Where do mollusc murderers hide during winter?

Mollusc farming has become a major portion of global aquaculture, with oysters considered second only to cyprinids in importance of aquaculture products. Recently however, cultivated European populations have been suffering from mass mortality episodes.  Mortality in the summer in sheltered areas, where these outbreaks generally occur, can reach 80/100%. The causes behind these outbreaks are still poorly understood and are thought to be attributed to complex interactions among molluscs, pathogens and their environment. Two pathogens associated with the summer mortalities are Vibrio splendidus and Vibrio aestaurianus. Although some of their biology and toxicity within host organisms is now understood, little is known about the ecology of these pathogens outside of their bivalve hosts. The lack of information on the aquatic ecology and lifestyle limits the understanding of their role in the occurrence of mortality outbreaks. Vezzulli et. al. (2015) extensively investigated the ecology of these species in an aquatic brackish environment.

Using the strains LGP32 and 01/32 from V. splendidus and V. aestuarianus respectively, both related to diseases and mortality in Crassostrea gigas, they conducted laboratory microcosm experiments to assess the persistence of these strains in both seawater and sediment in 5°C and 25°C temperature and 20‰ and 35‰ salinity. The capability of LGP32 and 01/32 to interact (adhere) with both chitin particles and plankton crustaceans in vitro was analysed as well as the biofilm formation of both strains on PVC surfaces. Finally, in order to validate results obtained, the occurrence and temporal variations of V. splendidus and V. aestuarianus-clade bacteria were investigated in the Goro lagoon.

LGP32 generally lost culturability in all experiments in seaweater after a short incubation time (less than 5 days) and entered a viable but not culturable (VBNC) state. Whereas, 01/32 lost both viability and culturability in seawater within 5 days in almost all experimental settings suggesting 01/32 may be more demanding in its living requirements than LGP32. The short culture time for both strains also suggests the laboratory conditions of the experiment were not suitable for either strain. Both culturability and viability were higher in sediment experiments for the two Vibrio species, which may be due to sediment providing surfaces for biofilm development and the concentration of organic matter being higher than surrounding water. The presence and activity of two ligands (MSHA and GlcNAc) mediating the attachment of V. cholerae to chitin surfaces was also analysed. 01/32 was found to have both ligands and inhibition experiments supported their role in attachment of this strain to chitin-containing surfaces. However, although LGP32 contained the mshA gene, inhibition experiments pointed to a non-significant contribution of either ligand. Biofilm formations constitutes a successful survival strategy, however, although LGP32 showed a greater capability to form biofilms on PVC surfaces and possibly why LGP32 had a higher persistence in lab experiments, both strains showed lower capability than V. cholerae. When exposed to prolonged low temperatures, both strains entered a VBNC state. Interestingly though, VBNC cells of both strains retained virulence related factors and were smaller than 0.2 μm. This suggests they may remain undetected in some experiments and can reactivate under favourable conditions and infect hosts. The lagoon had an absence of a clear temporal trend in Vibrio spp. as well as the presence of culturable vibrios in seawater during cold periods. This could be due to a high nutrient content in the Goro lagoon. Similar to lab experiments, sediment samples also contained the highest proportion of viable and culturable vibrios.

The multitude of tests performed in this study allows us to understand more of the ecology and biology of these bivalve pathogens, however, some tests may be limited in their depth. Binding experiments only tested binding on chitin and copepods. The use of other substrates associated with bivalve morphology may have been beneficial to this study. Similarly in biofilm experiments, although PVC may allow tests to be easily repeatable and comparable, using a plastic may have less real world relevance. Therefore, biofilm formation of these strains may be different on organic surfaces in the natural environment. With VBNC bacteria found to retain virulence factors means monitoring these populations in locations of bivalve aquaculture could be important in determining disease risk in summer months. Monitoring will also have to account for the smaller than 0.2 μm size. As found in Goro lagoon though, culturable populations can survive through winter probably due to increased nutrients. The presence of aquaculture in habitats will probably also increase the amount of nutrients present in the habitat. Therefore, a significant proportion of culturable pathogens may remain in the environment. This could result in summer months having a large population of pathogenic bacteria and help cause the mass mortality of bivalves.

Vezzulli, L., Pezzati, E., Stauder, M., Stagnaro, L., Venier, P., & Pruzzo, C. (2014). Aquatic ecology of the oyster pathogens Vibrio splendidus and Vibrio aestuarianus. Environmental microbiology.

http://onlinelibrary.wiley.com/doi/10.1111/1462-2920.12484/abstract;jsessionid=AAD534EA8D57255E79EB5828EBFAD99B.f02t03?deniedAccessCustomisedMessage=&userIsAuthenticated=false