Aquaculture 199 2001.

229244
www.elsevier.nlrlocateraqua-online

An investigation into the relative resistance of Irish

flat oysters Ostrea edulis L. to the parasite
Bonamia ostreae Pichot et al., 1980/
F. Mulcahy
Sarah C. Culloty ) , Michelle A. Cronin, Maire
Department of Zoology and Animal Ecology, Uniersity College Cork, Lee Maltings, Prospect Row,
Cork, Ireland
Received 5 February 2000; received in revised form 17 November 2000; accepted 18 December 2000

Abstract

The parasite Bonamia ostreae has caused significant mortalities in the flat oyster Ostrea
edulis. To date, methods of control and eradication have proved largely unsuccessful. Research is
now concentrating on development of a population of oysters that has increased resistance to
bonamiasis. This study evaluated the relative resistance of three Irish strains of flat oysters to this
disease: two naive strains that had not previously been exposed to the parasite, and one that has
been exposed to B. ostreae since the 1980s and has been selectively bred from survivors. In both
field and laboratory trials, oysters from the selected strain showed lower prevalence of infection,
intensity of infection and mortalities compared to the two naive strains. Development of a strain
showing some resistance to B. ostreae would allow oysters to be grown to market size before
significant mortalities occurred, and would also allow restocking of areas that have been
decimated by the disease. q 2001 Elsevier Science B.V. All rights reserved.

Keywords: Bonamia ostreae; Ostrea edulis; Resistance; Selective breeding; Cork harbour

1. Introduction

The flat oyster Ostrea edulis is the native species of oyster found in Ireland. This
species had been the basis for oyster production until the parasite Bonamia ostreae was
diagnosed in the 1980s. Bonamiasis was initially diagnosed in Rossmore oysters in Cork
harbour in 1987 but may have been present for a number of years previous to this

)
Corresponding author. Tel.: q353-21-4904348; fax: q353-21-4270562.
E-mail address: s.culloty@ucc.ie S.C. Culloty..

0044-8486r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved.
PII: S 0 0 4 4 - 8 4 8 6 0 1 . 0 0 5 6 9 - 5
230 S.C. Culloty et al.r Aquaculture 199 (2001) 229244

McArdle et al., 1991; Rogan et al., 1991.. Control and eradication of this disease have
been largely unsuccessful Van Banning, 1985, 1987.. In a number of other shellfish
diseases, the only potential long-term method of control has been development of
resistance in the host species. Resistance in bivalves generally refers to relatively
greater survival, implying a reduced susceptibility to the presence of the parasite. In
strains of oysters exhibiting some resistance, for example Crassostrea irginica Gmelin,
1791. infected by Haplosporidium nelsoni Haskin, Stauber and Mackin. survival
mechanisms involved restriction of parasite development and tolerance of infection
Ford, 1988.. In practical terms, selected strains were considered successful if they
reached market size before significant losses occurred Ford, 1988..
Following mortalities of over 90% in 1987 in Cork harbour caused by B. ostreae, the
decision to breed from survivors was taken. Broodstock are selected from those oysters
alive after 4 years on the beds. They are placed in spatting ponds and spawn when
temperatures become optimal. Following settlement on culch and several months growth
in the ponds, spat are re-laid to designated beds, where different age groups are kept
segregated. It appears that a degree of resistance or reduced susceptibility to the parasite
may have developed, as a viable percentage of oysters are reaching market size in recent
years. The objective of the present study was to determine if the selective breeding of
Rossmore oysters, the strain present in Cork harbour, has resulted in the emergence of a
stock exhibiting some resistance or reduced susceptibility to B. ostreae. This was
carried out by comparing susceptibility of the Rossmore F2 generation oysters to that of
a number of other naive Irish oyster strains, following exposure to the pathogen in both
the field and the laboratory. Bonamia has also been recorded more recently in Galway
bay 1989. McArdle et al., 1991., but it was thought unlikely that natural selection
would have occurred in the 7 years to the beginning of this trial. Furthermore, no
breeding selection had been carried out in Galway; therefore, it was decided not to
include this strain.

2. Materials and methods

2.1. Field trials

2.1.1. Oysters
Naive oysters were obtained from Tralee and Lough Foyle B. ostreae has never
been diagnosed in oysters from these areas., and selected oysters from Rossmore in
December 1996 Fig. 1.. Two-year groups of each strain were used for the trial: 1995
1 q years old. and 1994 2 q years old.. The main criteria in picking the age groups
for the trial were twofold.
i. That oysters pre- and post-initial spawning were included. It has been shown
previously that infection and mortality levels are negligible up to 2 years and then
increase significantly in the months after the oysters initially spawn during the second
`
summer Figueras, 1991; Cacares-Martinez et al., 1995; Culloty and Mulcahy, 1996..
ii. As the field trial was to be carried out over a 2-year period, it was considered best
to include age groups that would be reaching market size and age when the trial
 S.C. Culloty et al.r Aquaculture 199 (2001) 229244 231

Fig. 1. Map of Ireland showing provenance of three Irish oyster strains used in field and laboratory trials.

finished, as it appears that prevalence of infection can be age and size related Figueras,
`
1991; McArdle et al., 1991; Cacares-Martinez et al., 1995; Culloty and Mulcahy, 1996..
Oysters were placed in bags at a density of 300 per bag for 1 q oysters and 150 per
bag for 2 q oysters. Oysters 400, 600 and 600 1 q . were available from Rossmore
R1 q ., Tralee T1 q . and Lough Foyle LF1 q ., respectively. Oysters 710, 450 and
1050 2 q . were available from Rossmore R2 q ., Tralee T2 q . and Lough Foyle
LF2 q ., respectively. Following heavy mortalities in late March 1997 in the T2 q
group, a further 600 Tralee oysters T2 q) . were relaid in the harbour in April 1997.

2.1.2. Trial site and monitoring

The three strains were placed on trestles, 40 cm off the substrate, in the North
Channel of Cork harbour, where exposure was only at spring tide. Thirty oysters from
232 S.C. Culloty et al.r Aquaculture 199 (2001) 229244

each age group and each strain were taken for screening both at the start December
1996., and during the trial period: in April, June, August, and November 1997, and
March, and June 1998. Cumulative mortality %. was estimated according to Tige and
Grizel 1984.:
No. oysters placed in bags at T0 y No. alive at T1 = 100
No. oysters placed in bags at T0

where T0 s time 0 and T1 s time of particular sample.

2.1.3. Screening of oysters

Following cleaning, oysters were weighed on an analytical balance to the nearest 0.1
g. All oysters were screened for B. ostreae infection by ventricular heart smears
` et al., 1982.. Infection was classified as follows:
Bachere

Class 0: no infection observed within 5 min of examination;

Class 1: 110 parasites observed within 5 min of observation;
Class 2: 10100 parasites observed within 5 min of observation;
Class 3: parasites observed within all fields of view;
Class 4: heavy infection observed in all blood cells.

2.2. Laboratory-based trials

2.2.1. Experimental injection and monitoring of oysters

Laboratory trials were set up in February 1998 following evaluation of field results.
Three- and four-year-old Rossmore oysters were selected for the trial, as were 3-year-old
oysters from Tralee. These age groups were chosen as i. younger oysters were used in
the field trial and this would allow us to look at all age groups up to market size; and ii.
previous studies have shown that infection levels increase with age Figueras, 1991;
McArdle et al., 1991; Culloty and Mulcahy, 1996.. All three groups were screened for
infection, using heart smears, prior to experimental injection in the laboratory, to
determine baseline prevalence of infection.
Bonamia cells were isolated from Rossmore oysters with class 3 infections, which
were the heaviest infections available, according to the method of Mialhe et al. 1988.
and Hervio et al. 1995.. All oysters to be injected were initially anaesthetised with 3.5%
MgCl 2 P 6H 2 O Culloty and Mulcahy, 1992.. Thirty to forty control oysters from each
strain and each age group were injected with 50 ml of filtered seawater 0.45 mm..
Ninety experimental oysters from each strain and each age group were injected with
4.03 = 10 4 B. ostreae cellsr50 ml of filtered seawaterroyster. The numbers of oysters
injected were the maximum that could be held under the laboratory conditions available
and were within the range of sample sizes used in laboratory trials in a number of other
studies Poder et al., 1982; Elston et al., 1987; Martin et al., 1993; Hervio et al., 1995;
Renault et al., 1995; Culloty et al., 1999..
 S.C. Culloty et al.r Aquaculture 199 (2001) 229244 233

Following injection, the oysters were returned to fresh aerated seawater, where they
recovered after 30 to 60 min. Experimental and control groups were held in 500 l and
250 l tanks, respectively, with aeration and biofilters. Approximately half the water in
the tanks was exchanged two to three times per week. All oysters were fed daily on a
diet of Tetraselmis suecica. The temperature in the tanks varied from 118C to 188C over
the course of the trial. Oysters were monitored on a daily basis, and all moribund
animals removed and screened for infection. A sub-sample of oysters was removed 11
weeks post-injection to check prevalence of infection and 20 weeks post-injection in
July 1998. All remaining animals were removed and screened.

2.2.2. Statistical analysis

Analysis of variance general linear model, ANOVA. was carried out to examine
weight and infection with respect to strain, age and seasonality. A test for homogeneity
of variance indicated that a transformation natural log. of the weight data was required.
Tukey multiple comparison tests were run following the ANOVAs.

3. Results

3.1. Field trials

3.1.1. Whole wet weights

The weights of all age groups and all three strains increased over 18 months in the
field with a highly significant strain = age = month interaction Fig. 2a and b.. Both the
Rossmore 1 q and 2 q groups had the highest mean whole wet weights both at the
beginning and the end of the trial. The largest increase in weight over the trial period in
the 1 q group occurred in the Tralee group, with the final weight being 3.8 times the
initial weight. In the 2 q groups, the Lough Foyle oysters had the largest increase in
weight twice the initial weight. by June 1998. The last results for weights for Tralee
2 q available in November 1997 showed that the mean weight had increased to 1.5
times its initial weight. The mean whole weight of the Tralee 2 q) group increased by
1.6 times from June 1997June 1998.

3.1.2. Prealence of infection

When the field-based trials began in December 1996, no parasites were detected in
the 1 q Rossmore group but the 2 q group already had a 6.0% prevalence of infection
Fig. 3a and b.. No infection was found in either the Tralee or Lough Foyle strains.
There was a significant strain = age = month interaction for infection levels in the three
strains. There were very highly significant differences in infection levels between the
Rossmore 1 q group and the other two strains in March and June 1998 P - 0.001.
37.0% in the Rossmore group compared to 80% in the Tralee 1 q strain and 100% in
the Lough Foyle 1 q strain.. There was a very highly significant difference P - 0.001.
between infection levels in the Rossmore 2 q group and the Lough Foyle 2 q group in
234 S.C. Culloty et al.r Aquaculture 199 (2001) 229244

Fig. 2. a and b. Mean whole weights g. SD. of three oyster strains of two age classes re-laid to Cork
harbour Dec. 1996June 1998..

November 1997 20.0% and 96.7%, respectively. and June 1998 30.2% and 100.0%,
respectively. and between the Rossmore 2 q group and the other two 2 q groups in
March 1998 7.1% in Rossmore group compared to 100.0% in the Tralee 2 q group and
80.0% in the Lough Foyle 2 q group..
 S.C. Culloty et al.r Aquaculture 199 (2001) 229244 235

Fig. 3. a and b. Prevalence of infection of three oyster strains re-laid to Cork harbour when one and a half
years of age a. and two and a half years of age b. Dec. 1996June 1998..

3.1.3. Intensities of infection

Intensity or class of infection followed a progression over the trial period with
infections starting off as class 1 or 2 and then progressing to classes 3 and 4. A similar
development of infection occurred in both naive strains, so only one group is represented
236 S.C. Culloty et al.r Aquaculture 199 (2001) 229244
 S.C. Culloty et al.r Aquaculture 199 (2001) 229244 237

Fig. 5. Cumulative mortality %. from Dec. 1996June 1998 in three strains of oysters of two age
groups1q a. and 2q b. re-laid to Cork harbour.

in the figures. It is clear from Fig. 4ad that, as infection developed over the course of
the trial, the naive strains suffered much heavier infections, i.e. classes 3 and 4, than did
the Rossmore oysters, particularly in the latter months of sampling.

Fig. 4. a and d. Some examples of the difference in intensity of infection between the selected strain
Rossmore and one of the naive populationsLough Foyle during the field trial in 1q a and b. and 2q c
and d. age groups.
 238
S.C. Culloty et al.r Aquaculture 199 (2001) 229244
Table 1
Prevalence of infection %. in 3- and 4-year-old Rossmore and Tralee oysters before, during and after injection with either B. ostreae experimental groups. or
filtered seawater controls.
Groups of oysters used in laboratory trial Initial prevalence No. injected Prevalence of infection Prevalence of infection %. Percentage of oysters
of infection at T0 10 weeks post-injection at the end of the trial alive at the end of
%.ypre-injection the trial
4-year-old Rossmore oysters Control 36.7 11r30. 30 0.0 0r5. 16.7 3r18. 60.0 26.7. ) )
Experimental 90 30.0 3r10. 17.8 10r56. 62.2 26.7. ) )
3-year-old Rossmore oysters Control 6.7 2r30. 30 0.0 0r5. 6.2 1r16. 53.3 30.0. ) )
Experimental 90 10.0 1r10. 64.6 31r48. 53.3 35.6. ) )
3-year-old Tralee oysters Control 0.0 0r30. 40 0.0 0r5. 0.0 0r26. 65.0 22.5. ) )
Experimental 90 50.0 5r10. 63.1 29r46. 51.1 37.8. ) )

Numbers in brackets denote actual numbers screened.

))
Percentage of oysters that could not be screened during the trial number in brackets..
 S.C. Culloty et al.r Aquaculture 199 (2001) 229244
Table 2
Percentage of oysters with various classes of infection observed in the experimental and control groups throughout the laboratory trial
Groups of oysters used Class of infection %. at the beginning Class of infection 10 weeks Class of infection at the end of the trial
in the laboratory trial of the trial pre-injection post-injection
0 1 2 3 4 0 1 2 3 4 0 1 2 3 4
4-year-old Control 63.3 19 .) 26.7 8. 6.7 2. 3.3 1. 0.0 100.0 5. 0.0 0.0 0.0 0.0 83.3 15. 16.7 2. 0.0 0.0 0.0
Rossmore oyster Exp. 70.0 7. 10.0 1. 20.0 2. 0.0 0.0 82.2 46. 8.9 5. 8.9 5. 0.0 0.0
3-year -old Control 93.3 28. 6.7 2. 0.0 0.0 0.0 100.0 5. 0.0 0.0 0.0 0.0 93.8 15. 0.0 6.2 1. 0.0 0.0
Rossmore oyster Exp. 90.0 9. 10.0 1. 0.0 0.0 0.0 35.4 17. 33.3 16. 14.6 7. 12.5 6. 4.2 2.
3-year -old Control 100.0 30. 0.0 0.0 0.0 0.0 100.0 5. 0.0 0.0 0.0 0.0 100.0 26. 0.0 0.0 0.0 0.0
Tralee oyster Exp. 50.0 5. 40.0 1. 10.0 1. 0.0 0.0 36.9 17. 45.7 21. 6.5 3. 8.7 4. 2.2 1.

Exp.Experimental group.
)
Numbers in brackets denote actual numbers of oysters screened.

239
240 S.C. Culloty et al.r Aquaculture 199 (2001) 229244

3.1.4. Percentage cumulatie mortality

Cumulative mortalities in the 1 q groups at the end of the trial were 68.2%, 96.0%
and 96.2% in the Rossmore, Tralee and Lough Foyle 1 q groups, respectively.
Cumulative mortalities in the 2 q group were 63.9%, 100.0%, 89.6% and 92.0% in the
Rossmore, Tralee, Tralee ) and Lough Foyle groups, respectively Fig. 5a and b..

3.2. Laboratory experiments

3.2.1. Prealence and intensity of infection throughout the laboratory trial

The 4-year-old Rossmore oysters had 36.7% prevalence of infection before injection
Tables 1 and 2.. The 3-year-old Rossmore oysters had a lower prevalence of 6.7%. No
parasites were observed pre-injection in the Tralee sample as expected Table 1.. In
May, 10 weeks post-injection, the samples screened from the three control groups were
all negative for B. ostreae n s 5 for each group.. Prevalence of infection had dropped
slightly in the 4-year-old Rossmore experimental group to 30.0% n s 10. and had risen
to 10.0% in the 3-year-old Rossmore oysters. The Tralee group had 50.0% prevalence of
infection. At this stage, all infections observed in the three experimental groups were
class 1 or 2.
At the end of the trial, 20 weeks post-injection, only the Tralee control group
remained uninfected. A prevalence of infection of 16.7% was observed in the 4-year-old
Rossmore control oysters compared to 17.8% in the experimental group and 6.2% in the
3-year-old control group compared to 64.6% in the experimental group. The Tralee
experimental group also showed a further increase in prevalence to 63.1%. Class of
infection in the 4-year-old Rossmore experimental group had decreased, particularly the
class 2 infections from 20.0% in May to 8.9% in July. Table 2.. In the 3-year-old
Rossmore experimental group, all classes of infection were observed, which was not the
case in the control group. In the Tralee experimental group, all classes of infection were
also observed Table 2..
Even though oysters were monitored daily, a certain percentage of oysters from each
group could not be screened during the trial period. This was generally a result of dead
oysters not being observed and removed quickly enough from the tanks, so that tissues
had degenerated and heart smears could not be successfully carried out. The cause of
these mortalities as a result cannot be confirmed Table 1..

4. Discussion

Over the 18 months of the field trial in Cork harbour, lower prevalence and intensity
of infection and cumulative mortalities were observed in the Rossmore strain than in
either the Lough Foyle or Tralee strains in both age groups. In the Tralee and Lough
Foyle strains, prevalence of infection remained low over the summer months of 1997 but
increased dramatically in November 1997, 11-months post-relaying. Prevalence and
intensity of infection continued to develop rapidly over the remaining months of the
trial.
In the laboratory trial both Rossmore groups had a certain level of infection at the
beginning of the trial, in contrast to the Tralee group in which no parasites were
 S.C. Culloty et al.r Aquaculture 199 (2001) 229244 241

observed. By 10 weeks post-injection no parasites were observed in any of the control

groups. At the end of the trial the Tralee control group still remained disease-free and
the 4-year-old Rossmore control oysters had a heavier level of infection than the
3-year-old controls. In contrast, in the experimental groups, the Tralee oysters had the
highest prevalence of infection 10 weeks post-injection. However, by the end of the trial,
the 4-year-old Rossmore oysters had a very low prevalence of infection in comparison to
that found in the 3-year-olds from either Tralee or Rossmore, which had very similar
high infection levels.
Previous studies have shown that older oysters in a population experience higher
prevalence of infection McArdle et al., 1991; Culloty and Mulcahy, 1996.. The results
obtained here, wherein older oysters had much lower prevalence of infection, would
indicate that more susceptible oysters had possibly died off when the oysters were
younger and more resistant individuals remained. Another factor that may have con-
tributed to the prevalences of infection observed, though not to the differences between
the strains, was the low number of parasites injected per oyster, due to the low number
of heavily infected oysters available as a source of parasites. A number of previous
studies have used varying numbers of parasites to induce infection and it appears that
the rate of development of bonamiasis is dose dependent Hervio et al., 1995; Culloty et
al., 1999.. Martin et al. 1993. used a dose of 50,000 parasites per oyster. Hervio et al.
1995. determined that the 50% infectious dose was 80,000 parasites per oyster. Only
40,000 parasites per oyster were available in this study. In Hervio et al.s 1995. study,
injections of 10,000 parasites showed 1633% prevalence of infection in a series of
experiments after 4 months; however, they also noted that doses as low as one parasite
resulted in some prevalence of infection. In this study, all experimental groups received
the same dose so relative results are valid.
It appears that the Rossmore strain may be able to tolerate or eliminate a certain
number of parasites, as infection was not always followed by mortalities. Hervio et al.
1995. found individual variability in susceptibility to bonamiasis in oysters, when
injected with a known number of B. ostreae cells. MSX resistant oysters became
infected with H. nelsoni but parasites were rarely observed beyond the gill epithelium
and some infections were entirely eliminated at this stage Myrhe and Haskin, 1970;
Ford, 1986..
Cumulative mortalities were high for all groups at the end of the field trial, but lowest
in both Rossmore groups. By the end of the field trial, most of the Tralee and Lough
Foyle oysters had died. The husbandry techniques employed in this study may have
contributed to mortalities, as these oysters are normally grown sub-tidally without
interference until they reach market size. However, all strains were handled in the same
way therefore relative results are valid. Other studies looking at the relative resistance of
O. edulis strains have also observed high mortalities in field trials but lowest mortalities
in populations selected for resistance, indicating that the conditions under which these
trials are carried out can induce high stress related mortalities in the animals involved
Baud et al., 1997; Naciri-Graven et al., 1998, 1999..
In the field trial, all strains showed good growth rates over the trial period, with most
growth occurring in the younger oysters during the summer, as has been found
previously Walne, 1956.. Overall, the Tralee 1 q group performed best with its final
242 S.C. Culloty et al.r Aquaculture 199 (2001) 229244

weight being nearly four times the initial weight. In the older oysters assessed during the
course of the trial, the Lough Foyle oysters had the highest growth rates, but were
smallest when the trial began. Walne 1956. measured the weight increments during a
single season for O. edulis of the same age but different initial weight, and found that
larger individuals grew less rapidly, with individuals exhibiting a decreased growth
efficiency as their size increases.
There did not appear to be a relationship between faster growth rates and decreased
susceptibility to infection in this study. However, Rossmore oysters, which had the
highest mean weights, had the lowest levels of infection. The other two strains, which
had lower mean weights than the Rossmore strain when the trial began, grew to a greater
degree over the trial period but were very susceptible to the disease. Barber et al. 1998.
and Davis and Barber 1999. found that a group of oysters selected for increased
resistance to juvenile oyster disease grew faster than a wild population, indicating that
improved growth in bivalves occurs as a result of selective breeding. Similar results
have also been found in other studies evaluating the relative resistance of O. edulis
selectively bred against B. ostreae Martin et al., 1993; Baud et al., 1997; Naciri-Graven
et al., 1999.. To determine if similar enhanced growth rates occur in the Rossmore
group, a further study would be required, with all strains to be assessed ideally having
the same starting weight and size.
Production of a strain that is sufficiently resistant, that it can be grown to market size
prior to large mortalities occurring, would be of significant economic importance. A
number of major oyster growing areas throughout Europe are still devastated by
bonamiasis, and the industry has not recovered due to continuing high losses attributed
to the parasite. These areas could benefit from restocking with a strain that was
sufficiently tolerant of the parasite that the oysters could be grown to market size. In so
far as comparisons allowed, the results of both field and laboratory trials presented here
support the idea that a degree of AresistanceB has developed in the Rossmore oysters. It
must be remembered however, that the two groups of Rossmore oysters had already
been exposed for either one and a half or two and a half years to B. ostreae by the time
the field trials began. Furthermore, the fact that the life cycle of B. ostreae is still
unknown and little is known of the biology of the host, hampers efforts to determine the
basis of this resistance. Further trials are now being undertaken to further assess the
reduced susceptibility of these Rossmore oysters and to compare them with a number of
oyster strains from other European countries.

Acknowledgements

This study was supported by FORBAIRT grant HEr96r113 and VOKES Ltd. The
authors would like to thank all at Atlantic Shellfish for their help during the study.

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