source alone. The six treatments offered to U. marinum included two concentrations of sterile I. gulbana, two concentrations of bacteria, sterile oyster tissue, and sterile sea water as the control. Triplicates of the six treatments were sampled at Days 3, 5 and 7. Initially, 54 test tubes contained 5 ml of sterile sea water and out of these, five groups of nine tubes were inoculated as follows: 0.2 ml of a bacteria-free culture of I. gulbana in log phase growth; 1 ml of the same culture; one drop of a 24-h bacterial culture; two drops from the same culture; and, finally, oyster heart rinsed in sterile sea water. All test tubes received 0.3 ml of a sterile ciliate culture at a density of 10 000 per ml and then were incubated at room temperature in the dark. On Day 3, triplicates of each treatment were killed by adding three drops of 10% (u/u) formalin to each test tube. The ciliates were counted under a compound microscope after staining with Lugol’s solution. On Day 5 and Day 7 the process was repeated with the remaining live cultures. The results were computer analysed using programs from the Statistical Package for the Social Sciences. ANOVA tables were constructed and the Duncan’s multiple range test applied to the results. The second experiment investigated the possibility that bacterized (lysed) oyster tissue might function as a food source. The three treatments offered were sterile oyster tissue, bacteria, and the combination of oyster tissue and bacteria. Growth was designated as the increase in the number of ciliates per unit volume per unit time with experimental cultures compared to the unfed control cultures. Triplicates of each of three treatments were inoculated as follows: four drops of an 18-h bacterial culture; a single oyster heart; and lastly, four drops of an 18-h bacterial culture plus a single oyster heart. All tubes received 0.3 ml of a sterile ciliate culture and were incubated at room temperature in the dark. On Day 6, tests determined that the tissue treatment was indeed sterile. The contents of all test tubes were killed, stained, counted and analyzed as in the first experiment. RESULTS
The ciliate isolated from infested juvenile oysters was identified as U. as redescribed by Thompson (1964). This strain was slightly smaller than the type species, averaging 30 X 15 pm rather than 33 X 17 pm and exhibited an average of ten kineties. The usual number for this species ranges from 13 to 16. marinum
223
Feulgen’s nuclear reaction was applied three times to this strain of U. marinum with poor results. The material was lightly stained never achieving the clarity which normally characterizes this reaction. This strain, however, was unquestionably amicronucleate; and is the first reported amicronucleate strain of U. marinum. Of the five food sources tested in the first experiment only the two bacterial treatments resulted in growth of the ciliates (Fig. 1). There were no
Fig. 1. Numbers of Uronema marinum in response to several food sources as follows: Control 0 l ; Isochrysis galbana (A) l - - - - l ; Isochrysis galbana (B) l Bacterial (A) n - - - - n ; Bacterial (B) 0 -------0 ; Oyster tissue l - - - -4.
l
;
significant differences among treatments on Day 3, but significant differences were apparent in the bacterial treatments on Day 5 and 7. Only bacterial treatment B was significantly different from the control on Day 5, while on Day 7, both bacterial treatments A and B were significantly different from the control as revealed by the Duncan’s multiple range test. These results were confirmed in the second experiment where, again, only the bacterial treatments were associated with ciliate reproduction (Fig. 2). The combination of bacteria plus oyster tissue did not result in additional reproduction of the ciliates; thus it is apparent that the ciliates did not derive benefit from oyster tissue that may have been lysed by bacterial action. DISCUSSION
This investigation defined a specific trophic niche for U. marinum as a bacteriophage, not as a histophage, in our oyster tank environment. Thus, the consistently high mortalities accompanying the early juvenile stage of hatchery oysters cannot be attributed primarily to the ciliate infestation. Rather, these infestations are a symptom of different problems possibly including: shell damage during removal from the cultch and/or high bacterial concentration. Oyster shell damage which occurs on removal is the most plausible explanation. Invariably, as the metamorphosed juveniles are removed from the cultch they are damaged to some extent. The endemic population of bacteria
224
A=Bactsria B=Oystsr
tissue
C=Bacterio
8
Oyster
tissue
IOOO-
0
A TRE&NTC
Fig. 2. Numbers of Uronema marinum in response to bacteria and oyster food sources, alone and in combination, after 6 days of culture.
then, no doubt, responds to the damaged oyster tissue. Bacteriophagic ciliates, which appear to be ubiqitous, respond to the expanding bacterial population and enter an exponential growth phase. These speculations agree with the findings of L. Leibovitz (personal communication, 1975) who experimentally determined that ciliates characteristically appear in larval cul tures as the bacterial concentrations increase. During the Spring of 1974 and 1975 all of the ciliate isolates were identified as U. marinum, but other unidentified species were observed in low numbers in cultures of larval and juvenile oysters. It is not unreasonable to assume that one or more of these species might initiate an infestation. The ciliate infestations occurring in the hatchery are not unique. Other workers have noted Uronema infesting recently killed crustacea and have speculated on the feeding type of the ciliate. Finenko and Zaika (1970) placed dead Arcartiu clausi Giesbrecht in a sea water bath and made observations on the microorganisms which infiltrated the dead individuals. After 3 h, ciliates of the genera Euplotes and Uronemu as well as nematodes were visible in the animals. Although no controlled experiments were performed and no bacterial counts taken, high numbers of Uronema seemed to correspond with low concentrations of bacteria and low numbers of Uronemu seemed to correspond with high concentrations of bacteria. From these observations Finenko and Zaika speculated that Uronemu was not a histophage but a bacteriophage, a conclusion supported by these experiments.
225
ACKNOWLEDGEMENTS
We thank Dr. David Dean and Samuel R. Chapman for significant advice and support. Dr. Arthur Borror of the University of New Hampshire kindly provided much technical information with the Protozoa. The study was supported by NOAA, Office of Sea Grant, Project No. NG40-72.
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