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Potential resistance of a number of populations of the oyster Ostrea edulis to the parasite Bonamia ostreae
Aquaculture 237 (2004) 41 – 58 www.elsevier.com/locate/aqua-online
Potential resistance of a number of populations of the oyster Ostrea edulis to the parasite Bonamia ostreae S.C. Culloty *, M.A. Cronin 1, M.F. Mulcahy Department of Zoology, Ecology and Plant Science, University College Cork, Lee Maltings, Prospect Row, Cork, Ireland Received 17 March 2003; received in revised form 1 April 2004; accepted 4 April 2004
Abstract The study investigated the susceptibility of a number of European populations of Ostrea edulis to the protistan parasite Bonamia ostreae. The study was carried out at oyster-growing regions in three European countries where Bonamia is endemic. The oyster populations screened during the trial were either: naı¨ve populations, which had never previously been exposed to the parasite; populations of oysters, which had been exposed to the parasite for a number of years and where no management of the population to try to reduce infection had occurred; and an infected population, where selective breeding has taken place to try to reduce susceptibility to the parasite. Results of the study indicated that this latter population ‘Rossmore’ performed significantly better in some trials than the other populations in terms of prevalence and intensity of infection. Another population from Lake Grevelingen performed best in one trial when cumulative mortality rates were compared. D 2004 Elsevier B.V. All rights reserved. Keywords: Ostrea edulis; Bonamia ostreae; Resistance; Field trial; Parasite
1. Introduction The European flat oyster Ostrea edulis L. is the native species of oyster found throughout Europe and in the past was the basis for oyster production in a number of European countries including France, Ireland, UK, The Netherlands, and Spain (Culloty
* Corresponding author. Tel.: +353-21-4904198; fax: +353-21-4270562. E-mail address: [email protected] (S.C. Culloty). 1 Current address: Coastal and Marine Resources Centre, Environmental Research Institute, University College Cork, Naval Base, Haulbowline, Cobh, County Cork, Ireland. 0044-8486/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2004.04.007
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and Mulcahy, 1996). However, since the 1970s, large losses have been sustained in this industry, initially due to Marteilia refringens and then to the protistan parasite Bonamia ostreae (Pichot et al., 1980; Van Banning, 1985; Howard, 1994; Ca´cares-Martinez et al., 1995; Culloty and Mulcahy, 1996). It would appear that there may have been two independent introductions of B. ostreae into European oysters stocks—into Brittany, France, and Asturias, Spain from which the disease spread (Cigarrı´a and Elston, 1997). In France, the first observations of abnormal mortalities due to Bonamia were made in Ile Tudy (southern Brittany) in July 1979 (Tige´ and Grizel, 1984). In the Netherlands, Bonamia was first described in the Yerseke Bank in 1980, following exposure of oysters to stocks imported from France, and in the Stampersplaat area of Lake Grevelingen in 1988 (Balouet and Poder, 1983; Van Banning, 1990, 1991). Bonamiasis was first diagnosed in England in October 1982 following an investigation into an unexplained mortality in the creeks of the rivers Fal and Helford in Cornwall (Bucke and Feist, 1985). In Ireland, Bonamia is present at a number of sites, including Cork harbour on the south coast, where the disease was initially diagnosed in 1987 (McArdle et al., 1991). To date, there have been no records of Bonamia in either Scotland or Wales. Control and eradication of bonamiasis have been largely unsuccessful. A number of modified husbandry techniques have been employed to try to minimise disease effects and allow the animals to grow to market size before mortalities have become unsustainable (Grizel, 1983; Van Banning, 1985, 1987; Le Bec et al., 1991). However, these have not been successful on a continuing basis. Alternative species including Ostrea angasi and Tiostrea lutaria (Hutton) have been investigated as possible replacement species for O. edulis (Grizel, 1983; Bucke et al., 1984; Bougrier et al., 1986). However, these species have been found to be equally susceptible to B. ostreae infection. 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, which allows the oysters to be grown to market size before disease-induced mortalities occur (Ford, 1986). Selective breeding in some strains has been used to try to increase resistance to Bonamia (e.g., in Rossmore oysters in Cork harbour, Ireland). Previous studies would indicate that field exposure to naturally occurring disease appears to be the best method for large-scale selection programs (Gaffney and Bushek, 1996). Over the past 16 years, a selective breeding program has been undertaken at Cork harbour, with broodstock being selected from 3- to 4-year-old survivors on the oyster beds. Controlled spawning is carried out in spatting ponds, and oysters are on-grown in designated beds in the harbour. In a previous study, Rossmore oysters were found to have lower prevalence of infection and percentage mortality than a number of other Irish strains, when exposed to the parasite in the laboratory and the field (Culloty et al., 2001). This study was carried out to examine the performance of the Rossmore oysters in comparison to oysters from other European sources. The objectives were to compare native flat oysters from different geographic regions in Europe, when relaid at a number of Bonamia endemic sites, for (a) growth and (b) susceptibility to bonamiasis, in order to identify the oyster sources with the optimal potential for restoring oyster-growing areas decimated by this disease.
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2. Materials and methods 2.1. Oysters Oysters to be tested in the trials had a starting mean weight of 57 – 67 g and were from the following locations: Rossmore, Cork harbour, Ireland (code IER)— oysters that had been selectively bred for resistance to B. ostreae for over 10 years Lough Foyle, Ireland (code IELF)—a public fishery where Bonamia has never been diagnosed Tralee, Ireland (code IET)—a public fishery where Bonamia has never been diagnosed Lough Kishorn, Scotland (code UKLK)—a fishery where Bonamia has never been diagnosed Mull, Scotland (code UKM)—a fishery where Bonamia has never been diagnosed Lake Grevelingen, Holland (code NLG)—a public fishery where Bonamia has been present for 10 years Brittany, France (Fb)—oysters known to have been infected with Bonamia for over 10 years. 2.2. Trial sites and holding conditions Field trials were set up at three sites, in all of which bonamiasis has occurred, in June 1998 and these ran until January 2000 or April 2000. The sites were (1) Cork harbour, Ireland; (2) Lake Grevelingen, The Netherlands; and (3) Brittany, France. At the Irish site, the five strains were placed on the lower shore, in oyster bags, on trestles in the North Channel of Cork harbour. This is a site where the trestles and oysters are exposed only at spring tide. The trestles held the oyster bags approximately 40 cm off the substrate, which is muddy. In Lake Grevelingen, oysters were placed in bags on trestles, which were continually covered. Lake Grevelingen, a former estuary is now a saline lake closed off from the North Sea in 1971. In Brittany, oysters were placed in bags on trestles on the upper shore and so were exposed at low tide. Approximately 700 oysters from each strain were held in oyster bags (70 oysters per bag) at each site, except from the Lake Grevelingen and French strains, which could not be moved from their own sites due to EU Directive 91/67. Heavy mortalities occurred at Lake Grevelingen in the first trial, and so a second trial was carried out from March 1999 to April 2000. Due to further heavy mortalities in the Lake Grevelingen strain during this second trial, another batch of this strain was introduced into the trial in October 1999. Continuous temperature monitors (Guernsey Sea farms) were placed at all sites and temperatures were recorded on an hourly basis throughout the trials. 2.3. Monitoring of oysters A sample of 30 oysters was withheld from each strain before relaying at each site to determine baseline wet weights and prevalence of infection of Bonamia. Samples of
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oysters from six of the seven strains (none available from France) were available for this prescreening. In September and December 1998; March, June, and September 1999; and January and April 2000, subsamples of 30 oysters were taken from each strain at each site. This was hampered in some cases by high mortalities in some strains and by loss of bags at some sites, so some samples were not available for later samplings. 2.4. Percentage mortality and percentage cumulative mortality In July 1998, one month after the start of the trial, mortalities were monitored at all sites, except the French site, to assess losses attributable to transport and handling stress in the weeks following transfer to the different sites. Mortalities from August 1998 onward were recorded to assess mortality due to ‘natural causes’ including bonamiasis. All the oysters, both live and dead, were counted each time they were sampled. Percentage mortality was estimated as the percentage of dead oysters among the oysters remaining at a particular sampling period. Cumulative mortality (%) was estimated according to Tige´ and Grizel (1984): Number of oysters placed in bags at T0 Number of oysters alive at T1 100=Number of oysters placed in bags at T0 where T0=Time 0 and T1=Time of particular sample. 2.5. Wet weights Mud and fouling agents were removed from all oysters, on return to the laboratory. Oysters were then weighed on an analytical balance to the nearest 0.1 g to determine wet weight. 2.6. Monitoring for infection with B. ostreae All oysters were screened for B. ostreae infection by ventricular heart smears as previously described (Bache´re et al., 1982; Culloty et al., 1999): Class Class Class Class Class
0: 1: 2: 3: 4:
no Bonamia cells observed after 5 min of screening 1 –10 Bonamia cells observed within 5 min of screening 11– 100 Bonamia cells observed within 5 min of screening Bonamia cells observed in all fields of vision a heavy parasite burden in all host cells.
For the purposes of comparing the intensity of infection in the different strains of oysters at the different sites, class 1 and 2 infections were deemed to be ‘light’ infections, and class 3 and 4 infections were deemed to be ‘heavy’ infections.
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2.7. Condition To measure condition index, the flesh and shell of each oyster were separated and each placed in preweighed tinfoil cases, which had been individually numbered. The cases were placed in an oven at 60 jC for 48 h. The cases were reweighed and the condition index calculated using the following formula (Walne and Mann, 1975; Lucas and Beninger, 1985): Dry flesh weight ðgÞ 100=Dry shell weight ðgÞ 2.8. Statistics Data were tested for homogeneity of variance and were found to be ‘non-normal.’ Analysis of variance (general linear model) was carried out to examine weight and condition index with respect to strain and seasonality following a natural log transformation of the weight and infection data and ArcSineM transformation of the condition index data. Tukey multiple comparison tests were run following analysis of variance (ANOVA).
3. Results 3.1. Prevalence of infection Prior to relaying, IER had an initial prevalence of infection of 28%, which was highly significantly different ( P<0.01) from all other strains which appeared uninfected (Table 1). At the Cork harbour site, all strains except UKM had picked up infection by September 1998, 3 months postrelaying, but the latter strain was also infected by December 1998 (6 months postrelaying). By December, IELF had 79.3% prevalence of infection, which was very highly significantly different from that observed in IER (17.9%) and UKM (3.9%) ( P<0.001). From June 1999 (12 months postrelaying), all naı¨ve strains had similarly high prevalence of infection, which continued up to the end of the trial. The lowest prevalence continued to occur in IER throughout the rest of the trial up to January 2000, and there was a very highly significant difference between IER and all other strains in June and October 1999 ( P<0.001). At Lake Grevelingen in Trial 1, no infection was observed in NLG when screened at the beginning of the trial in June 1998. By September 1998, no infection was observed in IER, which had had 28.0% prevalence of infection in June 1998. The only strain to show any infection was IELF with 8.0% prevalence. By December 1998 (6 months postrelaying), all strains were infected, with IET and UKLK having 100% prevalence of infection. IER had the lowest prevalence of infection at 40.6%, which was very highly significantly different ( P<0.001) from all other strains. In March 1999, IER again had the lowest prevalence of infection. By June 1999 (12 months postrelaying), few oysters remained and prevalence had decreased in NLG and IELF (8.0% and 22.2%, respectively) and increased in IER (54.6%).
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Table 1 Prevalence of infection (%) in the European populations of native oysters at the three field sites from June 1998 to April 2000 June 1998
September
28.0 0.0 0.0 0.0 0.0
10.7 17.2 10.7 0.0 16.0
Lake Grevelingen 1 NLG 0.0 IER 28.0 IELF 0.0 IET 0.0 UKM 0.0 UKLK 0.0 Lake Grevelingen NLG NLG (9/1999) IER IET UKLK
2 – – – – –
Brittany FB IER IELF IET UKM UKLK
28.0 0.0 0.0 0.0 0.0
Cork harbour IER IELF IET UKM UKLK
b
December
Mar 1999
June
October
Jan 2000
17.9 79.3 32.1 3.9 53.3
44.8 78.6 26.7 48.3 89.7
25.0 96.7 92.6 76.7 100.0
10.3 87.5 100.0 100.0
50.0 100.0 88.9 50.0
0.0 0.0 8.0 0.0 0.0 0.0
76.7 40.6 68.0 100.0 63.0 100.0
36.7 14.3 36.4 44.4 25.9 25.0
– – – – –
– – – – –
70.0 – 0.0 0.0 0.0
0.0 – 0.0 0.0 0.0
74.1 9.1 19.4 31.0 6.9 6.7
28.6 14.3 0.0c 0.0c 0.0c 12.5c
26.7 23.8 86.2 16.0 18.5 30.0
14.3 5.6 20.0 4.3 54.6 0.0
April a a a a
a
a
a
8.0 54.6 22.2
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
93.1 0.0 76.7 96.7 82.8
a
a
13.3 26.0 73.3 90.0
0.0 20.0 100.0 92.9
a
a
28.0
a
a a
a
a
a
16.7 60.0 72.8
a
a
a
a
a
a
( – ) Trial had not begun. a No samples available (all oysters dead). b No initial sample screened. c Heart smears could not be read; results were determined from screening of tissue sections.
In the second trial at Lake Grevelingen, NLG had 70% prevalence of infection at the beginning of the trial in March 1999. However, by June, 3 months postrelaying, no infection was observed in oysters of this strain nor any of the other strains. By October, all strains were infected except NLG (9/99), which had been relayed that month. Prevalence of infection varied from 76.7% to 96.7% in IER and IET, respectively, which was very highly significantly different ( P<0.001). By January and April 2000, IET and UKLK had continuing high prevalence of infection (100% and 92.9% respectively), which was very highly significantly different from the other two strains ( P<0.001) NLG (09/99) and IER (0% and 20%, respectively). NLG had all died at this stage. At the French site, it appears that IER again was the only group to be infected at the beginning of the trial but the status of the French strain was unknown because no sample was available. By September 1998, all strains were infected with prevalence of infection
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varying from 6.7% to 74.1% in UKLK and Fb, respectively. A number of heart smears could not be read in December 1998 because of degradation of cells, and tissue sections were screened. No infection was detected in these tissue sections. By March 1999, the highest prevalence of infection was in IELF at 86.2%, but levels decreased in all strains in June 1999 except UKM. Prevalence of infection in UKM (54.6%) during this month was very highly significantly different from IET and UKLK, which had the lowest levels at 4.3% and 0.0%, respectively ( P<0.001). By October, the highest prevalences of infections in the remaining oysters were in the UKM and UKLK strains at 60.0% and 72.8%, respectively. Prevalence of infection in UKLK was very highly significantly different ( P<0.001) from IER and IET, which had the lowest prevalence of infection at 28.0% and 16.7%, respectively. 3.2. Intensities of infection In Cork harbour, a high prevalence of infection had developed in some strains by December 1998, 6 months postrelaying. Heavy infections (i.e., class 3 and 4 infections) were observed in some populations [i.e., IELF (13.8%) and IER and IET (both 10.7%)] at this stage. In March 1999, the percentage of oysters with class 3 and 4 infections varied from 0.0% (IER) to 20.7% (UKLK) with all strains except IER, which showed heavy infections. By June 1999, all the populations showed ‘heavy’ infections ranging from 7.0% (IER) to 63.4% (IELF). In October 1999, only two strains had class 3 and 4 infections—UKM (88.9%) and IET (56.0%). All of the UKLK oysters were dead. On the final sampling date in January 2000, 33.3% of IELF oysters had heavy infections as had 3.3% of IER oysters, but ‘heavy’ infections had decreased in UKM and IET (0.0% and 27.8%, respectively). In June 1998, during Trial 1 at Lake Grevelingen, 8% of the IER group had heavy infections (i.e., classes 3 and 4). No further heavy infections were observed until December 1998, 6 months postrelaying, when the percentage of oysters with heavy infections varied from 0.0% (IER) to 30.0% (IET). By March 1999, there was a large reduction in these percentages in all strains with heavy infections varying from 0.0% (IER) to 11.1% (IET). In June 1999, only three strains—IELF, NLG, and IER—had surviving oysters and were available for sampling and, in these, heavy intensities of infection varied from 0.0% to 9.1%. When the second trial began at Lake Grevelingen in March 1999, NLG had a high prevalence of infection (70%). Among NLG, 10.0% of oysters had heavy infections. Infections were not observed in the other strains until October 1999 when ‘heavy’ infections varied from 0.0% (NLG (09/99) and IER) to 30.0% (IET). In January 2000, IER still did not have any heavy infections, in comparison to 43.3% of oysters with heavy infections from UKLK. In April 2000, at the end of the trial, IER and NLG (9/99) still did not have any heavy infections; the highest prevalences were observed in IET (55.9%). At the Brittany site, IER was the only strain with heavy infections in June 1998. By September, Fb had 11.1% of oysters with class 3 infections and IELF had 3.2%. The heaviest intensities of infection occurred in IELF (48.2%) in March 1999 and UKLK (45.0%) in October 1999. Most infections observed throughout the trial at this site were light (i.e., classes 1 and 2).
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3.3. Percentage cumulative mortality At Cork Harbour, when the trial finished in January 2000, percentage cumulative mortality varied from 86.5% to 100%, with IER having the lowest level and UKLK the highest (Fig. 1A – D). During Trial 1 at Lake Grevelingen, by June 1999, cumulative mortality had risen to 100% in IET, UKM, and UKLK, and in the other three strains varied from 79.8% (NLG), 83.5% (IER), to 97.9% (IELF) (Fig. 1B). At the end of the second trial at Lake Grevelingen in April 2000, cumulative mortality varied from 36.5% (IER) to 92.5% (IET). The NLG (9/99) group relayed in October 1999 had 7.9% cumulative mortality. The trial in Brittany terminated in October 1999 and cumulative mortality there varied from 76.6% (IER) to 100% (Fb and IELF) (Fig. 1D).
Fig. 1. (A – D) Cumulative mortalities (%) in all populations of oysters at the three field sites from June 1998 to April 2000. A, Cork Harbour; B, Lake Grevelingen #1; C, Lake Grevelingen #2; D, Brittany.
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Fig. 1 (continued).
3.4. Whole wet weights All strains had started the field trial with mean weights within 10 g of each other at each site (Fig. 2). It can be seen from Fig. 2 that the largest increases in whole mean wet weights took place in oysters held in Lake Grevelingen. In the first trial at this site from June 1998 to June 1999, the best performers were the native strain (NLG), with an increase in weight of 102.5% over the 12 months of the trial, which was significantly different from all other strains ( P<0.001). IET and UKM had the lowest increases at 23.1% and 17.9%, respectively. In the second trial at this site from March 1999 –April 2000, all oysters grew well—IER performed the best with an increase of 106.1% in its mean whole weight. In the second batch of oysters from Lake Grevelingen, introduced in October 1999, the mean weight decreased from this date to April 2000. At Cork harbour, all strains increased in weight over the trial period from June 1998 to January 2000. Over the 18 months of the trial, IELF had the highest increase in mean
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Fig. 2. Percentage change in whole wet weight of each population over the trial period at each field site.
weight at 59.5% and was very significantly different from all other strains ( P<0.001). This increase was followed by IER at 40.3% and IET at 38.1%. UKLK had a percentage increase of 33.3% and the lowest was in UKM at 23.8%. It can be seen from Fig. 2 that oysters performed worst at the French site. Over the trial period from June 1998 to October 1999, a decrease in mean weight was recorded in UKM (9.2%). Of the other strains, IER performed best with an increase of 37.7% compared to 25.6% for IELF and increases of 9.5%, 10.8%, and 10.9% for UKLK, IET, and Fb, respectively. 3.5. Condition indices The highest condition indices were recorded in Cork harbour in June 1998 when the trial began, and there was a very highly significant difference between values in this month and all other months except June 1999 ( P<0.001) (Fig. 3A – D). Condition for all strains available were at their lowest level when the trial finished in January 2000, with values ranging on this month from 0.7 to 3.6 (UKM and IER, respectively) and being very highly significantly different from all other months ( P<0.001). Throughout the trial, UKM and IELF consistently had the lowest condition indices each time the oysters were sampled, and the condition indices of both these strains were very highly significantly different from all others ( P<0.001). In Trial 1 at Lake Grevelingen, oysters were from the same group relayed in Ireland, and so were starting with the same condition indices (Fig. 3B). Over the subsequent months, there was no clear pattern, with the condition of some strains increasing and others decreasing. UKM had a lower condition index than the other populations and there was a very highly significant difference between this strain and all others ( P<0.001). In June 1999, at the end of the trial, of the populations available, conditions ranged from 5.1F1.5 (IER) to 3.9F1.0 (NLG). In the second trial at this site, which started in March 1999, conditions ranged from 2.9 to 4.4 (IET and NLG, respectively) (Fig. 3C). The condition of NLG dropped over the trial, but other strains showed some increases. When the trial finished in April 2000, condition indices varied from 4.4 to 5.9 (IER and UKLK, respectively). Condition index for UKLK was very highly significantly different from all others ( P<0.001).
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At Brittany, no initial condition index was available for the French population (Fig. 3D). Condition indices ranged from 2.9 to 5.7 in June 1998 in the other populations and were lowest in March and October 1999, with levels on these months being very highly significantly different from all other months ( P<0.001). UKM had the lowest condition indices throughout the trial, being very highly significantly different from all other strains ( P<0.001). Final condition indices varied from 1.5 to 4.1, with IET having the highest and UKM the lowest. 3.6. Temperature data In Cork harbour, mean monthly temperatures varied from a low in January 2000 (7.7 jC) to a high of 17.6 jC in August 1999. Peaks in temperature occurred in August of both 1998 and 1999 and the lowest temperatures occurred in January 1998 and January 1999. In Lake Grevelingen, not all recordings were available due to the loss of some recorders. Temperatures for the dates available varied from 3.1 jC in November 1998 to 20.4 jC in July 1999. In France, mean temperatures varied from 9.9 jC in March 1999 to 16.3 jC in
Fig. 3. (A – D) Mean condition indices for all populations at each field site over the trial period (June 1998 – April 2000). A, Cork Harbour; B, Lake Grevelingen #1; C, Lake Grevelingen #2; D, Brittany.
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Fig. 3 (continued).
September 1998. It appeared that the least variation in mean temperatures occurred at the French site and most at Lake Grevelingen over the months when temperatures were recorded.
4. Discussion Temperature data were collected from each site over the study period. Most variations occurred at Lake Grevelingen, with the highest and lowest monthly mean temperatures being observed here. This was also the only site where the oysters were continually submerged throughout the trials. Intersite differences were observed when analysis of growth rates of each strain at each site was undertaken, with the greatest growth rates in Lake Grevelingen. This may be due to a combination of optimal food levels and environmental factors, or the fact that oysters were continually submerged throughout the trial. The lowest growth rates occurred at the site in Brittany, and this along with the other factors would indicate that this site is not
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suitable for growth of this oyster species. No one population performed best at all sites. The only consistent result was that UKM had the lowest increases in weight in Cork harbour, Brittany, and Lake Grevelingen, followed by UKLK. The bad performance of the Scottish oysters at all sites may indicate that these strains do not adapt well to the combination of the environmental factors that they experienced at these sites. 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 decreased growth efficiency as their size increases. In this study, all oysters started the trial with a mean wet weight within 10 g of each other. Over the trial period, condition indices fluctuated and all groups lost condition. Conditions showed some reductions during the winter months possibly related to spawning, cessation of feeding, lower water temperatures, and high levels of infection (Walne and Mann, 1975; Baud et al., 1997). No one population showed consistently higher condition over the study. However, UKM consistently had the lowest condition when monitored in Cork harbour, Brittany, and in Trial 1 in Lake Grevelingen. Walne and Mann (1975) found that in the spring, a high rate of growth of the flesh weight and slower growth of the shell led to an improvement in the condition, with increased shell growth in summer related to an increase in water temperature. Barber et al. (1988) found that MSX reduced condition in C. virginica and the reduction was related to the severity of the infection. The results of this study suggest that differences exist between European populations of O. edulis in susceptibility to infection with B. ostreae. The field trials indicated that Rossmore oysters showed some tolerance to infection with Bonamia compared to other populations. When comparing prevalence and intensity of infection in the different populations over the 22 months of the trial, lower levels were observed in the Rossmore group, and significant differences in prevalence of infection were found between the Rossmore oysters and the other populations tested. However, when comparing mortality, the picture was not as clear, with the Lake Grevelingen population performing better in one trial. The Rossmore oysters had been exposed to the parasite throughout life, having been selectively bred in an area where the parasite has been present for over 16 years. Intraspecific genetic variation in disease susceptibility is indirectly demonstrated by the evolution of resistance in disease-challenged natural populations (Gaffney and Bushek, 1996). Bushek (1994) in Gaffney and Bushek (1996) found that oysters from different geographical regions showed distinct differences in their response to infection with P. marinus. Populations with previous exposure to the pathogen showed lower body burden than oysters originating from less exposed populations. A program undertaken with the aim of breeding faster-growing Sydney rock oysters Saccostrea glomerata demonstrated that after one generation, a small improvement in resistance to QX disease was observed (Nell et al., 2000). All other populations (whether naı¨ve or previously exposed to Bonamia) appeared to be equally susceptible to infection, when comparing prevalence of infection and intensity, with initial infections being observed several months postrelaying in agreement with previous observations (Hine, 1991; Culloty and Mulcahy, 1996). There was no evidence of increased resistance with respect to prevalence and intensity of infection in the Lake Grevelingen oysters. All previously unexposed populations (i.e., Tralee, Lough Foyle, Mull, and Lough Kishorn) were equally susceptible when exposed to the parasite.
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Cumulative mortalities were high for all populations by the end of each field trial. However, the Rossmore oysters had the lowest percentage in each trial, except in Trial 1 at Lake Grevelingen when the native population had the lowest cumulative mortality at the end of the trial. By the end of each trial, most of the oysters from the other populations had died. Similar studies looking at resistance to bonamiasis have also observed generally high mortalities in field trials, but the lowest mortalities in populations selected for resistance (Baud et al., 1997; Naciri-Graven et al., 1998, 1999). The husbandry techniques employed in this study, such as holding the oysters in bags on trestles, sorting and handling every couple of months, and placing them in the intertidal areas, may have contributed to mortalities as these oysters are normally grown subtidally without interference until they reach market size. However, all populations were handled in the same way at each site and at similar intervals, so the comparative results are valid. In this study, the oysters in Cork harbour and Brittany were exposed on the trestles at spring tides and were handled frequently. At Lake Grevelingen, oysters were continually submerged, except when samples were being removed, but this factor did not appear to reduce mortalities. Martin et al. (1993) in similar trials found that oysters in intertidal areas are more susceptible to development of B. ostreae disease than those in subtidal areas; exposing oysters in the intertidal zone of a estuary boosted infestation. However, when infection and mortality patterns are compared, it is clear that most heavy mortalities, particularly in the naı¨ve populations, occurred in the months after heavy prevalence and intensity of infection were noted, particularly in Cork harbour and Lake Grevelingen, indicating that these peaks in mortality were related to disease. Montes et al. (2002) found a relationship between the level of Bonamia and high mortality rates. Analysis of results from Brittany indicated that prevalence of infection was only one factor contributing to the high mortalities observed: it appears that this area is not suitable for growth of this species, and that other factors contributed to the heavy losses observed. In the field trials, a decrease in prevalence of infection was not always related to an increase in mortalities in the Rossmore group. It appears that this strain may be able to tolerate a certain level of infection and, in some cases, overcome the parasite. Hervio et al. (1995) found individual variability in susceptibility to bonamiasis in oysters, when inoculated with a known number of B. ostreae cells. Elston et al. (1987) noted that populations of oysters exposed to Bonamia for a number of years had a substantial degree of resistance to infection compared to populations with no prior exposure history, and resistant oysters had lesions, which indicated that they could contain the infection. In the case of MSX-resistant oysters, it has been found that the animals did become infected with Haplosporidium 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). Differences in susceptibility to H. nelsoni were associated with geographic origin of oysters tested, as it was thought that the parasite interferes with energy acquisition, storage, and conversion cycles, which differ seasonally with geographic origin (Ford et al., 1990). Physiological variation, whether due to environmental influences, heredity, or both, may result in differences in susceptibility to infection as may more specific biochemical or cellular responses (Gaffney and Bushek, 1996). For example, Oliver et al. (2000) produced 10 families of Crassostrea virginica with high interfamily and intrafamily variability. In the laboratory, three families, when challenged with Perkinsus marinus, had the highest
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survival rate, lowest numbers of parasites, and highest average protease inhibitory activity, and they also performed best in field studies in terms of cumulative mortality. Romstead et al. (2002) also associated highest protease inhibitory activities with resistance of Crassostrea gigas to infection with P. marinus. Anderson and Beaven (2001) in a comparative study of anti-Perkinsus activity in bivalve sera found that sera from mussels, that are resistant to Perkinsus, contained high antiprotease activity compared to C. virginica that are susceptible. How this increased resistance to Bonamia could be exploited to improve O. edulis stocks has to be considered carefully. At present, breeding of the Rossmore-‘resistant’ oysters involves a large randomly mating population of older oysters, that are known to have survived 4 years on the oyster beds, which are removed to spatting ponds to spawn several hundred broodstock per pond. Launey et al. (2001) looked for evidence of inbreeding and population bottlenecks in three hatchery-propagated populations of the flat oyster as part of a selection program for resistance to Bonamia. A reduction in the effective population size following a population bottleneck is correlated with a decrease in heterozygosity and a loss of rare alleles (Launey et al., 2001; Evans et al., 2004). It would appear that bottlenecks may have a stronger and more immediate effect on allelic diversity than on heterozygosity. In the Launey et al. study, which looked at Bonamia-resistant populations of O. edulis, although heterozygosity was still high in the resistant and control populations, the number of alleles in the selected population was significantly reduced compared with the control population, and this was due in large part to the loss of rare alleles. As a result, a decrease in performance was expected as soon as the second generation in relation to both growth and survival. However, Marsic-Lucic and David (2003) found a lack of heterozygote deficiency and multilocus heterozygosity –fitness correlations within five natural populations of O. edulis from the Adriatic Sea. They considered that this was due to very low inbreeding within the populations. The objectives in relation to management of the Rossmore population in the future are to maintain this resistance without loss of other traits associated with fitness. Gaffney and Bushek (1996) concluded that field exposure to naturally occurring disease appears to be the best approach for large-scale selection programs, but they do suffer from annual variation in disease pressure plus other variables such as temperature, salinity, etc. During these field trials, oysters were exposed to non-native Bonamia parasites at the sites to which they were relaid. However, in the case of the Rossmore group, these oysters still performed better at the other sites apart from cumulative mortality results from Trial 1 in Lake Grevelingen. The possibility of different strains of Bonamia being present at different sites may, however, require further investigation. The existence of different strains has to be taken into consideration due to the fact that two foci of infection may have initially been responsible for the spread of Bonamia within Europe (Cigarrı´a and Elston, 1997). Bushek and Allen (1996) examined the resistance of four genetically distinct populations of C. virginica that had different histories of exposure to P. marinus with four geographically distinct isolates of P. marinus of varying virulence. They found that in the oysters, resistance was related to length of exposure. Different isolates of Perkinsus showed different degrees of virulence; however, no statistically significant interaction was detected between oyster populations and parasite isolates, indicating that mechanisms of resistance and virulence were general, not race-specific. The fact that tolerance is not
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complete as, for example, has been observed in populations of C. virginica exposed to H. nelsoni. This would indicate that the ability to actually resist rather that just tolerate infection suggests that the oyster’s repertoire of genetic variability is not sufficient to provide complete protection against the disease (Gaffney and Bushek, 1996). However, the extent to which the parasite has adapted itself to take advantage of different environmental factors present in different regions remains undetermined. In conclusion, in so far as one can compare the susceptibility of different oyster strains to B. ostreae, the results of these field trials support the idea that a degree of ‘‘resistance’’ has developed in the Rossmore oysters. It would appear that the Lake Grevelingen population also performed well in terms of overall survival. Previous exposure in these populations has conferred some reduced susceptibility to the parasite compared to naı¨ve populations. In relation to the life cycle of B. ostreae, it is clear from a number of studies that direct transmission of the parasite can occur from oyster to oyster (Culloty et al., 1999) and there is some evidence to suggest that entry of the parasite may be initially through the gills (Montes et al., 1994). Van Banning (1990) suggested a presumptive life cycle in which an infectious phase might be involved in the ovarian tissue of O. edulis. However, the possibility of other life cycle stages such as a spore stage or an intermediate or secondary host being involved cannot, at this stage, be discounted. Phylogenetic analysis of DNA sequence data confirmed Bonamia to be a member of the Haplosporidia, of which a spore stage is typical (Carnegie et al., 2000; Cochennec et al., 2000). To date, a spore stage has not been observed in infected oysters. As questions still arise in relation to aspects of the life cycle and further study is required of the immune mechanisms of the host, both of these factors hamper efforts to determine the basis of the ‘resistance’ observed during this study.
Acknowledgements This study was supported by EU CRAFT CT98-910. The authors would like to acknowledge the contribution of David Hugh Jones, Atlantic Shellfish, Cork, Dennis Gowland, Orkney, Jan Bol, and Dr. Aad Smaal (RIVO, The Netherlands) to the field work. References Anderson, R.S., Beaven, A.E., 2001. A comparative study of anti-Perkinsus marinus activity in bivalve sera. J. Shellfish Res. 20 (3), 1011 – 1018. Bache´re, E., Durand, J., Tige´, G., 1982. Bonamia ostreae (Pichot et al., 1979) parasite de l’huıˆtre plate comparison de deux methodes de diagnostic. ICES CM, F 28, 1 – 11. Balouet, G., Poder, M., 1983. Bonamia—a threat to oyster stocks. Proceedings of the XIVth Conference on Shellfish, 74 – 83. Barber, B.J., Ford, S.E., Haskin, H.H., 1988. Effects of the parasite MSX (Haplosporidium nelsoni) on oyster (Crassostrea virginica) energy metabolism: 1. Condition index and relative fecundity. J. Shellfish Res. 7 (1), 25 – 31. Baud, J.P., Ge´rard, A., Naciri-Graven, Y., 1997. Comparative growth and mortality of Bonamia ostreae-resistant and wild flat oysters Ostrea edulis in an intensive system. Mar. Biol. 130, 71 – 79.
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Bougrier, S., Tige´, G., Bache´re, E., Grizel, H., 1986. Ostrea angasi acclimatization to French coasts. Aquaculture 58, 151 – 154. Bucke, D., Feist, S.W., 1985. Bonamiasis in the flat oyster, Ostrea edulis with comments on histological techniques. Fish and Shellfish Pathology. Academic Press, London, pp. 387 – 392. Bucke, D., Hepper, B., Key, D., Bannister, R.C.A., 1984. A report on Bonamia ostreae in Ostrea edulis in the UK. ICES CM, K 9, 1 – 7. Bushek, D., 1994. Dermo disease in American oysters: genetics of host parasite interactions. PhD Dissertation. Rutgers, New Brunswick, New Jersey. 189 pp. Bushek, D., Allen, S.K., 1996. Host – parasite interactions among broadly distributed populations of the eastern oyster Crassostrea virginica and the protozoan Perkinsus marinus. Mar. Ecol. Progr. Ser. 139, 127 – 141. Ca´cares-Martinez, J., Robledo, J.A.F., Figueras, A., 1995. Presence of Bonamia and its relation to age, growth rates and gonadal development of the flat oyster Ostrea edulis in the Ria de Vigo, Galicia (NW Spain). Aquaculture 130, 15 – 23. Carnegie, R.B., Barber, B.J., Culloty, S.C., Figueras, A.J., Distel, D.L., 2000. Development of a PCR assay for detection of the oyster pathogen Bonamia ostreae and support for its inclusion in the Haplosporidia. Dis. Aquat. Org. 42, 199 – 206. Cigarrı´a, J., Elston, R., 1997. Independent introduction of Bonamia ostreae, a parasite of Ostrea edulis, to Spain. Dis. Aquat. Org. 29, 157 – 158. Cochennec, N., Le Roux, F., Berthe, F., Gerard, A., 2000. Detection of Bonamia ostreae on small subunit ribosomal probe. J. Invertebr. Pathol. 76 (1), 26 – 32. Culloty, S.C., Mulcahy, M.F., 1996. Season-, age-, and sex-related variation in the prevalence of bonamiasis in flat oysters (Ostrea edulis) L. on the south coast of Ireland. Aquaculture 144, 53 – 63. Culloty, S.C., Novoa, B., Pernas, M., Longshaw, M., Mulcahy, M.F., Feist, S.W., Figueras, A., 1999. Susceptibility of a number of bivalve species to the protozoan parasite Bonamia ostreae and their ability to act as vectors for this parasite. Dis. Aquat. Org. 37, 73 – 80. Culloty, S.C., Cronin, M.A., Mulcahy, M.F., 2001. An investigation into the relative resistance of Irish flat oysters Ostrea edulis L. to the parasite Bonamia ostreae (Pichot et al., 1980). Aquaculture 199, 229 – 244. Elston, R., Kent, M.L., Wilkinson, M.T., 1987. Resistance of Ostrea edulis to Bonamia ostreae infection. Aquaculture 64, 237 – 242. Evans, F., Matson, S., Brake, J., Langdon, C., 2004. The effects of inbreeding on performance traits of the adult Pacific oyster (Crassostrea gigas). Aquaculture 230, 89 – 98. Ford, S.E., 1986. Comparison of hemolymph proteins from resistant and susceptible oysters Crassostrea virginica exposed to the parasite Haplosporidium nelsoni (MSX). J. Invertebr. Pathol. 47, 283 – 294. Ford, S.E., Figueras, A.J., Haskin, H.H., 1990. Influence of selective breeding, geographic origin and disease on gametogenesis and sex ratios of oysters Crassostrea virginica exposed to the parasite Haplosporidium nelsoni (MSX). Aquaculture 88, 285 – 301. Gaffney, P.M., Bushek, D., 1996. Genetic aspects of disease resistance in oysters. J. Shellfish Res. 15 (1), 135 – 140. Grizel, H., 1983. Impact de Marteilia refringens et de Bonamia ostreae sur l’ostreiculture Bretonne. ICES CM, Gen. 9 (30 pp.). Hervio, D., Bache´re, E., Boulo, V., Cochenne, N., Vuillemin, V., Le Coguic, Y., Gailletaux, G., Mazurie, J., Miahle, E., 1995. Establishment of an experimental infection protocol for the flat oyster Ostrea edulis with the intrahaemocytic protozoan parasite Bonamia ostreae: application in the selection of parasite resistant oysters. Aquaculture 132 (3/4), 183 – 194. Hine, P.M., 1991. The annual pattern of infection by Bonamia sp. in New Zealand flat oysters Tiostrea chilensis. Aquaculture 93 (3), 241 – 252. Howard, A.E., 1994. The possibility of long distance transmission of Bonamia by fouling on boat hulls. Bull. Eur. Assoc. Fish Pathol. 14 (6), 211 – 212. Launey, S., Barre, M., Gerard, A., Naciri-Graven, Y., 2001. Population bottleneck and effective size in Bonamia ostreae resistant populations of Ostrea edulis as inferred by microsatellite markers. Genet. Res. 78, 259 – 270. Le Bec, C., Mazurie, J., Cochennec, N., le Coguic, Y., 1991. Influence of Crassostrea gigas mixed with Ostrea edulis on the incidence of Bonamia disease. Aquaculture 93, 263 – 272.
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Lucas, A., Beninger, P.G., 1985. The use of physiological condition indices in marine bivalve aquaculture. Aquaculture 44, 187 – 220. Marsic-Lucic, J., David, P., 2003. Relationship between multiple-locus heterozygosity and growth rate in Ostrea edulis populations. J. Molluscan Stud. 69, 319 – 323. Martin, A.G., Ge´rard, A., Cochennec, N., Langlade, A., 1993. Selecting flat oysters, Ostrea edulis for survival against the parasite Bonamia ostreae: assessment of the resistance of a first selected generation. In: Barnabe´, G., Kelmont, P. (Eds.), Production, Environment and Quality. Bordeaux Aquaculture, vol. 92. European Aquaculture Society, Ghent, Belgium, pp. 547 – 554. Special Publication No. 18. McArdle, J., McKiernan, F., Foley, H., Hugh-Jones, D., 1991. The current status of Bonamia disease in Ireland. Aquaculture 93, 273 – 278. Montes, J., Anado´n, R., Azevedo, C., 1994. A possible life cycle for Bonamia ostreae on the basis of electron microscopy studies. J. Invertebr. Pathol. 63 (1), 1 – 6. Montes, J., Ferro-Soto, B., Conchas, R.F., Guerra, A., 2002. Determining culture strategies in populations of the European flat oyster. Ostrea edulis affected by bonamiosis. Aquaculture 220 (1 – 4), 1 – 8. Myrhe, J.L., Haskin, H.H., 1970. MSX infections in resistant and susceptible oyster stocks. Proc. Natl. Shellfish Assoc. 60, 9. Naciri-Graven, Y., Martin, A.G., Baud, J.P., Renault, T., Ge´rard, A., 1998. Selecting the flat oyster Ostrea edulis (L.) for survival when infected with the parasite Bonamia ostreae. J. Exp. Mar. Biol. Ecol. 224, 91 – 107. Naciri-Graven, Y., Haure, J., Ge´rard, A., Baud, J.P., 1999. Comparative growth of Bonamia ostreae resistant and wild flat oyster Ostrea edulis in an intensive system: II. Second year of the experiment. Aquaculture 171, 195 – 208. Nell, J.A., Smith, I.R., McPhee, C.C., 2000. The Sydney rock oyster Saccostrea glomerata (Gould, 1850) breeding programme: progress and goals. Aquacult. Res. 31, 45 – 49. Oliver, J.L., Gaffney, P.M., Allen, S.K., Faisal, M., Kaattari, S.L., 2000. Protease inhibitory activity in selectively bred families of eastern oysters. J. Aquat. Anim. Health 12 (2), 136 – 145. Pichot, Y., Comps, M., Tige´, G., Grizel, H., Rabouin, M., 1980. Recherches sur Bonamia ostreae Gen. N. sp. N. parasite nouveau de l’huıˆtre plate Ostrea edulis L. Rev. Trav. Inst. Peches Marit. 43 (1), 131 – 140. Romstead, B., Corbier, F., Roch, P., 2002. Protease inhibitors and haemagglutinins associated with resistance to the protozoan parasite, Perkinsus marinus, in the Pacific oyster, Crassostrea gigas. Parasitology 125, 323 – 329. Tige´, G., Grizel, H., 1984. Essai de contamination d’Ostrea edulis Linne´ par Bonamia ostreae (Pichot et al., 1979) en rivie`re de Crach (Morbihan). Rev. Trav. Inst. Peches Marit. 46 (4), 307 – 314. Van Banning, P., 1983. Control of Bonamia in Dutch oyster culture. In: Ellis, A.E. (Ed.), Fish and Shellfish Pathology, pp. 393 – 396. September 20 – 23, 1983, Plymouth, Academis Press, London. Van Banning, P., 1987. Further results of the Bonamia ostreae challenge tests in Dutch oyster culture. Aquaculture 67, 191 – 194. Van Banning, P., 1990. The life cycle of the oyster pathogen Bonamia ostreae with a presumptive phase in the ovarian tissue of the European flat oyster Ostrea edulis. Aquaculture 84 (2), 189 – 192. Van Banning, P., 1991. Observations on bonamiasis in the stock of the European flat oyster Ostrea edulis in the Netherlands with special reference to the recent developments in Lake Grevelingen. Aquaculture 93, 205 – 212. Walne, P.R., 1956. Experimental rearing of the larvae of Ostrea edulis L. in the laboratory. Fish. Invest., Land Ser. 20, 1 – 23. Walne, P.R., Mann, R., 1975. Growth and biochemical composition in Ostrea edulis and Crassostrea gigas. In: Barnes, H. (Ed.), Proceedings of the 9th European Marine Biology Symposium. Aberdeen Univ. Press, Aberdeen, pp. 587 – 607.
Potential resistance of a number of populations of the oyster Ostrea edulis to the parasite Bonamia ostreae S.C. Culloty *, M.A. Cronin 1, M.F. Mulcahy Department of Zoology, Ecology and Plant Science, University College Cork, Lee Maltings, Prospect Row, Cork, Ireland Received 17 March 2003; received in revised form 1 April 2004; accepted 4 April 2004
Abstract The study investigated the susceptibility of a number of European populations of Ostrea edulis to the protistan parasite Bonamia ostreae. The study was carried out at oyster-growing regions in three European countries where Bonamia is endemic. The oyster populations screened during the trial were either: naı¨ve populations, which had never previously been exposed to the parasite; populations of oysters, which had been exposed to the parasite for a number of years and where no management of the population to try to reduce infection had occurred; and an infected population, where selective breeding has taken place to try to reduce susceptibility to the parasite. Results of the study indicated that this latter population ‘Rossmore’ performed significantly better in some trials than the other populations in terms of prevalence and intensity of infection. Another population from Lake Grevelingen performed best in one trial when cumulative mortality rates were compared. D 2004 Elsevier B.V. All rights reserved. Keywords: Ostrea edulis; Bonamia ostreae; Resistance; Field trial; Parasite
1. Introduction The European flat oyster Ostrea edulis L. is the native species of oyster found throughout Europe and in the past was the basis for oyster production in a number of European countries including France, Ireland, UK, The Netherlands, and Spain (Culloty
* Corresponding author. Tel.: +353-21-4904198; fax: +353-21-4270562. E-mail address: [email protected] (S.C. Culloty). 1 Current address: Coastal and Marine Resources Centre, Environmental Research Institute, University College Cork, Naval Base, Haulbowline, Cobh, County Cork, Ireland. 0044-8486/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2004.04.007
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and Mulcahy, 1996). However, since the 1970s, large losses have been sustained in this industry, initially due to Marteilia refringens and then to the protistan parasite Bonamia ostreae (Pichot et al., 1980; Van Banning, 1985; Howard, 1994; Ca´cares-Martinez et al., 1995; Culloty and Mulcahy, 1996). It would appear that there may have been two independent introductions of B. ostreae into European oysters stocks—into Brittany, France, and Asturias, Spain from which the disease spread (Cigarrı´a and Elston, 1997). In France, the first observations of abnormal mortalities due to Bonamia were made in Ile Tudy (southern Brittany) in July 1979 (Tige´ and Grizel, 1984). In the Netherlands, Bonamia was first described in the Yerseke Bank in 1980, following exposure of oysters to stocks imported from France, and in the Stampersplaat area of Lake Grevelingen in 1988 (Balouet and Poder, 1983; Van Banning, 1990, 1991). Bonamiasis was first diagnosed in England in October 1982 following an investigation into an unexplained mortality in the creeks of the rivers Fal and Helford in Cornwall (Bucke and Feist, 1985). In Ireland, Bonamia is present at a number of sites, including Cork harbour on the south coast, where the disease was initially diagnosed in 1987 (McArdle et al., 1991). To date, there have been no records of Bonamia in either Scotland or Wales. Control and eradication of bonamiasis have been largely unsuccessful. A number of modified husbandry techniques have been employed to try to minimise disease effects and allow the animals to grow to market size before mortalities have become unsustainable (Grizel, 1983; Van Banning, 1985, 1987; Le Bec et al., 1991). However, these have not been successful on a continuing basis. Alternative species including Ostrea angasi and Tiostrea lutaria (Hutton) have been investigated as possible replacement species for O. edulis (Grizel, 1983; Bucke et al., 1984; Bougrier et al., 1986). However, these species have been found to be equally susceptible to B. ostreae infection. 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, which allows the oysters to be grown to market size before disease-induced mortalities occur (Ford, 1986). Selective breeding in some strains has been used to try to increase resistance to Bonamia (e.g., in Rossmore oysters in Cork harbour, Ireland). Previous studies would indicate that field exposure to naturally occurring disease appears to be the best method for large-scale selection programs (Gaffney and Bushek, 1996). Over the past 16 years, a selective breeding program has been undertaken at Cork harbour, with broodstock being selected from 3- to 4-year-old survivors on the oyster beds. Controlled spawning is carried out in spatting ponds, and oysters are on-grown in designated beds in the harbour. In a previous study, Rossmore oysters were found to have lower prevalence of infection and percentage mortality than a number of other Irish strains, when exposed to the parasite in the laboratory and the field (Culloty et al., 2001). This study was carried out to examine the performance of the Rossmore oysters in comparison to oysters from other European sources. The objectives were to compare native flat oysters from different geographic regions in Europe, when relaid at a number of Bonamia endemic sites, for (a) growth and (b) susceptibility to bonamiasis, in order to identify the oyster sources with the optimal potential for restoring oyster-growing areas decimated by this disease.
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2. Materials and methods 2.1. Oysters Oysters to be tested in the trials had a starting mean weight of 57 – 67 g and were from the following locations: Rossmore, Cork harbour, Ireland (code IER)— oysters that had been selectively bred for resistance to B. ostreae for over 10 years Lough Foyle, Ireland (code IELF)—a public fishery where Bonamia has never been diagnosed Tralee, Ireland (code IET)—a public fishery where Bonamia has never been diagnosed Lough Kishorn, Scotland (code UKLK)—a fishery where Bonamia has never been diagnosed Mull, Scotland (code UKM)—a fishery where Bonamia has never been diagnosed Lake Grevelingen, Holland (code NLG)—a public fishery where Bonamia has been present for 10 years Brittany, France (Fb)—oysters known to have been infected with Bonamia for over 10 years. 2.2. Trial sites and holding conditions Field trials were set up at three sites, in all of which bonamiasis has occurred, in June 1998 and these ran until January 2000 or April 2000. The sites were (1) Cork harbour, Ireland; (2) Lake Grevelingen, The Netherlands; and (3) Brittany, France. At the Irish site, the five strains were placed on the lower shore, in oyster bags, on trestles in the North Channel of Cork harbour. This is a site where the trestles and oysters are exposed only at spring tide. The trestles held the oyster bags approximately 40 cm off the substrate, which is muddy. In Lake Grevelingen, oysters were placed in bags on trestles, which were continually covered. Lake Grevelingen, a former estuary is now a saline lake closed off from the North Sea in 1971. In Brittany, oysters were placed in bags on trestles on the upper shore and so were exposed at low tide. Approximately 700 oysters from each strain were held in oyster bags (70 oysters per bag) at each site, except from the Lake Grevelingen and French strains, which could not be moved from their own sites due to EU Directive 91/67. Heavy mortalities occurred at Lake Grevelingen in the first trial, and so a second trial was carried out from March 1999 to April 2000. Due to further heavy mortalities in the Lake Grevelingen strain during this second trial, another batch of this strain was introduced into the trial in October 1999. Continuous temperature monitors (Guernsey Sea farms) were placed at all sites and temperatures were recorded on an hourly basis throughout the trials. 2.3. Monitoring of oysters A sample of 30 oysters was withheld from each strain before relaying at each site to determine baseline wet weights and prevalence of infection of Bonamia. Samples of
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oysters from six of the seven strains (none available from France) were available for this prescreening. In September and December 1998; March, June, and September 1999; and January and April 2000, subsamples of 30 oysters were taken from each strain at each site. This was hampered in some cases by high mortalities in some strains and by loss of bags at some sites, so some samples were not available for later samplings. 2.4. Percentage mortality and percentage cumulative mortality In July 1998, one month after the start of the trial, mortalities were monitored at all sites, except the French site, to assess losses attributable to transport and handling stress in the weeks following transfer to the different sites. Mortalities from August 1998 onward were recorded to assess mortality due to ‘natural causes’ including bonamiasis. All the oysters, both live and dead, were counted each time they were sampled. Percentage mortality was estimated as the percentage of dead oysters among the oysters remaining at a particular sampling period. Cumulative mortality (%) was estimated according to Tige´ and Grizel (1984): Number of oysters placed in bags at T0 Number of oysters alive at T1 100=Number of oysters placed in bags at T0 where T0=Time 0 and T1=Time of particular sample. 2.5. Wet weights Mud and fouling agents were removed from all oysters, on return to the laboratory. Oysters were then weighed on an analytical balance to the nearest 0.1 g to determine wet weight. 2.6. Monitoring for infection with B. ostreae All oysters were screened for B. ostreae infection by ventricular heart smears as previously described (Bache´re et al., 1982; Culloty et al., 1999): Class Class Class Class Class
0: 1: 2: 3: 4:
no Bonamia cells observed after 5 min of screening 1 –10 Bonamia cells observed within 5 min of screening 11– 100 Bonamia cells observed within 5 min of screening Bonamia cells observed in all fields of vision a heavy parasite burden in all host cells.
For the purposes of comparing the intensity of infection in the different strains of oysters at the different sites, class 1 and 2 infections were deemed to be ‘light’ infections, and class 3 and 4 infections were deemed to be ‘heavy’ infections.
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2.7. Condition To measure condition index, the flesh and shell of each oyster were separated and each placed in preweighed tinfoil cases, which had been individually numbered. The cases were placed in an oven at 60 jC for 48 h. The cases were reweighed and the condition index calculated using the following formula (Walne and Mann, 1975; Lucas and Beninger, 1985): Dry flesh weight ðgÞ 100=Dry shell weight ðgÞ 2.8. Statistics Data were tested for homogeneity of variance and were found to be ‘non-normal.’ Analysis of variance (general linear model) was carried out to examine weight and condition index with respect to strain and seasonality following a natural log transformation of the weight and infection data and ArcSineM transformation of the condition index data. Tukey multiple comparison tests were run following analysis of variance (ANOVA).
3. Results 3.1. Prevalence of infection Prior to relaying, IER had an initial prevalence of infection of 28%, which was highly significantly different ( P<0.01) from all other strains which appeared uninfected (Table 1). At the Cork harbour site, all strains except UKM had picked up infection by September 1998, 3 months postrelaying, but the latter strain was also infected by December 1998 (6 months postrelaying). By December, IELF had 79.3% prevalence of infection, which was very highly significantly different from that observed in IER (17.9%) and UKM (3.9%) ( P<0.001). From June 1999 (12 months postrelaying), all naı¨ve strains had similarly high prevalence of infection, which continued up to the end of the trial. The lowest prevalence continued to occur in IER throughout the rest of the trial up to January 2000, and there was a very highly significant difference between IER and all other strains in June and October 1999 ( P<0.001). At Lake Grevelingen in Trial 1, no infection was observed in NLG when screened at the beginning of the trial in June 1998. By September 1998, no infection was observed in IER, which had had 28.0% prevalence of infection in June 1998. The only strain to show any infection was IELF with 8.0% prevalence. By December 1998 (6 months postrelaying), all strains were infected, with IET and UKLK having 100% prevalence of infection. IER had the lowest prevalence of infection at 40.6%, which was very highly significantly different ( P<0.001) from all other strains. In March 1999, IER again had the lowest prevalence of infection. By June 1999 (12 months postrelaying), few oysters remained and prevalence had decreased in NLG and IELF (8.0% and 22.2%, respectively) and increased in IER (54.6%).
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Table 1 Prevalence of infection (%) in the European populations of native oysters at the three field sites from June 1998 to April 2000 June 1998
September
28.0 0.0 0.0 0.0 0.0
10.7 17.2 10.7 0.0 16.0
Lake Grevelingen 1 NLG 0.0 IER 28.0 IELF 0.0 IET 0.0 UKM 0.0 UKLK 0.0 Lake Grevelingen NLG NLG (9/1999) IER IET UKLK
2 – – – – –
Brittany FB IER IELF IET UKM UKLK
28.0 0.0 0.0 0.0 0.0
Cork harbour IER IELF IET UKM UKLK
b
December
Mar 1999
June
October
Jan 2000
17.9 79.3 32.1 3.9 53.3
44.8 78.6 26.7 48.3 89.7
25.0 96.7 92.6 76.7 100.0
10.3 87.5 100.0 100.0
50.0 100.0 88.9 50.0
0.0 0.0 8.0 0.0 0.0 0.0
76.7 40.6 68.0 100.0 63.0 100.0
36.7 14.3 36.4 44.4 25.9 25.0
– – – – –
– – – – –
70.0 – 0.0 0.0 0.0
0.0 – 0.0 0.0 0.0
74.1 9.1 19.4 31.0 6.9 6.7
28.6 14.3 0.0c 0.0c 0.0c 12.5c
26.7 23.8 86.2 16.0 18.5 30.0
14.3 5.6 20.0 4.3 54.6 0.0
April a a a a
a
a
a
8.0 54.6 22.2
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
93.1 0.0 76.7 96.7 82.8
a
a
13.3 26.0 73.3 90.0
0.0 20.0 100.0 92.9
a
a
28.0
a
a a
a
a
a
16.7 60.0 72.8
a
a
a
a
a
a
( – ) Trial had not begun. a No samples available (all oysters dead). b No initial sample screened. c Heart smears could not be read; results were determined from screening of tissue sections.
In the second trial at Lake Grevelingen, NLG had 70% prevalence of infection at the beginning of the trial in March 1999. However, by June, 3 months postrelaying, no infection was observed in oysters of this strain nor any of the other strains. By October, all strains were infected except NLG (9/99), which had been relayed that month. Prevalence of infection varied from 76.7% to 96.7% in IER and IET, respectively, which was very highly significantly different ( P<0.001). By January and April 2000, IET and UKLK had continuing high prevalence of infection (100% and 92.9% respectively), which was very highly significantly different from the other two strains ( P<0.001) NLG (09/99) and IER (0% and 20%, respectively). NLG had all died at this stage. At the French site, it appears that IER again was the only group to be infected at the beginning of the trial but the status of the French strain was unknown because no sample was available. By September 1998, all strains were infected with prevalence of infection
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varying from 6.7% to 74.1% in UKLK and Fb, respectively. A number of heart smears could not be read in December 1998 because of degradation of cells, and tissue sections were screened. No infection was detected in these tissue sections. By March 1999, the highest prevalence of infection was in IELF at 86.2%, but levels decreased in all strains in June 1999 except UKM. Prevalence of infection in UKM (54.6%) during this month was very highly significantly different from IET and UKLK, which had the lowest levels at 4.3% and 0.0%, respectively ( P<0.001). By October, the highest prevalences of infections in the remaining oysters were in the UKM and UKLK strains at 60.0% and 72.8%, respectively. Prevalence of infection in UKLK was very highly significantly different ( P<0.001) from IER and IET, which had the lowest prevalence of infection at 28.0% and 16.7%, respectively. 3.2. Intensities of infection In Cork harbour, a high prevalence of infection had developed in some strains by December 1998, 6 months postrelaying. Heavy infections (i.e., class 3 and 4 infections) were observed in some populations [i.e., IELF (13.8%) and IER and IET (both 10.7%)] at this stage. In March 1999, the percentage of oysters with class 3 and 4 infections varied from 0.0% (IER) to 20.7% (UKLK) with all strains except IER, which showed heavy infections. By June 1999, all the populations showed ‘heavy’ infections ranging from 7.0% (IER) to 63.4% (IELF). In October 1999, only two strains had class 3 and 4 infections—UKM (88.9%) and IET (56.0%). All of the UKLK oysters were dead. On the final sampling date in January 2000, 33.3% of IELF oysters had heavy infections as had 3.3% of IER oysters, but ‘heavy’ infections had decreased in UKM and IET (0.0% and 27.8%, respectively). In June 1998, during Trial 1 at Lake Grevelingen, 8% of the IER group had heavy infections (i.e., classes 3 and 4). No further heavy infections were observed until December 1998, 6 months postrelaying, when the percentage of oysters with heavy infections varied from 0.0% (IER) to 30.0% (IET). By March 1999, there was a large reduction in these percentages in all strains with heavy infections varying from 0.0% (IER) to 11.1% (IET). In June 1999, only three strains—IELF, NLG, and IER—had surviving oysters and were available for sampling and, in these, heavy intensities of infection varied from 0.0% to 9.1%. When the second trial began at Lake Grevelingen in March 1999, NLG had a high prevalence of infection (70%). Among NLG, 10.0% of oysters had heavy infections. Infections were not observed in the other strains until October 1999 when ‘heavy’ infections varied from 0.0% (NLG (09/99) and IER) to 30.0% (IET). In January 2000, IER still did not have any heavy infections, in comparison to 43.3% of oysters with heavy infections from UKLK. In April 2000, at the end of the trial, IER and NLG (9/99) still did not have any heavy infections; the highest prevalences were observed in IET (55.9%). At the Brittany site, IER was the only strain with heavy infections in June 1998. By September, Fb had 11.1% of oysters with class 3 infections and IELF had 3.2%. The heaviest intensities of infection occurred in IELF (48.2%) in March 1999 and UKLK (45.0%) in October 1999. Most infections observed throughout the trial at this site were light (i.e., classes 1 and 2).
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3.3. Percentage cumulative mortality At Cork Harbour, when the trial finished in January 2000, percentage cumulative mortality varied from 86.5% to 100%, with IER having the lowest level and UKLK the highest (Fig. 1A – D). During Trial 1 at Lake Grevelingen, by June 1999, cumulative mortality had risen to 100% in IET, UKM, and UKLK, and in the other three strains varied from 79.8% (NLG), 83.5% (IER), to 97.9% (IELF) (Fig. 1B). At the end of the second trial at Lake Grevelingen in April 2000, cumulative mortality varied from 36.5% (IER) to 92.5% (IET). The NLG (9/99) group relayed in October 1999 had 7.9% cumulative mortality. The trial in Brittany terminated in October 1999 and cumulative mortality there varied from 76.6% (IER) to 100% (Fb and IELF) (Fig. 1D).
Fig. 1. (A – D) Cumulative mortalities (%) in all populations of oysters at the three field sites from June 1998 to April 2000. A, Cork Harbour; B, Lake Grevelingen #1; C, Lake Grevelingen #2; D, Brittany.
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Fig. 1 (continued).
3.4. Whole wet weights All strains had started the field trial with mean weights within 10 g of each other at each site (Fig. 2). It can be seen from Fig. 2 that the largest increases in whole mean wet weights took place in oysters held in Lake Grevelingen. In the first trial at this site from June 1998 to June 1999, the best performers were the native strain (NLG), with an increase in weight of 102.5% over the 12 months of the trial, which was significantly different from all other strains ( P<0.001). IET and UKM had the lowest increases at 23.1% and 17.9%, respectively. In the second trial at this site from March 1999 –April 2000, all oysters grew well—IER performed the best with an increase of 106.1% in its mean whole weight. In the second batch of oysters from Lake Grevelingen, introduced in October 1999, the mean weight decreased from this date to April 2000. At Cork harbour, all strains increased in weight over the trial period from June 1998 to January 2000. Over the 18 months of the trial, IELF had the highest increase in mean
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Fig. 2. Percentage change in whole wet weight of each population over the trial period at each field site.
weight at 59.5% and was very significantly different from all other strains ( P<0.001). This increase was followed by IER at 40.3% and IET at 38.1%. UKLK had a percentage increase of 33.3% and the lowest was in UKM at 23.8%. It can be seen from Fig. 2 that oysters performed worst at the French site. Over the trial period from June 1998 to October 1999, a decrease in mean weight was recorded in UKM (9.2%). Of the other strains, IER performed best with an increase of 37.7% compared to 25.6% for IELF and increases of 9.5%, 10.8%, and 10.9% for UKLK, IET, and Fb, respectively. 3.5. Condition indices The highest condition indices were recorded in Cork harbour in June 1998 when the trial began, and there was a very highly significant difference between values in this month and all other months except June 1999 ( P<0.001) (Fig. 3A – D). Condition for all strains available were at their lowest level when the trial finished in January 2000, with values ranging on this month from 0.7 to 3.6 (UKM and IER, respectively) and being very highly significantly different from all other months ( P<0.001). Throughout the trial, UKM and IELF consistently had the lowest condition indices each time the oysters were sampled, and the condition indices of both these strains were very highly significantly different from all others ( P<0.001). In Trial 1 at Lake Grevelingen, oysters were from the same group relayed in Ireland, and so were starting with the same condition indices (Fig. 3B). Over the subsequent months, there was no clear pattern, with the condition of some strains increasing and others decreasing. UKM had a lower condition index than the other populations and there was a very highly significant difference between this strain and all others ( P<0.001). In June 1999, at the end of the trial, of the populations available, conditions ranged from 5.1F1.5 (IER) to 3.9F1.0 (NLG). In the second trial at this site, which started in March 1999, conditions ranged from 2.9 to 4.4 (IET and NLG, respectively) (Fig. 3C). The condition of NLG dropped over the trial, but other strains showed some increases. When the trial finished in April 2000, condition indices varied from 4.4 to 5.9 (IER and UKLK, respectively). Condition index for UKLK was very highly significantly different from all others ( P<0.001).
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At Brittany, no initial condition index was available for the French population (Fig. 3D). Condition indices ranged from 2.9 to 5.7 in June 1998 in the other populations and were lowest in March and October 1999, with levels on these months being very highly significantly different from all other months ( P<0.001). UKM had the lowest condition indices throughout the trial, being very highly significantly different from all other strains ( P<0.001). Final condition indices varied from 1.5 to 4.1, with IET having the highest and UKM the lowest. 3.6. Temperature data In Cork harbour, mean monthly temperatures varied from a low in January 2000 (7.7 jC) to a high of 17.6 jC in August 1999. Peaks in temperature occurred in August of both 1998 and 1999 and the lowest temperatures occurred in January 1998 and January 1999. In Lake Grevelingen, not all recordings were available due to the loss of some recorders. Temperatures for the dates available varied from 3.1 jC in November 1998 to 20.4 jC in July 1999. In France, mean temperatures varied from 9.9 jC in March 1999 to 16.3 jC in
Fig. 3. (A – D) Mean condition indices for all populations at each field site over the trial period (June 1998 – April 2000). A, Cork Harbour; B, Lake Grevelingen #1; C, Lake Grevelingen #2; D, Brittany.
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Fig. 3 (continued).
September 1998. It appeared that the least variation in mean temperatures occurred at the French site and most at Lake Grevelingen over the months when temperatures were recorded.
4. Discussion Temperature data were collected from each site over the study period. Most variations occurred at Lake Grevelingen, with the highest and lowest monthly mean temperatures being observed here. This was also the only site where the oysters were continually submerged throughout the trials. Intersite differences were observed when analysis of growth rates of each strain at each site was undertaken, with the greatest growth rates in Lake Grevelingen. This may be due to a combination of optimal food levels and environmental factors, or the fact that oysters were continually submerged throughout the trial. The lowest growth rates occurred at the site in Brittany, and this along with the other factors would indicate that this site is not
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suitable for growth of this oyster species. No one population performed best at all sites. The only consistent result was that UKM had the lowest increases in weight in Cork harbour, Brittany, and Lake Grevelingen, followed by UKLK. The bad performance of the Scottish oysters at all sites may indicate that these strains do not adapt well to the combination of the environmental factors that they experienced at these sites. 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 decreased growth efficiency as their size increases. In this study, all oysters started the trial with a mean wet weight within 10 g of each other. Over the trial period, condition indices fluctuated and all groups lost condition. Conditions showed some reductions during the winter months possibly related to spawning, cessation of feeding, lower water temperatures, and high levels of infection (Walne and Mann, 1975; Baud et al., 1997). No one population showed consistently higher condition over the study. However, UKM consistently had the lowest condition when monitored in Cork harbour, Brittany, and in Trial 1 in Lake Grevelingen. Walne and Mann (1975) found that in the spring, a high rate of growth of the flesh weight and slower growth of the shell led to an improvement in the condition, with increased shell growth in summer related to an increase in water temperature. Barber et al. (1988) found that MSX reduced condition in C. virginica and the reduction was related to the severity of the infection. The results of this study suggest that differences exist between European populations of O. edulis in susceptibility to infection with B. ostreae. The field trials indicated that Rossmore oysters showed some tolerance to infection with Bonamia compared to other populations. When comparing prevalence and intensity of infection in the different populations over the 22 months of the trial, lower levels were observed in the Rossmore group, and significant differences in prevalence of infection were found between the Rossmore oysters and the other populations tested. However, when comparing mortality, the picture was not as clear, with the Lake Grevelingen population performing better in one trial. The Rossmore oysters had been exposed to the parasite throughout life, having been selectively bred in an area where the parasite has been present for over 16 years. Intraspecific genetic variation in disease susceptibility is indirectly demonstrated by the evolution of resistance in disease-challenged natural populations (Gaffney and Bushek, 1996). Bushek (1994) in Gaffney and Bushek (1996) found that oysters from different geographical regions showed distinct differences in their response to infection with P. marinus. Populations with previous exposure to the pathogen showed lower body burden than oysters originating from less exposed populations. A program undertaken with the aim of breeding faster-growing Sydney rock oysters Saccostrea glomerata demonstrated that after one generation, a small improvement in resistance to QX disease was observed (Nell et al., 2000). All other populations (whether naı¨ve or previously exposed to Bonamia) appeared to be equally susceptible to infection, when comparing prevalence of infection and intensity, with initial infections being observed several months postrelaying in agreement with previous observations (Hine, 1991; Culloty and Mulcahy, 1996). There was no evidence of increased resistance with respect to prevalence and intensity of infection in the Lake Grevelingen oysters. All previously unexposed populations (i.e., Tralee, Lough Foyle, Mull, and Lough Kishorn) were equally susceptible when exposed to the parasite.
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Cumulative mortalities were high for all populations by the end of each field trial. However, the Rossmore oysters had the lowest percentage in each trial, except in Trial 1 at Lake Grevelingen when the native population had the lowest cumulative mortality at the end of the trial. By the end of each trial, most of the oysters from the other populations had died. Similar studies looking at resistance to bonamiasis have also observed generally high mortalities in field trials, but the lowest mortalities in populations selected for resistance (Baud et al., 1997; Naciri-Graven et al., 1998, 1999). The husbandry techniques employed in this study, such as holding the oysters in bags on trestles, sorting and handling every couple of months, and placing them in the intertidal areas, may have contributed to mortalities as these oysters are normally grown subtidally without interference until they reach market size. However, all populations were handled in the same way at each site and at similar intervals, so the comparative results are valid. In this study, the oysters in Cork harbour and Brittany were exposed on the trestles at spring tides and were handled frequently. At Lake Grevelingen, oysters were continually submerged, except when samples were being removed, but this factor did not appear to reduce mortalities. Martin et al. (1993) in similar trials found that oysters in intertidal areas are more susceptible to development of B. ostreae disease than those in subtidal areas; exposing oysters in the intertidal zone of a estuary boosted infestation. However, when infection and mortality patterns are compared, it is clear that most heavy mortalities, particularly in the naı¨ve populations, occurred in the months after heavy prevalence and intensity of infection were noted, particularly in Cork harbour and Lake Grevelingen, indicating that these peaks in mortality were related to disease. Montes et al. (2002) found a relationship between the level of Bonamia and high mortality rates. Analysis of results from Brittany indicated that prevalence of infection was only one factor contributing to the high mortalities observed: it appears that this area is not suitable for growth of this species, and that other factors contributed to the heavy losses observed. In the field trials, a decrease in prevalence of infection was not always related to an increase in mortalities in the Rossmore group. It appears that this strain may be able to tolerate a certain level of infection and, in some cases, overcome the parasite. Hervio et al. (1995) found individual variability in susceptibility to bonamiasis in oysters, when inoculated with a known number of B. ostreae cells. Elston et al. (1987) noted that populations of oysters exposed to Bonamia for a number of years had a substantial degree of resistance to infection compared to populations with no prior exposure history, and resistant oysters had lesions, which indicated that they could contain the infection. In the case of MSX-resistant oysters, it has been found that the animals did become infected with Haplosporidium 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). Differences in susceptibility to H. nelsoni were associated with geographic origin of oysters tested, as it was thought that the parasite interferes with energy acquisition, storage, and conversion cycles, which differ seasonally with geographic origin (Ford et al., 1990). Physiological variation, whether due to environmental influences, heredity, or both, may result in differences in susceptibility to infection as may more specific biochemical or cellular responses (Gaffney and Bushek, 1996). For example, Oliver et al. (2000) produced 10 families of Crassostrea virginica with high interfamily and intrafamily variability. In the laboratory, three families, when challenged with Perkinsus marinus, had the highest
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survival rate, lowest numbers of parasites, and highest average protease inhibitory activity, and they also performed best in field studies in terms of cumulative mortality. Romstead et al. (2002) also associated highest protease inhibitory activities with resistance of Crassostrea gigas to infection with P. marinus. Anderson and Beaven (2001) in a comparative study of anti-Perkinsus activity in bivalve sera found that sera from mussels, that are resistant to Perkinsus, contained high antiprotease activity compared to C. virginica that are susceptible. How this increased resistance to Bonamia could be exploited to improve O. edulis stocks has to be considered carefully. At present, breeding of the Rossmore-‘resistant’ oysters involves a large randomly mating population of older oysters, that are known to have survived 4 years on the oyster beds, which are removed to spatting ponds to spawn several hundred broodstock per pond. Launey et al. (2001) looked for evidence of inbreeding and population bottlenecks in three hatchery-propagated populations of the flat oyster as part of a selection program for resistance to Bonamia. A reduction in the effective population size following a population bottleneck is correlated with a decrease in heterozygosity and a loss of rare alleles (Launey et al., 2001; Evans et al., 2004). It would appear that bottlenecks may have a stronger and more immediate effect on allelic diversity than on heterozygosity. In the Launey et al. study, which looked at Bonamia-resistant populations of O. edulis, although heterozygosity was still high in the resistant and control populations, the number of alleles in the selected population was significantly reduced compared with the control population, and this was due in large part to the loss of rare alleles. As a result, a decrease in performance was expected as soon as the second generation in relation to both growth and survival. However, Marsic-Lucic and David (2003) found a lack of heterozygote deficiency and multilocus heterozygosity –fitness correlations within five natural populations of O. edulis from the Adriatic Sea. They considered that this was due to very low inbreeding within the populations. The objectives in relation to management of the Rossmore population in the future are to maintain this resistance without loss of other traits associated with fitness. Gaffney and Bushek (1996) concluded that field exposure to naturally occurring disease appears to be the best approach for large-scale selection programs, but they do suffer from annual variation in disease pressure plus other variables such as temperature, salinity, etc. During these field trials, oysters were exposed to non-native Bonamia parasites at the sites to which they were relaid. However, in the case of the Rossmore group, these oysters still performed better at the other sites apart from cumulative mortality results from Trial 1 in Lake Grevelingen. The possibility of different strains of Bonamia being present at different sites may, however, require further investigation. The existence of different strains has to be taken into consideration due to the fact that two foci of infection may have initially been responsible for the spread of Bonamia within Europe (Cigarrı´a and Elston, 1997). Bushek and Allen (1996) examined the resistance of four genetically distinct populations of C. virginica that had different histories of exposure to P. marinus with four geographically distinct isolates of P. marinus of varying virulence. They found that in the oysters, resistance was related to length of exposure. Different isolates of Perkinsus showed different degrees of virulence; however, no statistically significant interaction was detected between oyster populations and parasite isolates, indicating that mechanisms of resistance and virulence were general, not race-specific. The fact that tolerance is not
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complete as, for example, has been observed in populations of C. virginica exposed to H. nelsoni. This would indicate that the ability to actually resist rather that just tolerate infection suggests that the oyster’s repertoire of genetic variability is not sufficient to provide complete protection against the disease (Gaffney and Bushek, 1996). However, the extent to which the parasite has adapted itself to take advantage of different environmental factors present in different regions remains undetermined. In conclusion, in so far as one can compare the susceptibility of different oyster strains to B. ostreae, the results of these field trials support the idea that a degree of ‘‘resistance’’ has developed in the Rossmore oysters. It would appear that the Lake Grevelingen population also performed well in terms of overall survival. Previous exposure in these populations has conferred some reduced susceptibility to the parasite compared to naı¨ve populations. In relation to the life cycle of B. ostreae, it is clear from a number of studies that direct transmission of the parasite can occur from oyster to oyster (Culloty et al., 1999) and there is some evidence to suggest that entry of the parasite may be initially through the gills (Montes et al., 1994). Van Banning (1990) suggested a presumptive life cycle in which an infectious phase might be involved in the ovarian tissue of O. edulis. However, the possibility of other life cycle stages such as a spore stage or an intermediate or secondary host being involved cannot, at this stage, be discounted. Phylogenetic analysis of DNA sequence data confirmed Bonamia to be a member of the Haplosporidia, of which a spore stage is typical (Carnegie et al., 2000; Cochennec et al., 2000). To date, a spore stage has not been observed in infected oysters. As questions still arise in relation to aspects of the life cycle and further study is required of the immune mechanisms of the host, both of these factors hamper efforts to determine the basis of the ‘resistance’ observed during this study.
Acknowledgements This study was supported by EU CRAFT CT98-910. The authors would like to acknowledge the contribution of David Hugh Jones, Atlantic Shellfish, Cork, Dennis Gowland, Orkney, Jan Bol, and Dr. Aad Smaal (RIVO, The Netherlands) to the field work. References Anderson, R.S., Beaven, A.E., 2001. A comparative study of anti-Perkinsus marinus activity in bivalve sera. J. Shellfish Res. 20 (3), 1011 – 1018. Bache´re, E., Durand, J., Tige´, G., 1982. Bonamia ostreae (Pichot et al., 1979) parasite de l’huıˆtre plate comparison de deux methodes de diagnostic. ICES CM, F 28, 1 – 11. Balouet, G., Poder, M., 1983. Bonamia—a threat to oyster stocks. Proceedings of the XIVth Conference on Shellfish, 74 – 83. Barber, B.J., Ford, S.E., Haskin, H.H., 1988. Effects of the parasite MSX (Haplosporidium nelsoni) on oyster (Crassostrea virginica) energy metabolism: 1. Condition index and relative fecundity. J. Shellfish Res. 7 (1), 25 – 31. Baud, J.P., Ge´rard, A., Naciri-Graven, Y., 1997. Comparative growth and mortality of Bonamia ostreae-resistant and wild flat oysters Ostrea edulis in an intensive system. Mar. Biol. 130, 71 – 79.
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