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A temperature dependent disease : impact of Vibrio harveyi on the abalone Haliotis tuberculata Agnè ès Travers1,2*, O. Basuyaux2, M. Garnier3, J.L. Agn Agnès Nicolas3, M. Koken1, S. Huchette4 and C. Paillard1 1 3 IUEM, Plouzané, France, 2 SMEL,Blainville sur mer, 3 Ifremer Brest France Haliotis, Plouguerneau, France. * [email protected] Introduction: Wild abalone stocks are declining worldwide due to over-exploitation or diseases affecting several Haliotis species, including the ormer Haliotis tuberculata, the most commercially-important abalone in Europe [1]. Vibrio carchariae has been isolated in H. tuberculata during the mass mortality that occurred in 1998, in France [2]. Mortalities seems to be directly link to seawater temperature (>16,5°C, Figure 1). This vibrio is a synonym of Vibrio harveyi, already known to be pathogenic for shark, shrimp and prawn [3]. Figure 1: Distribution of H. tuberculata summer mortalities along France coast and isotherm of summer temperature maximum. Red abalone correspond to witnessed mortalities The goal of this new research topic is to highlight direct implications of this bacterium in ormer mortalities and to elucidate mechanisms of interaction between Vibrio harveyi and Haliotis tuberculata. Huchette and Clavier [1] 1. Pathogenesis: Clinic signs Mortalities have been reported in the field and in hatcheries in Brittany and Normandy (France). Mortalities are associated with loss of muscular motility, pericardial inflammation and white pustules on the foot (corresponding to bacteria clumps, as shown by histology, bacterial isolation and PCR identification). Bacteria were principally found in muscle and haemolymph. a b Figure 2: Macroscopic and microscopic characteristics of mortalities associated with V. harveyi. a) Pericardial inflammation, b) White pustule, c) Hematoxilyn Eosin coloration of muscle section. c 2. Modulation of the interaction 1. Pathogenicity of different V. harveyi strains Different V. harveyi (isolated in field as in hatcheries during mass mortalities) were compared with collection strains. Their pathogenicity was tested in vivo. vivo : As temperature seems to be determinant for disease development in the field, experimental inoculations were performed at different temperatures on hatchery abalone. 3. Host physiology maturation effects 100 Spawn just before infection Spawn 2 months before infection Immature abalone 90 80 60 40 20 0 15°C 17°C 18°C 19°C 20°C Temperature 29 species , 808 sites (global gap removal) Neighbor Joining Method Jukes and Cantor distance Figure 4: 500 bootstrap replicates Figure 3: gyrB. Pathogenic V. harveyi are in yellow. 29 species , 808 sites (global gap removal), Neighbor Joining Method, Jukes and Cantor distance, 500 bootstrap replicates. 01_021 to 05_043 correspond to abalone isolated V. harveyi. → Pathogenic strains cannot be differentiated from non pathogenic ones on the basis of gyrB or rpoA sequences. Percentages of dead abalone 5 days after introduction of V. harveyi strain 02_001 into the surrounding water. (106 bact./ml during 24 hours, triplicate tanks, 20 abalone/tank). → Significant mortalities due to V. harveyi occurred only over 18°C. and As mortalities occur in the field simultaneously with spawning, effects of gonad maturation on susceptibility to V. harveyi was investigated on adult abalone at 19°C. Cumulative mortalities % P e rce n ta g e o f a b a lo n e d e a d a fte r 5 d a ys 100 in 100 V. harveyi 0.063 01_022 01_021 02_001 04_165 S20 S35 05_055 02_109 05_043 LMG19114 LMG10948 LMG16863 dq499007 IFO15634 LMG7890T LMG11755 LB5 V campbelli ab01495 LMG10946 LMG10948 dq345719 LMG10947 dq499009 V pelagia_ab014954 V alginolyticus V splendidus V hollisae V aestuarianus V diazotrophicus_ab0 2. Pathogenicity Temperature effect 80 70 60 50 40 30 20 10 0 -10 0 5 10 15 Time (days) 20 25 Figure 5: Evolution of mortalities after introduction of the pathogen V. harveyi strain 02_001 into the water (105 bact./ml during 24 hours at 19°C, triplicate tanks, 20 abalone/tank). → Mature abalone (spawning immediately before infection) are more sensitive than abalone that spawned 2 months before. Immature abalone, independently of their size and age, appeared insensitive to infection. Conclusion and perspectives: H. tuberculata mortalities associated with V. harveyi depends on V. harveyi strain, abalone age, gonad maturation level and water temperature. Abalone are affected by temperature for sexual maturation or growth. Temperature may also increase their sensitivity to bacteria. But it may also affect bacterial growth (optimum 28°C) and virulence. Results highlight the importance of 1°C variation on disease development (17°C vs 18°C). Thus, in view of the current global warming, the impact of temperature on V. harveyi growth and virulence, and on abalone immunity should be investigated further. Moreover, differentiating virulent and avirulent strains should be one of our priority. Useful references : 1. Huchette, S. M. H. and J. Clavier (2004). "Status of the ormer (Haliotis tuberculata L.) industry in Europe." Journal of Shellfish Research. 23(4) 951-5. 2. Nicolas, J. L. et al. (2002). "Vibrio carchariae, a pathogen of the abalone Haliotis tuberculata." Diseases of Aquatic Organisms 50(1): 35-43. 3. Austin, B. and Zhang, X.H. (2006) “Vibrio harveyi: a significant pathogen of marine vertebrates and invertebrates”. Letters in applied microbiology 43: 119-24.