Recipient Organization
OREGON STATE UNIVERSITY
(N/A)
CORVALLIS,OR 97331
Performing Department
College of Veterinary Medicine
Non Technical Summary
Bivalve shellfish are vital elements of healthy coastal food webs. These animals support a wide range of ecosystem services, and modest but stable shellfish aquaculture and harvest fisheries. Within the last several years, heavy and episodic larval and juvenile mortalities of Pacific oysters have been documented along the US West Coast that are linked to a re-emergence of vibriosis as well as near-term ocean acidification effects. These are serious and well-publicized problems that have resulted in increased efforts directed at water management in commercial hatcheries and further research to better understand the responses of shellfish to changes in water chemistry. However, the role of ocean chemistry on shellfish-associated pathogens in these complex interactions has been largely ignored. Accordingly, we hypothesize that the combination of upwelling of pathogen-laden waters and enhanced corrosivity may act as multiple stressors for natural and farmed shellfish populations. This proposal is then designed to examine the interactive effects of carbonate system parameters and larval disease susceptibility as well as the environmental regulation of Vibrio pathogenicity in relationship to OA.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
Among the regional ramifications of global climate changes are increasing pressures on vital ecosystem services. For example, for the past several years, shellfish hatcheries along the US West Coast have experienced massive mortalities, which have been linked to shifting ocean conditions (i.e. increased temperatures, changes in ocean chemistry, acidification) and/or the enhanced growth or virulence of marine pathogens. While research is now underway to mechanistically describe the consequences of OA for survival of native and farmed shellfish species, it has only recently been recognized that shifting ocean conditions may also favor growth of a potent shellfish pathogen, the facultatively anaerobic bacterium Vibrio tubiashii. V. tubiashii, known bacterial constituents of oceanic and estuarine waters, have been identified as a cause of massive mortalities of shellfish larvae and juvenile seed, both in hatcheries and in the marine environment. Despite the importance of this marine pathogen to shellfish health and survival, little is known about the relationship between oceanic conditions and susceptibility of shellfish larvae to V. tubiashii. Initial research however suggests that OA and V. tubiashii may act as multiple stressors on wild and farmed shellfish resources - a one-two punch of sorts that can synergistically and deleteriously impact larval development. The objectives of this research are to characterize the susceptibility of oyster larvae to the combined stressors of V. tubiashii toxicity and shifts in carbonate chemistry. Our research findings will allow us to unravel the impact of increasing ocean acidification and V. tubiashii infection on early oyster larval development.
Project Methods
We will examine the susceptibility of shellfish larvae to both V. tubiashii and enhanced water column corrosivity. For this, we will conduct a series of experiments to assess the impact of V. tubiashii on larvae reared under variable pCO2 and mineral saturation states. This is a critical step because of the often non-linear relationship between toxigenic microbe abundances and host mortality, in this case likely due to metalloprotease secretion by the bacteria (Hasegawa et al., 2008).Experimental seawater manipulation systems have been developed at OSU with prior NSF funding. In short, sterile filtered seawater is manipulated as follows. First a batch manipulation of a large water mass (~2000 L) is altered to a DIC concentration half ambient conditions, via concentrated HCl addition, vigorous aeration with ambient air, and measurements of pCO2, alkalinity, and DIC. Water from the mixing tank is then split to three separate lines and pumped with high precision pumps to another mixing manifold where solutions of NaCO3, NaHCO3, and HCl are added in exact proportions to the stock solution based on flow rate. By altering dissolved inorganic carbon (DIC) and alkalinity (Alk), the mineral saturation state (W) is varied from 0.5-4.0 for a variety of constant pCO2 levels (Table 1). Using this system, we will prepare seawater with varying W at two pCO2 levels (400, 600, and 800) to measure the response of healthy larvae and challenged larvae to various concentrations of V. tubiashii.Initial, paired experiments will test the survival of oyster larvae to the addition of V. tubiashii under simulated conditions of ocean acidification. Multiple sets of Pacific oysters will be spawned and eggs will be gathered and rinsed. Eggs will be distributed into closed, 500-ml BOD bottles filled with sterile seawater which has been prepared as in Table 1, with two pCO2 levels and 4 values of W (8 treatments) and an unmanipulated control. Previous experience has shown that we can maintain carbonate chemistry at initial conditions in these bottles as long as we use low larval concentrations (<10 larvae/ml) (Chris Langdon, pers. communications). Algae (Isochrysis galbana, T-ISO) will be added at 50,000 cells ml-1 once a day to provide larvae with food and to encourage ingestion of bacterial cells. When larvae reach the D-veliger stage (approximately 24h old), a range of concentrations (104 to 106 cells per ml) of V. tubiashii, will be added to one set of replicates from each treatment. In these experiments, V. tubiashii (strain RE22) will be grown in marine broth at 25°C using qPCR to quantify any bacteria that have entered a viable-but-non-culturable (VBNC) state (Gharaibeh et al., 2009). Bacteria will be then washed by x3 centrifugation with sterile seawater to remove culture medium and then re-suspended in sterile seawater (adjusted as per Table 1) to produce a stock suspension for use in larval susceptibility bioassays. A second set of replicates will be maintained at each pCO2 / W level without added V. tubiashii in order to discriminate between the effects of OA and vibriosis. Replicate wells will be sampled every 24 hours over a period of 72 hours and the proportion of living and dead larvae will be determined by adding neutral red dye (an indicator for living larvae) and via analysis of larval shell lengths and morphology, using an inverted microscope (Estes et al., 2004). All experiments will be performed in triplicate. We note here that the mechanistic response of oyster larvae to shifts in carbonate chemistry is already being well explored. By using the same experimental systems, and perhaps shifting treatments to align with experimental findings of the effect of OA on larval development independent of vibriosis, we can address the synergistic effects of OA and V. tubiashii on a key shellfish species.Table 1. Calculated treatment combinations that can be manipulated in existing experimental chambers. These conditions can all be experienced currently in Pacific Northwest coastal ecosystems during extreme upwelling events.pCO2 \ W4210.54002950202514001000DIC, mmol/kg3345224015151065Alk, mmol/kg8.188.037.887.74pH8004000280019601370DIC, mmol/kg4350298020471405Alk, mmol/kg8.037.887.737.58pHAnalysis of outcomes. Significant differences between larval growth and survival at different pCO2 / W and Vibrio levels will be determined by Student's t-test. Analyses of variance (ANOVA) will be performed to test the null hypotheses that there are no effects of the tested parameters on larval survival. Tests will be performed at the 0.01 level of significance using Statistical Analysis Systems Software.