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Handling enhances the development of signs of Brown ring disease in Ruditapes philippinarum.

ABSTRACT Brown ring disease (BRD) in the Manila clam is characterized by the formation of a brown deposit of conchiolin on the inner surface of the shell that gives the disease its name. The development of the signs of BRD may be favored by the entry of bacteria in the extrapallial compartments via mechanical disruptions of the periostracal lamina and/or chipping of the shell margin. To test this hypothesis, we conducted an experiment simulating clam handling under aquaculture conditions and we checked for prevalence of BRD signs. Our results assess that rough handling of Ruditapes philippinarum in presence, of the bacterium Vibrio tapetis significantly increase the prevalence of BRD signs. As a consequence, our results show that minimizing manipulation and transfer of clams during culture is beneficial to avoid the development of BRD signs.

KEY WORDS: Ruditapes philippinarum, brown ring disease, handling, shellfish farming


The Manila clam Ruditapes philippinarum was introduced during the 1970s in a number of European countries for aquaculture purposes (Flassch & Leborgne 1992) and is now a widespread species. Brown ring disease (BRD) in R. philippinarum was first reported in North Finistere (France) in 1987 (Paillard et al. 1989). The disease is characterized by a brown deposit on the inner surface of the valves (Paillard et al. 1989) that gave the disease its name. These signs go in hand with the proliferation of the etiological agent of BRD, the bacterium Vibrio tapetis (Paillard & Maes 1990, Borrego et al. 1996). BRD has been responsible for mortalities of Manila clam in several European countries such as France, Italy, Spain, Portugal, and England (Paillard et al. 1989, Paillard & Maes 1990, Castro et al. 1992, Paillard et al. 1994, Figueras et al. 1996, Allam et al. 2000). Infection disrupts the production of the periostracal lamina and causes an anomalous deposition of periostracum on the inner shell of infected clams (Paillard et al. 1994, Paillard & Maes 1995a, Paillard & Maes 1995b). Although postcolonization processes (i.e., after penetration of V. tapetis into the extrapallial compartment) have been widely described (Paillard 2004), mechanisms of entry of V. tapetis into the extrapallial fluids remain poorly understood. A previous study (FlyeSainte-Marie et al. 2008) suggested that the pathogen V. tapetis may benefit from mechanical disruption of the periostracal lamina or chipping of the valve margins to colonize the Manila clam extrapallial compartment. These results suggest that rough handling of clams in aquaculture conditions may enhance BRD development.

The current experiment was designed to test this hypothesis. For this purpose we simulated rough handling of clams as it may occur during clam cultivation purposes and transport (e.g., between nursery and growing plot, or when small individuals are harvested and reseeded after harvest of commercially mature clams). We combined this treatment with exposure to V. tapetis to assess its effect on the development of BRD signs.


Specimens and Experimental Plan

More than 700 Manila clams larger than 25 mm were collected at low tide by hand on January 30, 2008, on the Lanveur mudflat, Bay of Brest, France. Particular care was taken to avoid any effect of handling on clams. During collection in the field, clams were stocked cautiously, one after the other, in boxes containing rags and were transferred to the Laboratoire des Sciences de l'Environnement Marin (Brest, France). At the laboratory, clams were gently rinsed individually in seawater. Initial prevalence of the BRD signs was estimated by sacrificing and evaluating 100 clams chosen at random.

Six hundred clams were randomly subdivided into 12 batches of 50 clams each. Throughout the experiment, each batch was maintained in 20-L tanks of aerated filtered (0.5 gm) seawater at 16oC, which is near optimal temperature for BRD development (Paillard 2004). A pump generated a smooth current in each tank. Filtered seawater was renewed every fifth day throughout the trial. Clams were fed with 1 L cultured Isochrysis aff. galabana (concentration of 50 cells/gm) per week and per tank. Tanks were checked on a daily basis for mortalities and moribund clams. Any gaping clams were presumed moribund and removed.

After 1 wk of acclimation, the 12 batches were randomly distributed into 4 triplicates; each triplicate was then attributed to one of the following experimental conditions:

* Untreated control

* Handling simulation (hereafter, "handled")

* Exposed to Vibrio strain (hereafter, "exposed").

* Handling simulation and exposed to Vibrio strain ("handled and exposed").

To simulate the rough handling of shellfish farming, handled clams were placed in a closed tank without water and manually shaken roughly for 30 sec. The tanks were then filled with 20 L fresh, filtered seawater. Handled and exposed clams were submitted to experimental infection immediately after simulation of handling, as described in the next section.

Experimental Infection

Vibrio tapetis strain CECT 4600 was grown in marine agar (Difco 2216) at 18[degrees]C for 48-72 h. Bacterial colonies were resuspended in filtered seawater. Bacterial suspension was added in V. tapetis-exposed tanks to reach a final concentration of 106 cells/mL, which is the same order of magnitude as used by Drummond et al. (2007). A first exposition was performed on February 8, 2008 (day 8), and water was renewed after 24 h. A second exposure experiment was conducted on February 22, 2008 (day 22), and water was renewed after 5 days. During exposures, clams were regularly monitored to verify that their shells were opened and that they were actively filter feeding. After each infection experiment, the water was drained and the clams remained out of water for 1 h to induce the closure of the valves and incorporation of V. tapetis in the pallial cavity. The tanks were then filled with 20 L fresh, filtered seawater.

In control tanks, clams were treated as described, except that the bacterial suspension was not added.

Characterization and Classification of Brown Ring Disease

The experiment last for about 6 wk, which is an intermediate duration between experiments by Paillard et al. (2004) and Drummond et al. (2007) that allows for the development of visible signs of BRD at the chosen experimental temperature. On day 40 of the experiment, clams were killed, the flesh was removed, the valves were cleaned under a trickle of water, and then they were then left to dry until further analysis.

All shells (including those of moribund individuals sampled throughout the trial) were retained and left to dry. Disease intensity was estimated by the extent of the symptomatic deposit according to the criteria of Paillard and Maes (1994), in which conchiolin deposit stages (CDS) range from a microscopic brown spot on the inner face of the shell in the earliest stages (CDS 1) to a thick brown deposit covering most of the inner shell in the most advanced stage (CDS 7).

Statistical Analyses

Variations of prevalence and mortality among treatments were tested using analysis of variance (ANOVA). Tank effect was always neglected, because it was never significant when we tried to take it into account. When ANOVA was significant, Tukey's HSD test (Yandell 1997) was used to assess pairwise differences among groups. Statistical analyses were conducted using R statistical software version 2.6.2 (R Development Core Team, 2006).


Mortality of Clams

Repeated mechanical disturbance is known to stress small R. philippinarum (Marin et al. 2005). However, our experimental handling of adult R. philippinarum, even those associated with exposure to V. tapetis, did not result in significant increased mortality. In total, mortality was low and only 10 clams died during the experiment: 6 of them were derived from handled and exposed clams, 3 from the handled group, and 1 from the control group (Table 1). ANOVA showed no clear significant difference among treatments (F = 4.0, df- 3, P = 0.052). Moreover, none of the dead clams exhibited BRD signs in the current trial. It can thus be hypothesized that the observed mortality was independent of the infection and handling challenge during this 40-day trial.

Prevalence and Intensity of Brown Ring Disease

At the beginning of the experiment, the initial prevalence of BRD signs, estimated using 100 clams, was null. On day 40, at the end of the experiment, there were a total of 22 clams presenting signs of BRD out of a possible 600 clams, of which 20 were derived from handled and exposed tanks (Table 1). ANOVA showed a significant effect of treatment on prevalence (F - 20.83, df = 3, P < 0.05). According to the Tukey HSD pairwise comparisons, the handled and exposed clams were significantly more susceptible to development of BRD signs than clams in any other group (Table 2).

Our results show that the sole exposure to V. lapetis does not lead to a higher prevalence of BRD than control clams. The influence of handling associated with V. tapetis exposure is obvious after a 40-day trial (Table 2). This result confirms that mechanical disruption of the periostracal lamina and shell edge enhances the development of BRD signs. Our results support the hypothesis that V. tapetis may benefit from mechanical disruptions to enter the extrapallial compartment (Flye-Sainte-Marie et al. 2008). Furthermore, this strong contrast between handled and exposed clams and other treatments should thus be taken into account for future experiments, especially during the growing season. As mentioned by Flye-Sainte-Marie et al. (2008), disruptions of the periostracal lamina and chipping of valve margins may occur more easily during this period because of the fragility of the newly calcified layers on valve margins.

The increased vulnerability of handled R. philippinarum exposed to V. tapetis may have implications for clams culture. Thus, our results suggest (1) limiting manipulations, including reseeding practices, in cultured clam beds and (2) minimizing transfers of clam seed, even for seed coming from non-BRD-affected regions, as these manipulations imply an increased sensitivity to BRD.


We thank Robert Marc and Eglantine Michalon for valuable help during fieldwork.


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* Corresponding author. E-mail: DOI: 10.2983/035.030.0103


Universite Europeenne de Bretagne, France; Universite de Brest, CNRS (CNRS/INSU) UMR 6539 Lemar, IUEM, place N. Copernic, 29280 Plouzane, France
Distribution of Manila clams R. philippinarum presenting
or not presenting signs of BRD among different treatments,
and distribution of dead clams among treatments.

                                                   Handled and
                     Control   Handled   Exposed     Exposed

Asymptomatic clams     150       149       149         130
Symptomatic clams       0         1         1          20
Dead clams              1         0         3           6

Numbers are the sum of individuals among triplicates in each

Tukey HSD pairwise comparisons of prevalence of
BRD among experimental groups.

          Exposed     Handled     Handled and Exposed

Control   D = 0.33    D = 0.33         D = 6.67
          p = 0.986   p = 0.986        p = 0.001

Exposed               D = 0.00         D = 6.33
                      p = 1            p = 0.001

Handled                                D = 6.33
                                       p = 0.001

ANOVA showed a significant effect of treatment on prevalence of BRD
signs (F = 20.83, df = 3, P < 0.05).
D, value of the difference; p, associated P value.
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Article Details
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Author:Jean, Fred; Flye-Sainte-Marie, Jonathan; Oudard, Clemence; Paillard, Christine
Publication:Journal of Shellfish Research
Article Type:Report
Geographic Code:9PHIL
Date:Apr 1, 2011
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