Source: UNIVERSITY OF RHODE ISLAND submitted to
MICROBIAL SOLUTIONS TO IMPROVING LARVAL RESILIENCE IN SHELLFISH HATCHERIES
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
OTHER GRANTS
Reporting Frequency
Annual
Accession No.
1029304
Grant No.
2022-70007-38315
Cumulative Award Amt.
$324,112.00
Proposal No.
2022-06004
Multistate No.
(N/A)
Project Start Date
Sep 1, 2022
Project End Date
Aug 31, 2025
Grant Year
2023
Program Code
[AQUA]- Aquaculture Research
Project Director
Gomez-Chiarri, M.
Recipient Organization
UNIVERSITY OF RHODE ISLAND
19 WOODWARD HALL 9 EAST ALUMNI AVENUE
KINGSTON,RI 02881
Performing Department
(N/A)
Non Technical Summary
This investigation addresses the program area priority to solve critical disease issues impacting commercial aquaculture species. Bivalve hatcheries experience "crashes" that seriously compromise production of seed for an expanding aquaculture industry. Besides vibriosis, the cause/s of many crashes of bivalve larvae are unknown. There is growing evidence that the "hatchery microbiome" is an important factor in production outcomes. Our overall goal is to develop novel tools to increase larval resilience and improve production in shellfish hatcheries. Our team has previously discovered and developed safe and effective commercial formulations of probiotic bacteria that reduce the threat of vibriosis in shellfish hatcheries. We now propose to work with industry partners to develop additional microbial products that improve larval resilience to threats beyond vibriosis. We will develop a novel multi-step process to identify and isolate candidate probionts, (1) using an 'omics' guided strategy to isolate and characterize bacteria and microbial assemblages associated with improved or decreased larval performance, and (2) screening for novel probionts based on their ability to compete with pathogens and increase larval performance (survival, growth) when cultivated in water collected during unsuccessful hatchery runs (proof of concept). Expected Outcomes: We will develop a process to identify beneficial bacteria that, singly or in consortia, enhance larval resilience to unknown pathogens and environmental change. This process will be used in the future to create new microbial products to enhance sustainable and reliable production of seed. This research will further improve knowledge of probiont-microbiome-host-environmental interactions.
Animal Health Component
100%
Research Effort Categories
Basic
(N/A)
Applied
100%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
31108111100100%
Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
0811 - Shellfish;

Field Of Science
1100 - Bacteriology;
Goals / Objectives
The long-term goal of our investigation is to develop novel microbial based tools to increase larval resilience in shellfish hatcheries and minimize losses due to various causes, including unknown pathogens and toxin-producing microorganisms. We define larval resilience as the ability to prevent mortality and sustain growth and development during exposures to pathogens and suboptimal environmental conditions and stress.We will develop a novel multi-step process to identify and isolate candidate bacterial probionts that exploits a range of mechanisms of action against bacterial pathogens, including antibiosis, virulence repression via quorum quenching, and host immunostimulation. Our proposed strategy also targets the discovery of probionts that improve larval resilience against unknown causes of decreases in larval performance; this novel approach assesses probiont capacity for rescuing larval survival and growth when cultivated in water collected during failed hatchery runs. The hypotheses for our proposed research are as follows: (1) Lower larval performance in hatcheries results from multifactorial causes, including microbiome dysbiosis; and (2) A consortium of microbes, isolated through our novel screening process, can be used to improve overall larval resilience and production in shellfish hatcheries through increasing the resilience of the microbiome (microbiome engineering).Specific Objectives: Isolation and characterization of bacteria and bacterial assemblages associated with improved or decreased larval performance. We will work with our commercial and research hatchery partners to collect water and larvae from a variety of larval runs, including "good" (successful performance) and "bad" (lower performance or crashes). Culture independent and dependent methods will be used to identify and isolate bacteria that are positively associated with larval performance. The results will inform the composition of healthy microbial assemblages and help guide the selection of beneficial bacteria for development.Selection of novel probionts that increase larval performance using a novel multi-step screening process. Candidate microbes isolated in objective 1 will be screened singly and in consortia using a combination of assays focused on microbe - microbe interactions, performed both in standard culture media and in a "hatchery-relevant" media (oyster derived nutrients), and in a range of environmental conditions experienced by eastern oyster hatcheries. The most promising candidates from the screens will be finally tested for their ability to increase the performance of larvae incubated in hatchery water collected during unsuccessful larval runs.This proposal responds to the FY2022 AQUA program by improving hatchery success, alleviating a major constraint on the bivalve aquaculture industry. In particular, it is responsive to the Program Area Priorities 2b and 2c to solve "Critical disease issues impacting commercial aquaculture species" and "Design and of environmentally and economically sustainable commercial aquaculture systems," respectively (FY 2022 SRGARP RFA).
Project Methods
Our novel approach to the rational development of microbial additives for aquaculture builds upon many years of research with industry to gain knowledge of host-microbe interactions that lead to disease resistance and prevention. While mechanisms of probiotic activity in aquaculture models are multifactorial and complex, traditional screening strategies for candidate probionts have primarily relied upon only one mechanism of action: antibiosis against well-characterized pathogens. Moreover, host-microbe interactions occur in the complex and variable environment of hatcheries. We propose an improved approach for the discovery of probiotic bacteria with the following strengths: (1) direct collaboration with shellfish hatcheries using recently collected samples; (2) targeting multiple mechanisms of action to increase the spectrum and redundancy of these prevention tools; (3) use of oyster relevant media and environmental conditions representative of the diverse environments in East Coast hatcheries; and (4) targeting both known and unknown causes of declines in larval performance.We will collaborate with hatcheries to collect water and larvae from multiple larval runs, including both "good" (successful or superior performance) and "bad" (lower performance or crashes). In objective 1, culture independent (sequencing of 16S rRNA gene) and dependent (culture in different media and environmental conditions that are relevant to oyster hatcheries) methods will be used to select promising taxa and isolate candidate probionts that may promote improved larval performance. Consistent association of particular isolates with lower or higher performance, combined with larval challenge experiments, will serve to identify those bacteria that may be pathogens. Isolates from objective 1 associated with higher performance will then be screened in objective 2 for attributes associated with increased larval performance and to identify strain combinations that provide superior results. The multi-step screening process includes the following steps: a) screening for inhibitory interactions against pathogens; b) screening for quorum-quenching activity against pathogens; c) screening of isolates from a and b for antibiosis against each other (identification of candidate probiotic isolates for those that can act in consortia); d) screening of candidates from previous steps for the ability to protect oyster larvae against challenge with oyster bacterial pathogens. The genome of the most promising candidates will also be sequenced to allow for future identification of mechanisms of action.

Progress 09/01/23 to 08/31/24

Outputs
Target Audience:This research is an integral part of efforts to aid in the growth of the local and national oyster aquaculture industry, represented in the United States of America by the East Coast Shellfish Growers Association (ECSGA) and the Pacific Coast Shellfish Growers Association (PCSGA). In addition to the oyster aquaculture industry, this research is targeted to a wide range of members of the bivalve shellfish community that are interested in shellfish health, characterizing host-microbial-environment interactions in shellfish hatcheries, and applying that knowledge for the management of wild and cultured populations of shellfish. These include hatchery managers that provide seed for public (restoration) and commercial aquaculture, aquatic pathologists, and companies involved in health management in aquaculture (vaccines, probiotics, health screening, antibiotics). This research is also of interest to regulators and managers looking for alternative options to the use of antibiotics in disease management in aquaculture. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?In addition to the training of the graduate student involved in the project in previous years, this project has trained one more undergraduate student in research. This project also continues to complement the project "The Northeast Bivalve Hatchery Health Consortium (NEBHHC): Managing larval mortalities in Northeast Hatcheries", funded by the Northeast Regional Aquaculture Center. How have the results been disseminated to communities of interest?Dissemination efforts included: a) direct outreach and participation of commercial hatcheries and companies involved in aquaculture health management in the research; b) scientific presentations at aquaculture meetings, including the Annual Meeting of the National Shellfisheries Association (March 2024), Aquaculture America 24 meeting (February 2024 in San Antonio Texas); and c) organization of a workshop to be held at the Northeast Aquaculture Conference and Exposition, Providence, Rhode Island, January 2024. What do you plan to do during the next reporting period to accomplish the goals?In 2025, analysis of the whole genome sequences from candidate pathogens and probionts will be completed. Publications are being prepared and outreach to stakeholders is ongoing. Outreach efforts being planned for this project and the BHHC NRAC project include one workshop at Aquaculture 2025 targeting researchers and one remote webinar targeting both researchers and hatchery managers.

Impacts
What was accomplished under these goals? From January to December 2024, we isolated an additional 414 isolates for a total of 536 from samples of eastern oysters (Crassostrea virginica), quahogs (Mercenaria mercenaria), and bay scallop (Argopecten irradians) larvae from 33 production runs from commercial and research hatcheries. About one third of these production runs were classified as "bad" runs (i.e., lower production than usual or a complete crash). These isolates corresponded to 41 genera and 236 unique species. The most abundant genera identified were Pseudoalteromonas (146, 46%), Vibrio (118, 38%), and Alteromonas (14%). Of these isolates, 43% were uniquely present in normal production runs, 31% were shared between normal and unproductive runs, and 26% were unique to unproductive runs. Isolates are being screened for antimicrobial activity, hemolytic activity, quorum quenching, biofilm formation, and toxicity to oyster hemocytes to evaluate the potential for pathogenicity or probiotic activity. After screening more than 300 isolates, in addition to five additional isolates of the known probiont of bivalve shellfish and turbot Phaeobacter inhibens, four isolates from other species were identified as potential probionts (Algoriphagus winogradskyi DEN5, Marinomonas gallaica Clam9, Glutamicibacter soli Clam16, and Pseudooceanicola nitratireducens NEH7). Larvae treated with these isolates and challenged with the bacterial pathogens Vibrio coralliilyticus RE22 and Aliiroseovarius crassostreae CV919-312 showed from 16% to 81% increase in relative percent survival (RPS) as compared to larvae only treated with the pathogens. Larvae treated with MgClam9 (46%) and AwDEN5 (52%) had higher RPS when challenged with the pathogen Vibrio tasmaniensis DEN41. Probiotic treatment did not improve survival (1-9% RPS) when larvae were challenged with the pathogen Vibrio neptunius DEN11, and high mortality rates indicated that VnDEN11 had significantly higher levels of pathogenicity that VcRE22 and Ar919-312. A combination of all selected probiotic strains, along with probiont Phaeobacter inhibens S4, resulted in better larval survival than S4 alone.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Samson, J.S., Kulesh, K., Rowley., D.C., Nelson, D.R., Gomez-Chiarri, M., 2024. Isolation, screening, and selection of potential pathogenic and probiotic bacteria from bivalve shellfishes. Northerast Aquaculture Conference and Exposition. Providence, RI, January 2024.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Samson, J.S., Kulesh, K., Rowley., D.C., Nelson, D.R., Gomez-Chiarri, M., 2024. Screening of potential pathogenic and probiotic bacteria from bivalve shellfishes. Aquaculture America 2024, San Antonio, TX, February 2024.


Progress 09/01/22 to 08/31/23

Outputs
Target Audience:This research is an integral part of efforts to aid in the growth of the local and national oyster aquaculture industry, represented in the United States of America by the East Coast Shellfish Growers Association (ECSGA) and the Pacific Coast Shellfish Growers Association (PCSGA). In addition to the oyster aquaculture industry, this research is targeted to a wide range of members of the bivalve shellfish community that are interested in shellfish health, characterizing host-microbial-environment interactions in shellfish hatcheries, and applying that knowledge for the management of wild and cultured populations of shellfish. These include hatchery managers that provide seed for public (restoration) and commercial aquaculture, aquatic pathologists, and companies involved in health management in aquaculture (vaccines, probiotics, health screening, antibiotics). This research is also of interest to regulators and managers looking for alternative options to the use of antibiotics in disease management in aquaculture Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has provided training in microbiology to one undergraduate (summer research) and one graduate student. The project also involved the participation of one extension specialist through Rhode Island Sea Grant, and to professional development opportunities for several bivalve hatchery managers that provided samples. As part of the sample collection, two graduate students, the extension specialist, and the Project Director visited two hatcheries and discussed issues affecting larval production with hatchery personnel. This project also complements another project recently funded by the Northeast Regional Aquaculture Center, titled "The Northeast Bivalve Hatchery Health Consortium (NEBHHC): Managing larval mortalities in Northeast Hatcheries", which has the goal of supporting the growth of the bivalve shellfish industry in the Northeast USA by ensuring reliable production of bivalve seed by hatcheries. The NEBHHC is developing a collaborative, stakeholder-driven, and proactive approach to collection and analysis of samples from both good (high survival) and bad (low survival) larval production runs. We are currently enrolling hatcheries in the NEBHHC. Numerous extension and professional opportunities will derive from this related project. How have the results been disseminated to communities of interest?Dissemination efforts included: a) direct outreach and participation of commercial hatcheries and companies involved in aquaculture health management in the research; b) scientific presentations at aquaculture meetings, including the Annual Meeting of the National Shellfisheries Association (March 2023 and 2024), Aquaculture America 24 meeting (February 2024 in San Antonio Texas); and c) organization of a workshop to be held at the Northeast Aquaculture Conference and Exposition, Providence, Rhode Island, January 2024. We have also established a NEBHHC site at the Aquaculture Information Exchange site. What do you plan to do during the next reporting period to accomplish the goals?We will continue the isolation and screening of bacteria from hatchery samples from January - June 2024 using the methods developed in this past reporting period. We also plan to add another assay based on the ability of the candidate probiont isolates to inhibit quorum sensing in pathogens, and potentially develop an assay test for improvement of water quality parameters and larval feeding. The genome of selected isolates will be sequenced in order to identify potential virulence factors, and genes involved in metabolism and the production of secondary metabolites. The interactions between candidate probionts and pathogens will then be tested using in vitro competition assays, as well as in vivo assays using oyster and clam hemocytes.

Impacts
What was accomplished under these goals? A total of 122 bacterial isolates from water, algae, and larval samples collected from bivalve (eastern oysters Crassostrea virginica and quahogs or hard clams Mercenaria mercenaria) hatcheries were identified using 16S rRNA sequencing. These isolates came from samples from normal production runs, as well as from samples of larvae experiencing mortality events. Among the 20 genera identified, Vibrio (34%), Pseudoalteromonas (23%), and Alteromonas (18%) were the most abundant, consistent with previous microbiome research in shellfish hatcheries. Other genera isolated included several Phaeobacter spp., Algoriphagus spp., and Pseudomonas spp. We screened these 122 bacterial isolates using a battery of assays, including: (1) antimicrobial activity against the bacterial pathogens of bivalves Aliiroseovarius crassostreae (causative agent of Juvenile or Roseovarius oyster disease), Vibrio coralliilyticus RE22 (a causative agents of vibriosis in bivalve larvae), and Vibrio parahaemolyticus PSU5579 (a causative agent of Acute HematoPancreatic Necrosis Disease in penaid shrimp), (2) antimicrobial activity against probionts we have previously isolated (Phaeobacter inhibens S4 and Bacillus pumilus RI0695), (3) hemolytic activity (indicative of potential pathogenicity), and (4) biofilm formation at different temperatures relevant to shellfish hatcheries (indicative of an ability to colonize surfaces in the hatchery and outcompete other microbes). We used a heat map to cluster bacterial isolates based on these traits, and identified several isolates with pathogenic potential, as well as several potential probionts to be used in shellfish aquaculture. The pathogenicity of the selected potential pathogenic and probiotic isolates was tested with oysters and clam larvae, and a newly developed high throughput screening colorimetric assay based on the effect of the bacterial isolates on oyster hemocyte viability. Results showed that potential probiotic isolates are generally safe while some potential pathogens (Clam15, DEN12, CH1, CH7, CH3, CH30, CH4, CH6) showed host-specific pathogenicity to either clam or oyster larvae. The lowest relative percentage survival in both clam and oyster larvae was observed in larvae challenged with Vibrio neptunius DEN11 (0%) and our control strain V. coralliilyticus RE22 (0.88%). Vibrio fortis CH6 caused higher levels of larval mortality in oysters (0% survival) than in clams (70% survival), while Vibrio tubiashii DEN12 and DEN41 were more pathogenic to clams (around 15% survival) than oysters (about 50% survival). Results from the high-throughput screening assay using oyster hemocytes were concordant with the results of the more labor-intensive larval assays. We have sequenced the genomes of Vibrio neptunius DEN11 and Vibrio alginolyticus DEN32 and deposited in GenBank (PRJNA758975).

Publications

  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Takyi, E., LaPorte, J., Sohn, S., Stevick, R.J., Witkop, E.M., Gregg, L.S., Chesler-Poole, A., Small, J., White, M.M., Giray, C., Rowley, D.C., Nelson, D.R., Gomez-Chiarri, M., 2023. Development and evaluation of a formulation of probiont Phaeobacter inhibens S4 for the management of vibriosis in bivalve hatcheries. Aquaculture, Fish and Fisheries 3, 256267. https://doi.org/10.1002/aff2.112