Developing a Thermal Shock Method to Control Disease and Biofouling on Oyster Farms

Progress report for GNE20-246

Project Type: Graduate Student
Funds awarded in 2020: $15,000.00
Projected End Date: 08/31/2022
Grant Recipient: Rutgers, the State University of New Jersey
Region: Northeast
State: New Jersey
Graduate Student:
Faculty Advisor:
Dr. David Bushek, PhD
Haskin Shellfish Research Laboratory, Rutger University
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Project Information

Summary:

Dermo disease, caused by the parasite Perkinsus marinus, is a prevalent problem in northeastern oyster stocks. We hope to exploit known temperature vulnerabilities via thermal shock treatments (a novel approach) to reduce disease loss. Oysters living intertidally in the Southeast are regularly exposed to temperatures exceeding the thermal tolerance of P. marinus—but not that of the oysters—which should limit the proliferation of the parasite within oysters.  If this hypothesis is correct, subjecting Northeastern oysters to similar conditions may help mitigate dermo disease, potentially providing a non-chemical therapeutic treatment for commercial stocks. Temperature can be readily elevated during low tide by temporarily covering oysters with clear plastic. Because oysters have a higher thermal tolerance, such exposures may reduce parasite loads without harming the oysters. Reducing biofouling may be a secondary benefit of this treatment. However, elevated temperatures could also disrupt the microbiome, potentially creating human health risks if pathogenic bacteria increase. Understanding how such temperature modulation impacts P. marinus and alters the oyster microbiome is critical to exploring if this could be a safe and effective treatment for aquaculture in the Northeast. Laboratory and field trials will be used to identify effective treatment regimens to reduce P. marinus loads and biofouling, while monitoring bacterial safety. If successful, this preliminary study will be used to design treatments for beta-tests on local oyster farms.

Project Objectives:
  1. Determine how environmental conditions (e.g., sun, clouds, wind) impact heat treatments.
  2. Model the changes in the marinus and bacterial populations in vitro when exposed to single or repeated acute heat shocks peaking at 45, 50 and 55 °C for periods of 2, 3 and 4 hrs.
  3. Confirm the tolerance and in vivo response of Northeastern oysters to temperatures of 45, 50 and 55 °C for periods of 2, 3 and 4 hrs as identified in objective 2.
  4. Assess the effect of heat treatment frequency and seasonality in a farm-like context on:
    1. Dermo infection intensity
    2. Biofouling
    3. The bacterial microbiome, including pathogens of human concern
    4. Oyster growth, condition, and mortality

Objectives 1, 2 and 3 will inform objective 4 and provide baseline data for further improvements should this technology prove fruitful in reducing dermo disease, minimizing biofouling, or impacting bacterial dynamics.  Testing both the frequency and seasonality of treatment will reveal if either factor has a particularly strong impact on Dermo infection intensity since P. marinus abundance changes seasonally, increasing exponentially from Spring to Fall as temperatures warm. Similarly, biofouling changes seasonally, as does the microbiome, which is defined in part by the bacterial community in the surrounding waters. If results indicated that heat treatments can provide an effective means of pest management on oyster farms, then we will pursue farm trials.

Introduction:

The purpose of this project is to develop a low-cost, low-tech method that reduces the impact of Dermo disease on oyster farms via a regimen of heat treatments conducted throughout the growing season. The intensity and frequency of treatments will be optimized for testing in the context of a real oyster farm. A secondary benefit may be reduced biofouling. Changes to human pathogens will be tracked to assess unintentional risks to human health.

Perkinsus marinus, the agent of Dermo disease, is a protistan parasite infecting the oyster as it feeds. Once ingested, P. marinus targets hemocytes—immune cells in the oyster hemolymph—which become infected and are destroyed as the parasite proliferates. The cascading effects of infection include compromised immunity, reduction in reproductive capacity, slowed growth, and increased mortality. The emaciation of the oyster meat degrades the quality of the oyster and mortality reduces farm production. Although P. marinus can be found along most of the Atlantic coast of the US, its impacts vary for reasons that are not fully understood, including climate, salinity, circulation, host resistance, and parasite virulence.  In the Southeast, intertidal oysters are routinely exposed to temperatures that exceed the thermal tolerance of P. marinus (> 35 ºC [1]) yet the oysters survive.  This thermal exposure may curtail the proliferation of P. marinus thereby reducing the impact of Dermo disease.  We propose testing the effectiveness of thermal treatments that mimic the conditions experienced by intertidal oysters in the Southeastern US to reduce Dermo-induced mortality (Figure 3).

Heat treatment may also reduce biofouling as such temperatures may exceed thermal tolerances for these organisms. Two mud worms are particularly problematic. Polydora websterii causes mud blisters within the oysters reducing quality and marketability. Polydora cornuta encases oysters and gear in mud, restricting water flow and potentially smothering oysters if not frequently removed. Farmers must invest significant portions of their time to keep oysters and farm gear free of these fouling organisms, therefore any treatment that reduces fouling will increase efficiency and improve marketability. 

Although P. marinus is harmless to humans, attempts to thermally control the impact that Dermo disease has on aquaculture output may have unintended effects on the oyster microbiome, including bacteria of concern to human health.  Warm temperatures, including those experienced on some intertidal farms can elevate levels of pathogenic vibrios [2, 3]. The effect of higher heat treatments may, however, be positive – V. parahaemolyticus, for example, has a low heat tolerance [4] and can be readily eliminated with heat as demonstrated on shrimp exoskeletons exposed to 50 ºC [5]. The effect of the proposed thermal treatment may be similar to dipping shellfish in boiling water—a method already being used to control biofouling [6]. The microbiome can be sensitive to disruptions such as heat shock, but this has mostly been studied in the context of naturally-occurring heat waves [7, 8], not the acute exposures proposed here as a method of disease treatment.

Figures

Research

Materials and methods:

Field Experiments

Large pieces of 20 gauge clear marine vinyl (www.MarineVinylFabric.com) were cut to temporarily cover oyster bags raised above an intertidal flat while exposed around mid-day by a strong low tide. For each replicate of the treated group, an MX2202 HOBO® Pendant MX Temperature/Light was positioned between the vinyl and the oyster bags. Once the threshold temperature of 40 °C was crossed, an hour timer was set and temperature was continuously monitored via phone app connection to the bluetooth-capable HOBO devices. 50 °C was the target temperature, so if the treatment approached 55 °C then the vinyl was vented (adjusted to allow more airflow or completely removed temporarily) to bring the temperature back down. At the end of the hour, the vinyl was removed and the bags secured to their racks.

 

 Table 1: Summary of tide and meteorological information for all treatment (both successful and failed attempts)

Attempt 

Treatment 

Date 2021 

Low tide time 

Low tide height (feet) 

Wind speed (knots) 

Ambient Temp (F) 

Barometric Pressure  (mb) 

Weather 

1 

Failed attempt 

6/21 

12:45 PM 

-0.13 

8, gusts of 12 

74.3 

1008.5 

Partly cloudy 

2 

A- Spring 

6/23 

2:33 PM 

-0.33 

7.39, gusts of 13.61 

68.7 

1022.9 

Sunny 

3 

B - Summer 

7/20 

12:23 PM 

0.05 

2.92, gusts of 4.08 

 

76.3 

1014.4 

Sunny 

4 

Failed attempt 

8/24 

5:16 PM 

0.23  

7, gusts of 7.8 

80.2 

1014.2 

Sunny 

5 

C- Fall 

9/7 

4:12 PM 

0.10  

4.28, gusts of 5.05 

75.4 

 

1016.1 

Sunny 

 

Meteorological observation (Cape May, NJ): https://tidesandcurrents.noaa.gov/met.html?bdate=20210823&edate=20210825&units=standard&timezone=GMT&id=8536110&interval=h 

Tide predictions (Bidwell Creek Entrance) : https://tidesandcurrents.noaa.gov/noaatidepredictions.html?id=8536581&units=standard&bdate=20210823&edate=20210825&timezone=LST/LDT&clock=12hour&datum=MLLW&interval=hilo&action=dailychart 

 

 

Figure 1: Treatment in progress as bags of oysters are covered (and sometimes wrapped, if windy) with vinyl. Photo from 6/23 treatment. 

 

Figure 2: NSF REU intern Grace Jackson (left) monitors the treatments in real-time from a dashboard on a phone that connect to the Bluetooth-capable temperature and light-loggers (right). 

 

Figure 3: Approximately one week after treatment oysters were removed from bags to count those that were alive or dead, as well as to collect a subsample to be sacrificed for molecular analysis.  

 

Figure 4: At the final timepoint, 30 oysters from each condition were sacrificed and a sample taken from the mantle and rectal tissue for analysis using RFTM to assess Dermo disease prevalence and intensity of infection.  

 

Molecular Methods to Study the Microbiome Composition  

 DNA extraction was optimized for these samples using the E.Z.N.A. Mollusc DNA Kit (Omega Bio-tek, Norcross GA). The majority of samples have had mantle tissue dissected (the remaining oyster is stored at –80C), DNA extracted, amplified, and sequencing using the Oxford Nanopore MinION is currently underway using R10 sequencing chemistry. Water samples collected at the high tide prior to each date of oyster sampling was filtered (0.2 micron) and the filter paper is undergoing phenol-chloroform extraction for subsequent amplification and sequencing by the same means as the mantle tissue. Following sequencing and bioinformatic analysis, these data will show if any microbiome differences persisted between groups a week after each treatment, and if there was any long-term change to the microbiome composition at the end of the season. Water samples also provide a point of comparison showing if any treatment groups diverged more significantly from the ambient bacterioplankton assemblage. If no differences are observed between treatment groups, then these data will at least show the seasonal trend for the microbiome of oysters grown at the Rutgers Cape Shore facility.  

 

Oven Experiments 

 Anticipating some difficulty performing consistent treatments on the farm, we ran a set of concurrent experiments to try thermal treatments of oysters in an oven. An initial test was performed to observe the mortality that resulted from a single exposure. In case mortalities may have been delayed, the oysters were maintained off of the dock at the Haskin Shellfish Research Laboratory in Port Norris, NJ for periodic checks. 

Figure 5: 8/28/21 check for the oyster mortalities treated in the oven a single time at 40, 50, and 60 C. 

 

As part of an NSF REU program hosted at Rutgers, we engaged an undergraduate student (Grace Jackson from the University of Dayton), to perform a set of experiments summarized in a poster.

RIOS poster Grace J

Her results from experiment 1 in which pure cultures of Perkinsus Marinus were subjected to the thermal treatment reaffirmed our foundational concept that the treatment should nearly eliminate the P. marinus population. However, we have yet to definitively show this in oysters in vivo. Experiment 2, in which oysters were treated daily over the course of a week, found the resulting dermo levels to be insignificant between the treated and untreated group. The team then wondered if the mortalities that occurred before the endpoint in this and experiment #3 may have been “culling” more highly infected oysters from group, resulting in an artificially low dermo result (as treated oysters seemed less infected, but not significantly so in experiment #3).   

To address the above question, as well as the concern that the temperature threshold was likely not being maintained when treatment was solely based on time, since adding oysters into the oven caused a precipitous drop in temperature, a second iteration of Experiment #2 was performed by the grantee graduate student Heidi Yeh. In this experiment, oysters were treated semi-daily over the course of a week in an oven that was pre-heated with a large mass of rocks to help maintain thermal inertia. Additionally, the treatment clock was not started until the oven with oysters had returned to the target temperature. For this iteration, oysters were then maintained in a raceway tank so that mortalities could be observed within hours and the oysters removed for RFTM sampling.  

Figure 12: Oysters were maintained in a raceway tank on a mesh platform that made daily transfer to the oven easier.  

 

In vitro experiments 

 The original plan to test oyster homogenate response to thermal treatment was modified to use just extra-pallial fluid (EPF) at the recommendation of Heidi’s dissertation committee. The EPF was then spread on bacterial plates, and samples preserved for DNA extraction and subsequent sequencing with the MinION to observe composition changes that may have occurred as a result of treatment.  

 

Research results and discussion:

Field Experiment

 

Treatment Key  

1,2, and 3 refer to the number of treatments  

A = spring (6/23/21) 

B = summer (7/20/21) 

C = fall (8/24/21) 

 

There was a clear negative impact on survival observed in all bags that were treated as part of the first round of fever in June (Figure 1). 

Figure 1: Final survival of oysters by treatment (N=3) 

 

Removing this potentially problematic round of treatment, survival rates were generally high, but all treatments had a deleterious effect on survival relative to the control (Figure 2).  

Figure 2: Final survival of oysters (N=3) with certain treatments omitted.  

Across treatments, final dermo levels were low to moderate, with a median of 0.5 or lower for all (Figure 3). There is no clear difference in the dermo outcomes between all of the treatment groups, despite the large disparity in their survival rates (Figure 1).  

Figure 3: All Dermo values by treatment 

 

Biofouling:  

There was no substantial difference in mud buildup on the oysters, and infestation with mud worms was non-existent to light across treatments

Figure 4: Cleaned shells from the control group show that mud worm infestation was not a baseline problem for the oysters grown at this site this year, so there is no biofouling problem for the heat treatment to have potentially addressed.  

 

Oven Experiments

Following a single oven treatment, all oysters that experienced a 60 °C heat shock eventually died, whereas oysters from the other treatments (40 °C, 50 °C, and the control) experienced minimal mortalities (Figure 5). 

Figure 5: Surviving oysters at each timepoint (1 = 8/24/21, 2= 8/25/21, 3= 8/28/21, 4=11/9/21). 

The Dermo levels observed at the end point were not significantly different between the conditions (Figure 6): 

Figure 6: Dermo disease intensity (mackin) 2+ months after a single oven treatment (N=9-13). 

Although exposure to a single heat treatment had been previously established to not have a deleterious effect on the oysters, mortalities quickly ramped up after a few days, and we decided to stop short of our goal of a week of treatments to preserve some survivors for the final sampling to occur a week later (Figure 7). The resulting record of mortality and associated levels of dermo infection are presented in the following figures:  

 

Timeline of the experiment (dates refeering to 2021):  

  • 1 week of acclimation to wet lab 
  • 5 days of oven treatments (8/24 (Heidi), 8/25 (Jenn), 8/26 (Jenn); 8/28 (Dave?), 8/29 (Heidi?) 
  • 5 days to recover/purge  
  • Final sacrifice on 9/3 

Figure 7: Surviving oysters at each timepoint (consecutive days between August 24 and September 1, 2021). 

At the starting point, there was a 90% Dermo prevalence among the oysters, with an average mackin rating of 2.4. There may have been a slight reduction in dermo held under the wet lab conditions (Figure 8), but this was not statistically significant.  

Figure 9: Dermo disease intensity at the start of the experiment (“Start”), compared to the final Mackin score for both Treated (“End-Treated”) and Control (“End-Control”) oysters. 

 

Participation Summary

Education & Outreach Activities and Participation Summary

Participation Summary:

Education/outreach description:

Results will be presented orally at the monthly Shellfish Growers' Forum hosted in Cape May, NJ. Following this project, any success achieved in this treatment will be built upon and adapted to real farm contexts through a round of beta-testing with local farm partners.

The results of the theoretical and applied components of this project may be submitted as manuscripts to scientific journals such as Aquaculture, Microbial Ecology, or the Journal of Invertebrate Pathology. Sequences generated by the project will be deposited in the Sequence Read Archive (SRA) of NCBI: https://www.ncbi.nlm.nih.gov/sra

Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture or SARE.