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: 04/30/2022
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:

Planning is underway and field work supplies will be ordered in late 2020/early 2021. The following experiments will be performed in 2021 during the spring-fall oyster growing season.

  1. The temperature spikes achieved using a clear plastic covering will be characterized in different weather conditions and arrangements of materials (e.g., laid on top only versus wrapped or draped). This will establish the baseline conditions that can be achieved under different weather conditions (e.g., sunny vs cloudy) to help guide treatment durations in the field. The experimental setup of plastic coverings over oyster aquaculture gear will be deployed on approximately 10 days throughout the spring, summer, and fall with a range of environmental conditions. Factors such as light, ambient air temperature, and windspeed will be recorded. The conditions experienced by oysters held within the experimental treatment will be monitored in real-time using a Bluetooth logger (MX2202 HOBO® Pendant MX Temperature/Light) measuring temperature (°C/°F) and light (lum/ft2). Exposures will be performed over the course of a 4-hour period, the maximum length of time that oystermen with intertidal farms may reasonably have during a low-tide to tend to their stocks.
  2. In vitro experiments will be used to quantify the changes expected in marinus and bacteria when subjected to different thermal regimes. Oyster tissue will be homogenized and combined with P. marinus cultures in a slurry, then subjected to thermal spikes under laboratory conditions mimicking a range of conditions to be achieved in the field. Replicate aliquots will be sampled at regular intervals before, during, and after the shock to provide temporal resolution than cannot be logistically completed in vivo as oyster sacrifice or invasive sampling is required to represent every time point. P. marinus viability will be assessed via optimized body burden analyses [1, 14, 15] and compared to control aliquots maintained under ambient conditions. Bacterial populations will be quantified via overnight plate incubations on marine agar and TCBS agar (selects for Vibrio genus) to determine if overall bacteria or Vibrio levels have increased as a result of the treatment. Results will inform the duration of heat treatment required for optimal effect in situ.
  3. To determine oyster survival and in vivo response, whole oysters will be sampled before, immediately after, and one week after thermal treatments and controls to test for survival, changes in dermo levels and microbiome composition. Before and after thermal treatments, oysters will be held in flow-through tanks with ambient seawater. Each oyster sampled will be shucked, homogenized, and analyzed using the same plate incubation and sequencing methods for the in vitro experiment from objective 2.  Likewise, a subsample of each homogenate will be assayed for viable marinus via the body burden assay [14].
  4. Using information from objectives 1, 2 and 3, field trials will be conducted at the Rutgers Cape Shore Laboratory, an intertidal experimental farm in the Delaware Bay, where oysters for this experiment will be grown alongside commercial stocks. The experimental design is illustrated in Figure 4, with each column representing a different treatment that will contain 3 replicate oyster bags of sub-market to market-size oysters. The month combinations to the left indicate the two-month period during which a treatment may occur—the actual timing of which will depend on the necessary confluence of weather and tides (e.g., low tide exposure on sunny days, as determined by objective #1). Following treatment and a week-long period for the oyster to purge any dead accumulated marinus cells, oysters will be sampled from each treatment and used for the following analyses (Figure 5):
    1. Ten oysters will be collected at each timepoint to assess Dermo infection intensity using Ray's Fluid Thioglycollate Medium body-burden analysis (Figure 5).
    2. The extent of biofouling by Polydora on the oysters collected for 4a will also be quantified by counting active P. websterii mud blisters and obtaining the dry weight of mud from P. cornuta that has accumulated on the shell surfaces (Figure 5).
    3. Three oysters will be sampled from each unique condition (note the redundancy in the first two timings of sampling) and all of the soft tissues homogenized. The homogenate will be suspended in sterile saline solution and spread on marine agar plates for overnight incubation and subsequent quantification of bacterial growth. DNA will be extracted from 25 mg of the homogenate for sequencing of the for 16S rRNA V3-V4 hypervariable region as well as qPCR quantification of marinus concentrations (to achieve greater precision than the methods used in 4a). A one ml aliquot with also be subject to body burden analysis to assist with relating microbiome to P. marinus infection status.
    4. At each time point, the number of oysters lost to mortality will be quantified. The size of the oysters collected for 4a will be measured to monitor growth differences among the treatments. The quality of the final oyster product will be assessed using a condition index, wherein the weight of the fresh oyster and its shell is compared to the weight of the meat following oven desiccation [15].
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.