Progress 09/01/19 to 08/31/22
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 involved the training of three undergraduate students and two egraduate students in research. These students gained experience in oyster larval maintenance, disease challenges, microbiological and diagnostic techniques, polymerase chain reaction, DNA and RNA extractions and library preparation for sequencing, coding in R Studio, and bioinformatics analysis for microbial community structure and function. Students also interacted with industry partners, including hatchery managers and scientists at a commercial company in aquatic animal health. These interactions included: discussion of experimental design after familiarization of the students with hatchery operations, challenges associated with larval production, and methods used to produce bacterial probiotic formulations at a commercial scale, protocol development in collaboration with all partners, training of hatchery managers in sample collection, and troubleshooting of experiments. Training also included preparation of scientific reports, including poster and oral presentations. Students are also working on the development of protocols for the effective delivery of probiotics in shellfish hatcheries in collaboration with all partners; this protocols will be disseminated to the industry as a practical manual. Two of the undergraduate students that particicipated in this project have been employed at institutions involved in aquaculture research, including the USDA Agricultural Research Services and an Aquatic Animal Health Diagnostic Laboratory. The third one is enrolled in a graduate program. The two doctoral students graduated and are now in postdoctoral positions. 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 (virtual in 2021) and the Aquaculture 2022 meeting (March 2022 in San Diego); and c) organization of a workshop on probiotics held at the Northeast Aquaculture Conference and Exposition (NACE), Portland, Maine, April 2022. A manual with recommendations on the use of probiotics in shellfish hatcheries is being developed in collaboration with industry partners. Conversations with the industry and other stakeholders at the NACE conference provided the seed for the establishment of a Shellfish Hatchery Health Collaborative. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Commercial formulations of probionts Bacillus pumilus RI0695 (RI) and Phaeobacter inhibens S4 (S4) were produced in collaboration with commercial partners, thanks to funding from the NOAA Saltonstall Kennedy program. These formulations were tested in a total of 8 trials: four hatchery trials performed at the Virginia Institute of Marine Sciences in Summer 2019 and Summer 2020 (VIMS, 2 trials per year), two trials at the Mook SeaFarm hatchery in Maine (January and June 2021), one two trials in hatcheries in Rhode Island (Matunuck, June 2020; Roger Williams University Blount Shellfish Hatchery, RWU, July 2021). In these trials, we compared the performance (growth and survival) of larvae in control and probiotic (RI, S4) treated tanks, the impact of probiotic treatment on the ability of larvae to survive a laboratory challenge with the bacterial pathogen Vibrio coralliilyticus RE22 (NOAA funding), and the effect of probiotic treatment on microbial composition through examination of microbial community structure and diversity using sequencing of 16S rRNA gene amplicons (this USDA funding). Bacterial communities were characterized by sequencing of amplicons from the V6 region of the 16S rRNA gene. Several measures were used to evaluate microbial community and structure: alpha diversity (Simpson's index), beta diversity (Bray Curtis), and differences in microbial composition at different levels, from order (broad composition) to amplified sequence variants (ASV, more detailed composition, sometimes at the species level), following methods we have previously used in hatchery trials of probiont RI (Stevick et al. 2019). Samples were also collected from two selected trials for examination of metatranscriptomes. Impact of probiotics on larval performance: The formulated probiotics were safe and effective in protecting larvae against bacterial challenge, similarly to the fresh probiotics that were tested in prior research. Treatment of larvae in the hatchery with the S4 or RI formulations did not significantly affect the survival and growth of the larvae. Formulated probiont S4 or RI treatment in the hatchery led to a significant increase in Relative Percent Survival (RPS) when larvae were subsequently challenged with the pathogen Vibrio coralliilyticus RE22 for 24 hours in a laboratory challenge, as compared to probiotic-untreated RE22-challenged larvae. High levels of variability were seen in larval performance between trials, with one of the trials (Trial 2) showing very low performance in both control and probiotic groups, suggesting that the probionts do not confer an advantage in highly adverse situations (e.g. in the summer, when larval crashes are more common). Impact of probiotics on microbial community: Probiont RI had a similar effect on oyster larvae as described in our previous research, showing a major significant effect on Oceanospirillales. Bacterial communities of larvae treated with S4 in the hatchery, were highly variable between hatcheries and trials, with only 34 out of 2,499 ASVs common to all trials. Overall, for all trials (except Trials 5 and 6, removed due to lack of replication) and treatments (control and S4), bacterial richness (Chao1 index) ranged from 70 ± 4 ASVs to 325 ± 17 ASVs and the alpha bacterial diversity (Simpson's diversity index) ranged from 0.76 ± 0.2 to 0.96 ± 0.01. There was no significant difference in bacterial or richness between control and S4-treated larvae in each trial. When examining trials independently for the effect of treatment, there was a significant difference between the structures of larval oyster bacterial communities of untreated and S4-treated larvae in Trials 1 and 7 only. Overall (for all trials), 18 ASVs showed significant differential abundance between untreated controls and S4-treated larvae (p < 0.05 and logfold change > 0.3). Most of these ASVs were present in low abundance, except for ASV3 (Alteromonas spp.; more abundant in S4 than control larvae) and ASV11 (Pseudomonas spp.; less abundant in S4 than control larvae), which were part of the most abundant and common taxa found in many of the trials. These resulst suggest that S4, similarly to RI, has subtle and targeted effects on specific members of the microbial community in larvae, and do not cause dysbiosis. Metatranscriptomics analysis of a subset of samples from these trials showed the ability to detect the presence of S4 in hatchery-treated larvae, and improved the ability to identify vibrios at the species level. Issues with the lack of annotation in marine species common in hatcheries prevented a thorough analysis of gene expression, which was limited to the most abundant and better annotated species. We also assessed the effect of ultraviolet (UV) treatment on the microbiota of oyster larvae in the hatchery in one of the trials (Trial 8). Results showed that UV treatment of incoming seawater led to a significant decrease in alpha diversity (Simpson's index) of the bacterial community in oyster larvae, as compared to larvae raised in nonUV-treated water. Treatment with probiont S4 restored alpha diversity in the bacterial community of larvae raised in UV-treated water; alpha-diversity was significantly higher in S4-treated larvae than in control larvae in UV-treated water but not in non-UV treated water. Interestingly, the effect of S4 treatment on community composition (beta diversity) was different depending on water treatment, with S4 treatment only having a significantly effect in UV water only. Finally, a dosage error for RI in from Trial 1 showed that probiotic RI treatment at about 1000X the recommended dose lead to dysbiosis, as shown by the significantly lower microbial alpha diversity (an index derived from the number of ASVs detected in each sample) observed in the larvae exposed to RI. Dysbiosis was probably due to the larvae being overwhelmed by the high amounts of probiotic provided to the tanks, as indicated by the high percentage of bacteria in the order Bacillales, the order to which probiont Bacillus pumilus RI0695 belongs, detected in the larvae (almost 75% compared to less than 25% in other treatments). This dysbiosis probably led to the negative impacts on larval survival and growth observed in the RI treatment tanks, which completely crashed on day 7. This research provides evidence on the effectiveness of a newly developed approach to formulation of marine gram-negative bacteria for use as probiotics in aquaculture. This formulation approach may be useful for developing formulations of other probionts, especially gram-negative bacteria for use in marine aquaculture. The S4 formulation was shown to be safe, easy to handle, and stable to use in the hatchery environment, and it may help manage the impact of vibriosis when used prophylactically in oyster hatcheries, although it may not offer protection against other largely uncharacterized causes of larval mortality. Evaluation suggests that use of S4 in hatcheries should not cause significant perturbations in larval microbiomes while also providing protective effects against mortality due to vibriosis, and may confer other benefits such as preventing bacterial dysbiosis in larvae due to the use of UV-treated water. It also revealed that S4 treatments may favor the abundance of potentially beneficial ASVs in taxa such as Alteromonas and Pseudoalteromonas while also limiting the proliferation of ASVs belonging to the genus Pseudomonas, for which some species have been associated with larval mortality. These findings further our understanding of how probiotic treatments may influence larvae-associated microbiomes in aquaculture systems and serve as safe additives to promote animal health.
Publications
- Type:
Journal Articles
Status:
Under Review
Year Published:
2023
Citation:
Takyi, E, LaPorte J., Sohn S., Stevick, R.J., Witkop, E.M., Gregg, L., Chesler-Poole A. Development and Evaluation of a Formulation of Probiont Phaeobacter Inhibens S4 for the Management of Vibriosis in Bivalve Hatcheries. Aquaculture, Fish, and Fisheries (under review)
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Gomez-Chiarri, M, Coppersmith J., Modak T., Witkop, E.M., Stevick, R.J., Takyi, E., Nelson, D.R., Rowley, D.C. (2022). A hologenomic approach to the management of infectious diseases affecting the American oyster, Crassostrae virginica. 1st Applied HoloGenomics Conference, Bilbao, Spain, September 2022.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Gomez-Chiarri, M, Coppersmith J., Schuttert., C., Takyi, E., Nelson, D.R., Rowley, D.C. (2022). A tale of two (or more) bacteria: Applying knowledge on vibrio-probiont interactions to manage bacterial diseases in bivalve hatcheries. Aquaculture 2022, San Diego, CA, February 2022.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Takyi, E., Gregg, L., Chesler-Poole, A., Moss Small, J., White, M.M., Hudson, R., Giray, C., Rowley, D.C., Nelson, D.R., Gomez-Chiarri, M. (2022). Effect of probiotic treatment on larval microbiomes of eastern oyster Crassostrea virginicca raised in different hatcheries. Aquaculture 2022, San Diego, CA, February 2022.
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2022
Citation:
Takyi, Evelyn. Probiotics for Eastern Oyster Hatcheries: Commercial Formulations and Effect on Microbial Communities. Ph.D. Dissertation, University of Rhode Island, 2022. https://www.proquest.com/docview/2671733072/abstract/B572135449F347B0PQ/2
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Takyi, E, LaPorte J., Sohn S., Stevick, R.J., Witkop, E.M., Gregg, L., Chesler-Poole A. Development and Evaluation of a Formulation of Probiont Phaeobacter Inhibens S4 for the Management of Vibriosis in Bivalve Hatcheries. bioRxiv, December 28, 2022. https://doi.org/10.1101/2022.12.27.522043
- Type:
Journal Articles
Status:
Under Review
Year Published:
2023
Citation:
Takyi, E., Stevick R.J., Witkop, E.M., Gregg, L., Chesler-Poole, A., Moss Small, J., White, M.M., Hudson, R., Giray, C., Rowley, D.C., Nelson, D.R., Gomez-Chiarri, M. (2023). Probiotic treatment in the hatchery shifts the composition of bacterial microbiomes in larval eastern oysters, Crassostrea virginica. Submitted to Frontiers in Marine Sciences (under revision).
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Progress 09/01/20 to 08/31/21
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:Hatcheries in 2021 continued to experience difficulties in producing larvae (i.e. experienced unusual larval mortalities and crashes due to unknown causes). Therefore, we were not able to perform as many large scale trials in commercial hatcheries as we hoped, since the commercial hatcheries could not afford to dedicate a run for our research purposes. Finally, we have not been able to present our results to industry stakeholders due to cancellation of the Northeast Aquaculture Conference and Exposition (NACE) in January 2021. We hope to be able to present the results of this research at Aquaculture 2022 and the NACE 2022 meeting. What opportunities for training and professional development has the project provided?This project involved the training of one more undergraduate student during this reporting period (summer and fall 2021) and two graduate students in research. These students gained experience in oyster larval maintenance, disease challenges, microbiological and diagnostic techniques, polymerase chain reaction, DNA and RNA extractions and library preparation for sequencing, coding in R Studio, and bioinformatics analysis for microbial community structure and function. Students also interacted with industry partners, including hatchery managers and scientists at a commercial company in aquatic animal health. These interactions included: discussion of experimental design after familiarization of the students with hatchery operations, challenges associated with larval production, and methods used to produce bacterial probiotic formulations at a commercial scale, protocol development in collaboration with all partners, training of hatchery managers in sample collection, and troubleshooting of experiments. Training also included preparation of scientific reports, including poster and oral presentations. Students are also working on the development of protocols for the effective delivery of probiotics in shellfish hatcheries in collaboration with all partners; this protocols will be disseminated to the industry as a practical manual. 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 (virtual in 2021) and the Aquaculture 2022 meeting (March 2022 in San Diego); and c) organization of a workshop on probiotics to be held at the Northeast Aquaculture Conference and Exposition, Portland, Maine, April 2022. A manual with recommendations on the use of probiotics in shellfish hatcheries is being developed in collaboration with industry partners. What do you plan to do during the next reporting period to accomplish the goals?Work to be performed during the next reporting period will include: 1) processing and analysis of larval and water samples for metatranscriptomic analysis; 2) evaluation of how variability in environmental parameters and hatchery practices at each of the different hatcheries may influence the efficacy of probiotics and their effect on microbial communities in the hatchery; 3) evaluation of potential associations between of members in the pre-existing microbial communities in the hatchery on probiotic efficacy; 4) using this information to develop a manual for probiotic delivery at shellfish hatcheries, 5) dissemination of results to industry in collaboration with industry partners, and 6) preparation of technical reports and publications.
Impacts
What was accomplished under these goals?
Commercial formulations of probionts Bacillus pumilus RI0695 (RI) and Phaeobacter inhibens S4 (S4) were produced in collaboration with commercial partners, thanks to funding from the NOAA Saltonstall Kennedy program. These formulations were tested in a total of 8 trials: four hatchery trials performed at the Virginia Institute of Marine Sciences in 2019 and 2020 (VIMS, 2 trials per year, Spring and Summer), two trials at the Mook SeaFarm hatchery in Maine (January and June 2021), and two trials in hatcheries in Rhode Island (Matunuck, June 2020; Roger Williams University Blount Shellfish Hatchery, RWU, July 2021). In these trials, we compared the performance (growth and survival) of larvae in control and probiotic (RI, S4) treated tanks (NOAA funding), the impact of probiotic treatment on the ability of larvae to survive a laboratory challenge with the bacterial pathogen Vibrio coralliilyticus RE22 (NOAA funding), and the effect of probiotic treatment on microbial composition through examination of microbial community structure and diversity using sequencing of 16S rRNA gene amplicons (this USDA funding). Bacterial communities were characterized by sequencing of amplicons from the V6 region of the 16S rRNA gene. Several measures were used to evaluate microbial community and structure: alpha diversity (Simpson's index), beta diversity (Bray Curtis), and differences in microbial composition at different levels, from order (broad composition) to amplified sequence variants (ASV, more detailed composition, sometimes at the species level), following methods we have previously used in hatchery trials of probiont RI (Stevick et al. 2019). Samples were also collected from two selected trials for examination of metatranscriptomes. Impact of probiotics on larval performance (NOAA funding): The results from these hatchery trials indicate that the formulated probiotics were safe and effective in protecting larvae against bacterial challenge, similarly to the fresh probiotics that were tested in prior research. No significant effects of probiotic treatment on larval survival or growth in the hatchery where seen in any of the 8 trials, but probiont-treated larvae at the hatchery consistently showed significantly higher survival to bacterial challenge with the pathogen Vibrio coralliilyticus RE22. High levels of variability were seen in larval performance between trials, with one of the trials (Trial 2) showing very low performance in both control and probiotic groups, suggesting that the probionts do not confer an advantage in highly adverse situations (e.g. in the summer, when larval crashes are more common). Overall characterization of the microbial community of oyster larvae in shellfish hatcheries (this funding): Microbial diversity in oyster larvae was high (Simpson's diversity index between 0.75 and 1) in all of the 8 trials; the number of ASVs detected in each trial ranged from around 500 (trials 2 at VIMS and 8 at RWU) to around 1200 (trial 3, VIMS). Significant differences in diversity were found between trials within hatcheries and between hatcheries, with slightly but significant lower diversity in Trial 1 (VIMS June 2019) and Trial 6 (Mook January 2021) than in any of the other trials. Microbial community structure (composition) also differed between trials and hatcheries. The largest effect on bacterial community structure was due to location (region), with oysters from all trials at VIMS (Virginia) showing a significantly different composition from oysters from all trials at Mook (Maine), and both showing a different composition from the bacterial community in oysters collected at the two hatcheries in Rhode Island (p <0.01; adonis2 Permutational Multivariate Analysis of Variance Using Distance Matrices test). Few ASVs (40 out of more than 1,000 ASVs detected) were shared between all the trials; these ASVs belonged to the most abundant ASVs in the whole dataset, including ASVs classified (from most abundant to least) as Alteromonoadales, Rhodobacterales, Vibrionales, unclassified bacteria, Pseumonodales, and Oceanospirillales. These core bacterial taxa differed in abundance between trials. Impact of probiotics on microbial community: Probiotic treatment (RI and/or S4) at the recommended dosage (104 CFU/mL) did not have a significant effect on microbial diversity in oyster larvae in any of the trials. However, a significant effect of probiotic treatment on bacterial community structure, as compared to control tanks, was observed for each trial. Overall, the effect of probiotic treatment on microbial community was highly variable between trials, suggesting that effect of the probiotic on microbial community depended on the original bacterial compositon characteristic of that trial. The effect of RI and S4 on community structure was restricted to a few taxa. ASVs that were affected by probiotic treatment in most trials included several ASVs in the Alteromonodales, Pseudomonodales, and Cellvibrionales. Probiont RI had a similar effect on oyster larvae as described in our previous research, showing a major significant effect on Oceanospirillales. Both RI and S4 had a significant effect on the relative abundance of Alteromonodales. Differences on the effect of each probiotic on microbial community structure within each trial suggests that these probiotic differ in their mechanism of action. With the exception of Trail 1, S4 treatment did not lead to a significant change in the abundance or composition of the vibrio community in larvae, a result similar to what we had observed in previous research. Finally, a dosage error for RI in from Trial 1 showed that probiotic RI treatment at about 1000X the recommended dose lead to dysbiosis, as shown by the significantly lower microbial alpha diversity (an index derived from the number of ASVs detected in each sample) observed in the larvae exposed to RI. Dysbiosis was probably due to the larvae being overwhelmed by the high amounts of probiotic provided to the tanks, as indicated by the high percentage of bacteria in the order Bacillales, the order to which probiont Bacillus pumilus RI0695 belongs, detected in the larvae (almost 75% compared to less than 25% in other treatments). This dysbiosis probably led to the negative impacts on larval survival and growth observed in the RI treatment tanks, which completely crashed on day 7. References cited: tevick, R.J., Sohn, S., Modak, T.H., Nelson, D.R., Rowley, D.C., Tammi, K., Smolowitz, R., Markey Lundgren, K., Post, A.F., Gómez-Chiarri, M., 2019. Bacterial Community Dynamics in an Oyster Hatchery in Response to Probiotic Treatment. Front. Microbiol. 10.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Gomez-Chiarri, M. 2021. Gesti�n de enfermedades infecciosas en poblaciones silvestres y cultivadas de organismos marinos a trav�s de probi�ticos y selecci�n gen�tica. Presented at: CONIPESCA Lima 2021: XVI Congreso Nacional y IX Congreso Internacional de Ingenieria Pesquera, Lima, Peru, October 25 29, 2021 (Invited Keynote)
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Progress 09/01/19 to 08/31/20
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:A few minor changes had to be made to the experimental design due to unforeseen circumstances. The hatchery trials in July 2019 and June 2020 were terminated on day 6 due to issues with water quality (2019) and COVID19 restrictions on hatchery personnel (2021). We were not able to travel to the participating hatcheries due to COVID-19 travel restrictions, so training of hatchery personnel had to be done virtually, and samples had to be collected and shipped to URI by hatchery personnel. The 2020 trials at one of the commercial hatcheries could not be performed due to issues with water quality that led to much lower larval performance, severely impacting production at the hatchery. Therefore, the hatchery manager decided they could not participate in the trials in 2020. These trials will be done in 2021. Finally, we have not been able to present our results to stakeholders due to cancellation of the National Shellfisheries Association meeting in 2020 and the Northeast Aquaculture Conference and Exposition in January 2021, but plan to do so later in 2021 through virtual meetings. What opportunities for training and professional development has the project provided?This project involved the training of one undergraduate (summer and fall 2019) and two graduate students in research. These students gained experience in oyster larval maintenance, disease challenges, microbiological and diagnostic techniques, polymerase chain reaction, DNA and RNA extractions and library preparation for sequencing, coding in R Studio, and bioinformatics analysis for microbial community structure and function. Students also interacted with industry partners, including hatchery managers and scientists at a commercial company in aquatic animal health. These interactions included: discussion of experimental design after familiarization of the students with hatchery operations, challenges associated with larval production, and methods used to produce bacterial probiotic formulations at a commercial scale, protocol development in collaboration with all partners, training of hatchery managers in sample collection, and troubleshooting of experiments. Training also included preparation of scientific reports, including poster and oral presentations. Students are also working on the development of protocols for the effective delivery of probiotics in shellfish hatcheries in collaboration with all partners; this protocols will be disseminated to the industry as a practical manual. How have the results been disseminated to communities of interest?We were unable to share results from this research with the community due to the canceling of targeted meetings attended by the industry due to COVID19. What do you plan to do during the next reporting period to accomplish the goals?Work performed during the next reporting period will include: 1) performance of at least two additional hatchery trials at two commercial hatcheries; 2) processing and analysis of the larval and water samples collected from the 2020 and 2021 hatchery trials for 16S rRNA gene amplicon sequencing and metatranscriptomic analysis; 3) evaluation of how variability in environmental parameters and hatchery practices at each of the different hatcheries may influence the efficacy of probiotics and their effect on microbial communities in the hatchery; 4) evaluation of potential associations between of members in the pre-existing microbial communities in the hatchery on probiotic efficacy; 5) using this information to develop a manual for probiotic delivery at shellfish hatcheries; 6) dissemination of results to industry in collaboration with industry partners; and 7) preparation of technical reports and publications.
Impacts
What was accomplished under these goals?
Commercial formulations of probionts Bacillus pumilus RI0695 (RI) and Phaeobacter inhibens S4 (S4) were produced in collaboration with commercial partners. Four hatchery trials were performed in June - July 2019 and May - June 2020, in which eastern oyster, Crassostrea virginica, larvae were treated daily in the hatchery for 12 days starting on day 2 after spawning with formulations of probiotics RI and S4. Results from these trials show that there was a high amount of variability in larval performance between trials, between treatments, and between tanks within a treatment. Despite this variability, commercial formulations of the RI and S4 probiotics showed similar performance to fresh probiotics tested in prior hatchery trials, significantly increasing survival of larvae to experimental challenge with the bacterial pathogen Vibrio coralliilyticus. The commercial formulations of these probiotics were safe to the larvae when provided at the recommended dose (104 colony forming units per mL), but treatment of larvae in the hatchery with probiont RI at about 1000x the recommended dose led to significantly higher mortality in treated larvae. Probiotic treatment did not lead to significant improvements on larval performance in the absence of a bacterial challenge. Moreover, they did not confer added benefits when larvae were highly compromised by suboptimal environmental conditions in the hatchery. We used larval samples and data on larval performance collected from the hatchery trials on two selected days to determine the impact of probiotics on microbial communities in larvae. Bacterial communities were characterized by sequencing of amplicons from the V6 region of the 16S rRNA gene. Several measures were used to evaluate microbial community and structure: alpha diversity (Simpson's index), beta diversity (Bray Curtis), and differences in microbial composition at different levels, from order (broad composition) to amplified sequence variants (ASV, more detailed composition, sometimes at the species level), following methods we have previously used in hatchery trials of probiont RI (Stevick et al. 2019). Probiotic treatment (RI and/or S4) at the recommended dosage did not have a significant effect on microbial diversity in larvae in either trial. Evaluation of the effect of probiotics on community structure using Non-metric Multidimensional Scaling (NMDS) analysis of the Bray Curtis index of beta diversity showed a significant effect of trial (p= 0.001; Adonis2, permutational multivariate analysis of variance) and treatment within trial (p=0.001). These analyses also showed a significant difference between communities in control and S4 treated tanks (p= 0.005, data from both trials), control and RI (p= 0.003, trial 2 only), control and probiotic mix (p= 0.001, trial 2 only), and RI and S4 (p= 0.049, trial 2 only). Two sets of analyses were performed to identify the bacterial amplicons (ASVs) that differentiate treatments from each other. These included a principal component analysis, as well as a differential abundance analysis using the DESeq method. Although variability between the two trials was observed, several orders were consistently associated with treatment with S4 in both trials, including Alteromonadales, Bradymonadales, and Pasteurellales (more abundant in S4) and Rhizobiales (less abundant in S4). Probiont RI had a similar effect on oyster larvae as described in our previous research, showing a major significant effect on Oceanospirillales. Both RI and S4 had a significant effect on the relative abundance of Alteromonodales. Differences on the effect of each probiotic on microbial community structure suggest that these probiotic have differences in their mechanism of action. Our results of the microbial composition analysis from Trial 1 also showed that probiotic RI treatment at about 1000X the recommended dose lead to dysbiosis, as shown by the significantly lower microbial alpha diversity (an index derived from the number of ASVs detected in each sample) observed in the larvae exposed to RI. Dysbiosis was probably due to the larvae being overwhelmed by the high amounts of probiotic provided to the tanks, as indicated by the high percentage of bacteria in the order Bacillales, the order to which Bacillus pumilus RI0695 belongs, detected in the larvae (almost 75% compared to less than 25% in other treatments). This dysbiosis probably led to the negative impacts on larval survival and growth observed in this trial, since larvae depend on a healthy microbial community structure to thrive.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Hart, K., Stevick, R.J., Roberts, E.M., Noori, B., Gomez-Chiarri, M., 2019. Effects of Phaeobacter inhibens S4 probiotic on larval oyster bacterial communities. University of Rhode Island Undergraduate Research Fellows Symposium. Poster Presentations. December 2019.
- Type:
Conference Papers and Presentations
Status:
Submitted
Year Published:
2021
Citation:
Takyi, E., Roberts, E.M., Gregg, L., Chesler-Poole, A., Moss Small, J., Rowley, D.C., Nelson, D.R., Hashman, T., Giray, C., Gomez-Chiarri, M., 2021. Probiotics for eastern oyster hatcheries: commercial formulations and effect on microbial communities. Annual Meeting of the National Shellfisheries Association, March 2021.
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