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Effect of Perkinsus olseni (Protozoa, Apicomplexa) on the weight of Tridacna crocea (Mollusca, Bivalvia) from Lizard Island, Great Barrier Reef
Aquaculture ELSEVIER
Aquaculture 141 (1996) 25-30
Effect of Perkinsus olseni ( Protozoa, Apicomplexa) on the weight of Tridacna crocea (Mollusca, Bivalvia) from Lizard Island, Great Barrier Reef C. Louise Goggin Department of Parasitology, The Uniuersity ofQueenslanrl. Brisbane, 4072 Qld., Australia
Accepted 22 October 1995
Abstract Protozoan parasites in the genus Perkinsus infect American oysters causing loss of condition and death of the host in heavy infections. Length-to-weight relationship was used to assess the
effect of Perkinsus olseni on wild tridacnid clams, Tridacna crocea, from around Lizard Island on the Great Barrier Reef. Perkinsus olseni infection intensity increased with increasing length of clams but did not induce a change in the length-to-weight relationship in 120 clams up to 100 mm shell length. Therefore, infection by Perkinsus parasites is unlikely to affect culture of tridacnid clams in this size range. Keywords: Tridacna cracea; Perkinsus; Clam; Weight
1. Introduction Protozoan parasites in the genus Perkinsus have been isolated from many Australian molluscs, including tridacnid clams (Goggin and Lester, 19871, but only one species, Perkinsus olseni, has been described from Australia (Lester and Davis, 1981). There is evidence that P. olseni, although described from blacklip abalone, Haliotis ruber (= rubra), also occurs in tridacnid clams (Goggin and Lester, 1995). Therefore, the parasite found in Triducna crocea in this study will be referred to as P. olseni. In the United States, Perkinsus marinus can cause up to 50% loss of weight in eastern oysters, Crassostrea virginica (Ray et al., 1953; Ray, 1954) but the effect of Perkinsus olseni on tridacnid clams was not determined. Giant clams are a major tourist attraction in Queensland and the focus of a budding mariculture industry in Australia. 0044-8486/96/$15.00 0 1996 Elsevier Science B.V. All rights reserved SSDI 0044-8486(95)0121 1-7
26
C.L. Goggin/Aquaculture
This study assessed the impact of infection Tridacna crocea, in Australia.
141 (1996) 25-30
by P. olseni on the weight of the clam,
2. Material and methods Triducna crocea were collected, by diving and snorkelling, from four sites around Lizard Island in September, 1988. Work was performed under Great Barrier Reef Marine Park Authority (GBRMPA) permit as part of a research project on the biology of Perkinsus species. Thirty clams were collected between South and Bird islands (site l), below Mt Cook (21, Research Beach (3) and Mermaid Cove (4) (Fig. 1). The length of the shell of each clam was measured, together with live weight, wet shell weight and wet tissue weight. Tissue from each mollusc, particularly from the gill, mantle, digestive gland and muscle was incubated in fluid tbioglycollate medium @TM), supplemented with 200 mg chloromycetin and 200 units of mycostatin ml-‘, for 5 days at room temperature (25-30°C). Tissues were flooded with Lugol’s iodine after incubation in
*Bird 0-
Island
m ‘000
Fig. 1. Sites where Tridacm crocea were collected from reefs around Lizard Island on the Great Barrier Reef.
C.L. Goggin/Aquuculture
141 (1996) 25-30
27
FTM to assess the infection intensity with Perkinsus olseni (Ray, 1966). Intensity of infection with P. olseni was ranked from uninfected (0) and 1 (light) to 6 (heavy) as described by Quick (1972). Intensities of 4, 5 and 6 were combined before analysis because few clams had heavy infections (> 4). 2.1, Statistical procedures The distribution of clams among the five infection intensities were compared for the four sites using a Kruskal-Wallis test. The difference in mean shell lengths from the four sites was compared in Analysis of Variance (ANOVA). The relationship between lengths of the shell and infection intensity of all clams was assessed by Spearman rank correlation. Quadratic regression equations were fitted to describe the relationship between wet tissue weight and shell length. Separate regressions were obtained for each infection intensity, and these were then compared between infection intensities using an F test (Zar, 1984). Where no significant difference was found between regression equations at different infection intensities, a combined regression equation for the length-to-weight relationship was calculated. Statistics were performed using SAS Version 6 package (SAS Institute Inc., Cary, NC, USA) and significance was set at the 0.05 level of probability for all tests.
3. Results There was no significant difference in the numbers of clams infected between sites (Kruskal-Wallis, x2 = 5.9111, df= 3). There was a significant difference, however, in the mean shell lengths of clams from the four sites (F = 3.55 with 3/l 16 df>. Shell length correlated positively with infection intensity (Spearman rank correlation coefficient = 0.601, n = 120). The mean shell lengths of clams increased with increasing intensities of infection (Table 1). The relationship is described by: I=
-4.081
+O.l17L
(r2=0.93,
n=5)
where I = infection intensity and L = shell length in mm. Thus, Tridacna crocea with longer shells have heavier infections of P. olseni than smaller clams. Mean wet tissue weights also increased with increasing infection intensity (Table 1). The relationship is described by: I = -0.025
+ 0.164WT
( r2 = 0.92,
n = 5)
where WT = wet tissue weight in g. There was no significant difference between quadratic regression equations for infection with P. olseni at intensities 0, 2, 3 and 4. The combined regression relationship is described by: WT = - 0.300492L
+ 0.008802L2
(R* = 0.973,
n = 89).
60.88
19.41 31.3-102.4
20.82 24.5-99.5
3 8 1 II 6 I 0 30
60.49
I 0 30
1
2 6 4 I6
20.62 22.4-98.1
50.54
30
4 IO 3 8 3 I I
17.73 17.9-89. I
47.41
0 30
0 0
I 7 19 3 13.3
36.5 41.9 50.0 67.1
Mean
4
Length 3
I
2
Site
19.4
8.5 13.7 13.7 19.0
S.D.
34.9- 102.4
27.2-49.1 17.9-76.9 25.0-89. I 28.3-99.5
Range
Weight, wet tissue weight (g); length, shell length (mm); n, total number of clams per site; S.D., standard deviation.
Mean shell length S.D. Range
I 2 3 4 5 6 n
0
Infection intensity
28.5
3.16 5.08 8.6 22.4
Mean
Weight
18.5
2.14 4.16 8.7 18.1
S.D.
Table I Numbers of Triducncr crocrtr infected with different intensities of Prrkinsus olseni from four sites around Lizard Island, Great Barrier Reef
2.7-62.7
0.5-6.2 0.2-22s 0.4-37.0 I .5-76.9
Range
C.L. Goggin/Aquuculrure
I4I (1996) 25-30
29
Thus, the presence of the parasite does not produce a noticeable weight loss in clams. However, the regression equation for the length-to-weight relationship at intensity 1 was significantly different from all other intensities of infection at the 5% level of significance but not at 1%. This relationship is described by: WT = - 0.103OL + 0.00484L2 The range different.
of shell
lengths
( R2 = 0.979,
at different
12= 31).
infection
intensities
were not obviously
4. Discussion Perkinsus olseni does not cause tridacnid clams, up to 100 mm shell length, to lose weight. In contrast, P. marinus is reported to produce up to 50% tissue weight loss in American oysters, Crussostrea virginicu (Ray et al., 1953; Ray, 1954). Weight loss, however, may not be an adequate indicator of pathogenicity of P. olseni to Australian clams; experiments in the laboratory found that infected clams, Triducnu gigus, are more affected by temperature stress than uninfected clams (Goggin and Lester, 1996). In these experimental tests, infected T. gigus die more rapidly and in larger numbers than uninfected clams. Triducnu croceu with longer shells have heavier infections of Perkinsus olseni than smaller clams. This may be a result of multiple infections and/or multiplication of P. olseni in the tissues. Similarly, Ray (1953) found that large, adult oysters, Crussostrea uirginicu, tended to have heavier infections of P. marinus than did small adults. A single quadratic regression equation described the length-to-weight relationship of uninfected clams and clams infected at all intensities except intensity 1. The size of clams were similar at all infection intensities. The different relationship of length-toweight for clams with infection intensity of 1 suggests that clams with new infections are more affected by the parasite than clams with chronic infections. Triducna crocea did not show any differences in infection intensity of Perkinsus olseni from four reefs around Lizard Island. Andrews and Hewatt (1957) found, however, that Perkinsus murinus had a patchy distribution in C. virginicu in Chesapeake Bay. The proximity of the four sites used here could account for the similarity in infection intensities of P. olseni. Perkinsus murinus is capable of infecting oysters in close proximity and oysters also can acquire infections across hundreds of metres or more (Mackin, 1951; Ray, 1954; Ray and Mackin, 1954; Andrews, 1965; Canzonier, 1966). Large numbers of bivalves from the reefs around Lizard Island harbour Perkinsus species (Goggin and Lester, 1987) and the ease of cross infection with these parasites was demonstrated in the laboratory (Goggin et al., 1989). In conclusion, Perkinsus olseni does not affect the wet tissue weight of Triducnu croceu, so that weight change cannot be used as an indicator of either the abundance of the parasite or its pathogenicity to the host. When an infection is found in a population of T. croceu, larger clams are likely to be more heavily infected with P. olseni than smaller clams and, as a result, more adversely affected by environmental stress.
C.L. Goggin/Aquaculture
141 (1996) 25-30
Acknowledgements I thank Drs Rob Adlard and Leslie Newman for assistance in the field and Dr Frank Roubal for help with statistical analyses. Field work was conducted at the Lizard Island Research Station. This project was supported by the Australian Research Council (MST A08600937 to Dr R.J.G. Lester and Mr P. Hunnam), the Great Barrier Reef Marine Park Authority and a Commonwealth Postgraduate Research Award.
References Andrews, J.D., 196.5. Infection experiments in nature with Dermocystidium marinum, in Chesapeake Bay. Chesapeake Sci., 6: 60-67. Andrews, J.D. and Hewatt, W.G., 1957. Oyster mortality studies in Virginia. II. The fungus disease caused by Dermocystidium marinum in oysters in Chesapeake Bay. Ecol. Monogr., 27: l-25. Canzonier, W.J., 1966. Dermocystidium in tray populations of oysters in Delaware Bay. Abstr. Proc. Natl. Shellfish. Assoc., 56: 1. Goggin, C.L. and Lester, R.J.G., 1987. Occurrence of Perkinsus species (Protozoa, Apicomplexa) in bivalves from the Great Barrier Reef. Dis. Aquat. Org., 3: 113-l 17. Goggin, C.L. and Lester, R.J.G., 1995. Perkinsus, a protistan parasite of abalone in Australia: a review. Mar. Freshwater Res., 46: 639-646. Goggin, C.L. and Lester, R.J.G., 1996. Pathogenicity of Perkinsus olseni (Protozoa, Apicomplexa) to Australian molluscs in the laboratory. Dis. Aquat. Org., submitted. Goggin, C.L., Sewell, K.B. and Lester, R.J.G., 1989. Cross-infection experiments with Australian Perkinsus species. Dis. Aquat. Org., 7: 55-59. Lester, R.J.G. and Davis, G.H.G., 1981. A new Perkinsus species (Apicomplexa, Perkinsea) from the abalone Haliotis ruber. J. Invert. Pathol., 37: 181- 187. Mackin, J.G., 1951. Histopathology of infection of Crassostrea uirginica (Gmelin) by Dermocystidium marinum Mackin, Owen and Collier. Bull. Mar. Sci. Gulf Caribb., 1: 72-87. Quick, J.A., Jr., 1972. Fluid thioglycollate medium assay of Labyrinthomyxa parasites in oysters. Fla. Dep. Nat. Resour. Mar. Res. Lab. Leafl. Ser., Vol. 6, Part 4, No. 3, 11 pp. Ray, SM., 1953. Studies on the occurrence of Dermocystidium mar&m, in young oysters. Proc. Natl. Shellfish. Assoc., 54: 55-69. Ray, S.M., 1954. Biological studies of Dermocystidium marinum, a fungus parasite of oysters. Rice Institute Pamphlet. Special Issue, 114 pp. Ray, S.M., 1966. A review of the culture method for detecting Dermocystidium marinum with suggested modifications and precautions. Proc. Natl. Shellfish. Assoc., 54: 55-69. Ray, S.M. and Mackin, J.G., 1954. Studies on the transmission and pathogenicity of Dermocystidium mar&m I. Tex. A. and M. Res. Found. Proj. 23, Tech. Rep. 11, 19 pp. Ray, S.M., Mackin, J.G. and Boswell, J.L., 1953. Quantitative measurement of the effect on oysters of disease caused by Dermocystidium marinum. Bull. Mar. Sci. Gulf Caribb., 3: 6-33. Zar. J.H., 1984. Biostatistical Analysis. 2nd edn., Prentice-Hall Inc., Englewood Cliffs, NJ, 718 pp.
Aquaculture 141 (1996) 25-30
Effect of Perkinsus olseni ( Protozoa, Apicomplexa) on the weight of Tridacna crocea (Mollusca, Bivalvia) from Lizard Island, Great Barrier Reef C. Louise Goggin Department of Parasitology, The Uniuersity ofQueenslanrl. Brisbane, 4072 Qld., Australia
Accepted 22 October 1995
Abstract Protozoan parasites in the genus Perkinsus infect American oysters causing loss of condition and death of the host in heavy infections. Length-to-weight relationship was used to assess the
effect of Perkinsus olseni on wild tridacnid clams, Tridacna crocea, from around Lizard Island on the Great Barrier Reef. Perkinsus olseni infection intensity increased with increasing length of clams but did not induce a change in the length-to-weight relationship in 120 clams up to 100 mm shell length. Therefore, infection by Perkinsus parasites is unlikely to affect culture of tridacnid clams in this size range. Keywords: Tridacna cracea; Perkinsus; Clam; Weight
1. Introduction Protozoan parasites in the genus Perkinsus have been isolated from many Australian molluscs, including tridacnid clams (Goggin and Lester, 19871, but only one species, Perkinsus olseni, has been described from Australia (Lester and Davis, 1981). There is evidence that P. olseni, although described from blacklip abalone, Haliotis ruber (= rubra), also occurs in tridacnid clams (Goggin and Lester, 1995). Therefore, the parasite found in Triducna crocea in this study will be referred to as P. olseni. In the United States, Perkinsus marinus can cause up to 50% loss of weight in eastern oysters, Crassostrea virginica (Ray et al., 1953; Ray, 1954) but the effect of Perkinsus olseni on tridacnid clams was not determined. Giant clams are a major tourist attraction in Queensland and the focus of a budding mariculture industry in Australia. 0044-8486/96/$15.00 0 1996 Elsevier Science B.V. All rights reserved SSDI 0044-8486(95)0121 1-7
26
C.L. Goggin/Aquaculture
This study assessed the impact of infection Tridacna crocea, in Australia.
141 (1996) 25-30
by P. olseni on the weight of the clam,
2. Material and methods Triducna crocea were collected, by diving and snorkelling, from four sites around Lizard Island in September, 1988. Work was performed under Great Barrier Reef Marine Park Authority (GBRMPA) permit as part of a research project on the biology of Perkinsus species. Thirty clams were collected between South and Bird islands (site l), below Mt Cook (21, Research Beach (3) and Mermaid Cove (4) (Fig. 1). The length of the shell of each clam was measured, together with live weight, wet shell weight and wet tissue weight. Tissue from each mollusc, particularly from the gill, mantle, digestive gland and muscle was incubated in fluid tbioglycollate medium @TM), supplemented with 200 mg chloromycetin and 200 units of mycostatin ml-‘, for 5 days at room temperature (25-30°C). Tissues were flooded with Lugol’s iodine after incubation in
*Bird 0-
Island
m ‘000
Fig. 1. Sites where Tridacm crocea were collected from reefs around Lizard Island on the Great Barrier Reef.
C.L. Goggin/Aquuculture
141 (1996) 25-30
27
FTM to assess the infection intensity with Perkinsus olseni (Ray, 1966). Intensity of infection with P. olseni was ranked from uninfected (0) and 1 (light) to 6 (heavy) as described by Quick (1972). Intensities of 4, 5 and 6 were combined before analysis because few clams had heavy infections (> 4). 2.1, Statistical procedures The distribution of clams among the five infection intensities were compared for the four sites using a Kruskal-Wallis test. The difference in mean shell lengths from the four sites was compared in Analysis of Variance (ANOVA). The relationship between lengths of the shell and infection intensity of all clams was assessed by Spearman rank correlation. Quadratic regression equations were fitted to describe the relationship between wet tissue weight and shell length. Separate regressions were obtained for each infection intensity, and these were then compared between infection intensities using an F test (Zar, 1984). Where no significant difference was found between regression equations at different infection intensities, a combined regression equation for the length-to-weight relationship was calculated. Statistics were performed using SAS Version 6 package (SAS Institute Inc., Cary, NC, USA) and significance was set at the 0.05 level of probability for all tests.
3. Results There was no significant difference in the numbers of clams infected between sites (Kruskal-Wallis, x2 = 5.9111, df= 3). There was a significant difference, however, in the mean shell lengths of clams from the four sites (F = 3.55 with 3/l 16 df>. Shell length correlated positively with infection intensity (Spearman rank correlation coefficient = 0.601, n = 120). The mean shell lengths of clams increased with increasing intensities of infection (Table 1). The relationship is described by: I=
-4.081
+O.l17L
(r2=0.93,
n=5)
where I = infection intensity and L = shell length in mm. Thus, Tridacna crocea with longer shells have heavier infections of P. olseni than smaller clams. Mean wet tissue weights also increased with increasing infection intensity (Table 1). The relationship is described by: I = -0.025
+ 0.164WT
( r2 = 0.92,
n = 5)
where WT = wet tissue weight in g. There was no significant difference between quadratic regression equations for infection with P. olseni at intensities 0, 2, 3 and 4. The combined regression relationship is described by: WT = - 0.300492L
+ 0.008802L2
(R* = 0.973,
n = 89).
60.88
19.41 31.3-102.4
20.82 24.5-99.5
3 8 1 II 6 I 0 30
60.49
I 0 30
1
2 6 4 I6
20.62 22.4-98.1
50.54
30
4 IO 3 8 3 I I
17.73 17.9-89. I
47.41
0 30
0 0
I 7 19 3 13.3
36.5 41.9 50.0 67.1
Mean
4
Length 3
I
2
Site
19.4
8.5 13.7 13.7 19.0
S.D.
34.9- 102.4
27.2-49.1 17.9-76.9 25.0-89. I 28.3-99.5
Range
Weight, wet tissue weight (g); length, shell length (mm); n, total number of clams per site; S.D., standard deviation.
Mean shell length S.D. Range
I 2 3 4 5 6 n
0
Infection intensity
28.5
3.16 5.08 8.6 22.4
Mean
Weight
18.5
2.14 4.16 8.7 18.1
S.D.
Table I Numbers of Triducncr crocrtr infected with different intensities of Prrkinsus olseni from four sites around Lizard Island, Great Barrier Reef
2.7-62.7
0.5-6.2 0.2-22s 0.4-37.0 I .5-76.9
Range
C.L. Goggin/Aquuculrure
I4I (1996) 25-30
29
Thus, the presence of the parasite does not produce a noticeable weight loss in clams. However, the regression equation for the length-to-weight relationship at intensity 1 was significantly different from all other intensities of infection at the 5% level of significance but not at 1%. This relationship is described by: WT = - 0.103OL + 0.00484L2 The range different.
of shell
lengths
( R2 = 0.979,
at different
12= 31).
infection
intensities
were not obviously
4. Discussion Perkinsus olseni does not cause tridacnid clams, up to 100 mm shell length, to lose weight. In contrast, P. marinus is reported to produce up to 50% tissue weight loss in American oysters, Crussostrea virginicu (Ray et al., 1953; Ray, 1954). Weight loss, however, may not be an adequate indicator of pathogenicity of P. olseni to Australian clams; experiments in the laboratory found that infected clams, Triducnu gigus, are more affected by temperature stress than uninfected clams (Goggin and Lester, 1996). In these experimental tests, infected T. gigus die more rapidly and in larger numbers than uninfected clams. Triducnu croceu with longer shells have heavier infections of Perkinsus olseni than smaller clams. This may be a result of multiple infections and/or multiplication of P. olseni in the tissues. Similarly, Ray (1953) found that large, adult oysters, Crussostrea uirginicu, tended to have heavier infections of P. marinus than did small adults. A single quadratic regression equation described the length-to-weight relationship of uninfected clams and clams infected at all intensities except intensity 1. The size of clams were similar at all infection intensities. The different relationship of length-toweight for clams with infection intensity of 1 suggests that clams with new infections are more affected by the parasite than clams with chronic infections. Triducna crocea did not show any differences in infection intensity of Perkinsus olseni from four reefs around Lizard Island. Andrews and Hewatt (1957) found, however, that Perkinsus murinus had a patchy distribution in C. virginicu in Chesapeake Bay. The proximity of the four sites used here could account for the similarity in infection intensities of P. olseni. Perkinsus murinus is capable of infecting oysters in close proximity and oysters also can acquire infections across hundreds of metres or more (Mackin, 1951; Ray, 1954; Ray and Mackin, 1954; Andrews, 1965; Canzonier, 1966). Large numbers of bivalves from the reefs around Lizard Island harbour Perkinsus species (Goggin and Lester, 1987) and the ease of cross infection with these parasites was demonstrated in the laboratory (Goggin et al., 1989). In conclusion, Perkinsus olseni does not affect the wet tissue weight of Triducnu croceu, so that weight change cannot be used as an indicator of either the abundance of the parasite or its pathogenicity to the host. When an infection is found in a population of T. croceu, larger clams are likely to be more heavily infected with P. olseni than smaller clams and, as a result, more adversely affected by environmental stress.
C.L. Goggin/Aquaculture
141 (1996) 25-30
Acknowledgements I thank Drs Rob Adlard and Leslie Newman for assistance in the field and Dr Frank Roubal for help with statistical analyses. Field work was conducted at the Lizard Island Research Station. This project was supported by the Australian Research Council (MST A08600937 to Dr R.J.G. Lester and Mr P. Hunnam), the Great Barrier Reef Marine Park Authority and a Commonwealth Postgraduate Research Award.
References Andrews, J.D., 196.5. Infection experiments in nature with Dermocystidium marinum, in Chesapeake Bay. Chesapeake Sci., 6: 60-67. Andrews, J.D. and Hewatt, W.G., 1957. Oyster mortality studies in Virginia. II. The fungus disease caused by Dermocystidium marinum in oysters in Chesapeake Bay. Ecol. Monogr., 27: l-25. Canzonier, W.J., 1966. Dermocystidium in tray populations of oysters in Delaware Bay. Abstr. Proc. Natl. Shellfish. Assoc., 56: 1. Goggin, C.L. and Lester, R.J.G., 1987. Occurrence of Perkinsus species (Protozoa, Apicomplexa) in bivalves from the Great Barrier Reef. Dis. Aquat. Org., 3: 113-l 17. Goggin, C.L. and Lester, R.J.G., 1995. Perkinsus, a protistan parasite of abalone in Australia: a review. Mar. Freshwater Res., 46: 639-646. Goggin, C.L. and Lester, R.J.G., 1996. Pathogenicity of Perkinsus olseni (Protozoa, Apicomplexa) to Australian molluscs in the laboratory. Dis. Aquat. Org., submitted. Goggin, C.L., Sewell, K.B. and Lester, R.J.G., 1989. Cross-infection experiments with Australian Perkinsus species. Dis. Aquat. Org., 7: 55-59. Lester, R.J.G. and Davis, G.H.G., 1981. A new Perkinsus species (Apicomplexa, Perkinsea) from the abalone Haliotis ruber. J. Invert. Pathol., 37: 181- 187. Mackin, J.G., 1951. Histopathology of infection of Crassostrea uirginica (Gmelin) by Dermocystidium marinum Mackin, Owen and Collier. Bull. Mar. Sci. Gulf Caribb., 1: 72-87. Quick, J.A., Jr., 1972. Fluid thioglycollate medium assay of Labyrinthomyxa parasites in oysters. Fla. Dep. Nat. Resour. Mar. Res. Lab. Leafl. Ser., Vol. 6, Part 4, No. 3, 11 pp. Ray, SM., 1953. Studies on the occurrence of Dermocystidium mar&m, in young oysters. Proc. Natl. Shellfish. Assoc., 54: 55-69. Ray, S.M., 1954. Biological studies of Dermocystidium marinum, a fungus parasite of oysters. Rice Institute Pamphlet. Special Issue, 114 pp. Ray, S.M., 1966. A review of the culture method for detecting Dermocystidium marinum with suggested modifications and precautions. Proc. Natl. Shellfish. Assoc., 54: 55-69. Ray, S.M. and Mackin, J.G., 1954. Studies on the transmission and pathogenicity of Dermocystidium mar&m I. Tex. A. and M. Res. Found. Proj. 23, Tech. Rep. 11, 19 pp. Ray, S.M., Mackin, J.G. and Boswell, J.L., 1953. Quantitative measurement of the effect on oysters of disease caused by Dermocystidium marinum. Bull. Mar. Sci. Gulf Caribb., 3: 6-33. Zar. J.H., 1984. Biostatistical Analysis. 2nd edn., Prentice-Hall Inc., Englewood Cliffs, NJ, 718 pp.