Techniques and Practices for Vibrio Reduction: Mote Marine Laboratory

Home , Brevetoxin, Velvet (fish disease)

Table of Contents Executive Summary ...... 2 Approach and Methodology ...... 3 Project Phases: ...... 3 Priority #1: ...... 3 Priority #2 ...... 5 References: ...... 11 Project Deliverables ...... 14 Project Management Approach...... 14 Definition of Roles...... 14 Data Management: ...... 16 Monitoring and Reporting: ...... 17 Project Timeline: ...... 17 Detailed and Itemized Pricing ...... 18 Budget Table ...... 18 Budget Justification ...... 18 Appendix: References ...... 20 Appendix: Project Team Staffing ...... 21 Andrea Tarnecki, PhD...... 21 Dana Wetzel, Ph.D...... 21 Tracy Sherwood Ph.D...... 21 Christelle Miller ...... 21 Appendix: Company Overview ...... 23 Company Information ...... 23 Key Contact ...... 23 Brief History of Mote Marine Laboratory ...... 23 Conflicts of Interest ...... 23

Executive Summary Foodborne vibriosis has increased nearly 5-fold since 2010. As climate change is expected to increase the dominance of vibrios in the world’s oceans, the incidence of vibrio diseases also is expected to continually increase. Despite the significance of these diseases, there is no rapid diagnostic test (RDT) available for detection of the pathogenic strains of the two main vibrio pathogens in the United States, Vibrio parahaemolytics (V.p) and V. vulnificus (V.v.). Although the total abundance of these pathogens is correlated with temperature and salinity, there are numerous confounding biotic and abiotic factors that likely contribute to the presence and abundance of pathogenic V.p. and V.v. strains. Unfortunately, data regarding these relationships are scarce; therefore, risk assessments for foodborne vibriosis may be missing vital factors that contribute to increased human illnesses.

Development of an RDT and improving predictability of virulent vibrios is essential to maintaining food safety standards and reducing risk to consumers. The ability to forecast surges in the abundance of pathogenic vibrios, paired with fast, inexpensive, sensitive testing for these clinical strains, will allow for better determination of the need for post-harvest processing.

We are proposing a two-part project to address three activities identified in the ISSC Request for Proposal for techniques and practices for Vibrio reduction. The first priority will develop, optimize, and validate monoclonal antibodies (mAbs) against virulent V.p. and V.v. for generation of an RDT. The second priority proposes an environmental monitoring program that includes parameters that are often ignored, including abiotic parameters such as total nitrogen and carbon, metals analysis and sediment grain size, as well as biotic factors including bacterial and eukaryotic community characterization, total lipid class composition, and fatty acid composition. We will examine oysters, sediments, and water to provide a clear picture of the relationship between these environmental influences and the abundance of both total and pathogenic V.p. and V.v., with final results summarized by statistical approaches that identify the key factors involved in determining vibrio abundances.

As a non-profit marine research institute, we have managed grants from multiple agencies including Sea Grant and the Gulf of Mexico Research Initiative, producing data, reports, and other deliverables according to strict timelines and budgets. We have a history of obtaining research funding and, with the results generated from this proposal through the ISSC, we will pursue continued state and federal funding to fully develop an RDT and dive deeper into environmental influences on distribution, occurrence, and prevalence of virulent vibrio.

Without understanding the environmental dynamics of these significant foodborne pathogens and being able to detect pathogenic strains quickly through an RDT, reduction of illness risks from these naturally occurring bacteria is difficult. A partnership with Mote Marine Laboratory can address the priorities outlined in the ISSC Request for Proposal, clarify risk factors for raw molluscan shellfish uptake of pathogenic vibrios, provide essential data for development of an RDT for the most virulent strains of these pathogens, and generate a lasting relationship for research and novel technology development leading to the reduction of vibriosis in raw shellfish.

Approach and Methodology

Project Phases:

This project will be completed in 3 Phases (Fig. 1). Phase 1 includes initiation of the contract and receival of funds, ordering required supplies, and finalizing protocols for the collection and analysis of samples. During Phase 2, all data collection will occur for the project’s two main priorities: 1) Development, optimization, and validation of monoclonal antibodies against virulent Vibrio parahaemolyticus (V.p.) and V. vulnificus (V.v.) for generation of a rapid diagnostic test, and 2) Environmental monitoring to characterize factors influencing total and pathogenic V.p. and V.v. abundances in the eastern Gulf of Mexico. Finally, Phase 3 will consist of statistical analyses and compilation of results to produce the project deliverables.

Figure 1. Phases of the proposed project.

Priority #1: Development, optimization, and validation of monoclonal antibodies against virulent V.p. and V.v. for generation of a rapid diagnostic test.

State-of-the-art in-situ biosensor devices have become extremely important for the detection of disease in multiple biological and environmental matrices (e.g. blood, tissue, mucus, urine, water and sediments). In addition, biosensors can provide a low-cost alternative to conventional methods for detecting diseases, be done in the field with minimal sample requirements, do not require skilled labor to perform, and provide results within minutes. A rapid diagnostic test (RDT) handheld biosensor, created using specific monoclonal antibodies (mAbs) to provide sensitive detection testing for the most virulent strains of V.p. and V.v. could be a valuable tool for monitoring of these pathogens in seafood and natural environments and assessing potential risks posed to human health (Fig. 2).

Figure 2. Example of antibody based lateral flow assay technology biosensor (adapted from Creative Diagnostics).

Available disease RDTs, such as for Vibrio cholerae (Crystal® VC, CDC), employ a lateral flow technology assay (LFA) based on the principles of immunochromatography and nanotechnology in which the main components used for detection are Abs. Antibody-based biosensors work similarly to enzyme linked immunosorbent assays (ELISA) where the sensor relies on the ability of Abs (biosensor) to recognize the target of interest (the antigen; Ag) [1], such as V.p. and/or V.v. An effective Ab-based biosensor should have high specificity in a very complex medium, well- characterized binding properties, high stability, and the potential for low-cost, large-scale production [2]. Immunoassays, based on antibodies developed in vivo, have been used for decades and are still among the most important diagnostic tools used in clinical and research areas [3]. Therefore, this priority of the proposal, which addresses ISSC activity I.A.(3) “Development of methods for detection of pathogenic vibrios that are faster, less expensive and more sensitive”, is aimed at developing and validating two pairs of mAbs, one pair each for the detection of V.p. and V.v., for their future use in a multiplex RDT biosensor.

Objectives for Priority #1:

1. Develop and generate mouse anti-V.p. and anti-V.v. mAbs through collaboration with a reputable commercial mAb service (Raybiotech, Inc.). 2. Test and optimize the anti-V.p. and anti-V.v. mAbs for best pairing in a sandwich ELISA format, the same format that will be used in the development of an RDT. 3. Validate the anti-V.p. and anti-V.v. mAbs for specificity (no false positives), selectivity (no false negatives), and sensitivity (lowest limit of detection) using other Vibrio species as well as non-Vibrio bacterial species.

Activities and techniques for Priority #1:

Activity 1. Culture naturally derived V.p. and V.v. for antigen (Ag) isolation. A source of Ag is fundamental in developing mAbs and is required as the immunogen for the generation of mouse Abs. To obtain a natural source of V. parahaemolyticus and V. vulnificus-Ag for use in the immunoassays of this project, virulent strains of V.p. and V.v. identified in the literature [4–6] will be obtained and cultured in a biosecure facility under standard ATCC protocols (i.e., growth in nutrient broth/agar supplemented with NaCl at 37° for V.p. and growth in marine broth/agar at 30°). Confirmation of identity will be performed upon receival of the isolate using species-specific

4 gene detection (see Priority #2). Long-term stock cultures will be maintained in glycerol stocks at -80°C.

Activity 2. Develop mouse anti-V.p. and anti-V.v. mAbs. Previous research has demonstrated the utility in developing mAbs in mice for the specific detection of either V.p. and V.v. [7–10], thus in collaboration with Raybiotech, Inc., we will develop several species-specific mouse mAbs. Following standard mAb development protocols, 5 mice for each Vibrio species will be immunized intraperitoneally with either heat killed V. parahaemolyticus or heat killed V. vulnificus in Freund’s complete adjuvant. The mice will be given booster shots at 2, 4 and 6 weeks. After 8 weeks the mice will be screened for Ab responses to the immunogens followed by removal of splenocytes for fusion to myeloma cells. The hybrid cells will be grown in mass cultures to screen for specific mAbs. The mAbs recognized to give the best specificity to the respective Ag will be selected for production and purification for further ELISA testing and validation.

Activity 3. Test for optimal Ab pairs using ELISA and mAb validation. The RDT will employ a LFA platform, based on a sandwich ELISA format that requires two Abs, one mAb will serve as the “capture” and the other as a “detector”. However, some Abs work better as the capture and others as the detector. It is anticipated that a minimum of 5 mAbs each for V.p. and V.v. will be produced and tested for optimal pairing using traditional ELISA. To do this, several combinations of the anti-V.p. and anti-V.v. mAbs will be used as either the capture or detector. The initial ELISA screening will use heat killed V.p. and V.v.as positive Ags and PBS as well as bovine serum albumin (BSA) as negative controls. Once the optimized pairs have been identified, they will be tested for species specificity and selectivity using several Vibrio and non-Vibrio species. Specifically, the species used for this activity will be comprised of bacterial species collected and identified during Priority #2: Environmental monitoring (see below) along with species that PI Tarnecki has archived in -80°C at Mote Marine Laboratory. Approximately 20 different Vibrio species and 10 non-Vibrio species are anticipated to be used in a sandwich ELISA for determination of the mAbs’ specificity and selectivity. Testing of the samples will be in duplicate, and the ELISA repeated in triplicate to assess reproducibility of the mAbs. For diagnostic purposes, the ELISA and future RDT will be qualitative, only requiring a simple positive or negative response for the presence of V.p. and V.v. For evaluation of both responses, optical density (OD) measurements obtained by the negative controls will determine the upper limit of negativity with a 99.9% confidence limit, where the positive value will be set at the mean OD of the negative controls plus three standard deviations of the mean [11].

Priority #2: Environmental monitoring to characterize factors influencing total and pathogenic V.p. and V.v. abundances in the eastern Gulf of Mexico.

The standard for identification and enumeration of total V.p. is based on detection of the thermolabile hemolysin (tlh) which is present in all V.p. strains. Determination of virulence is based on the presence/absence of thermostable direct hemolysin (tdh) and tdh-related hemolysin (trh) genes [12]. The standard for confirmation of V.v. is based on the species-specific hemolysin (vvhA) gene. To date, there has been no gene identified that is specific to virulent strains of V.v.; however, clinical strains do generally share common genetic and biochemical characteristics. V.v. is divided into three biotypes based on phenotypic properties, with biotype 1 most often found in clinical cases [13]. Biotype 1 can be differentiated from biotype 2 by its ability to convert tryptophan to indole (indole-positive). Biotype 3 strains are also indole-positive, but have to date

5 only been isolated in Israel [13]. There are also genetic markers that show promise for identifying virulent V.v. strains. Rosche et al. [14] determined that there are two groups of virulence correlated gene (vcg), with vcg type C found in 90% of clinical strains and vcg type E found in 93% of environmental strains. Nilsson et al. [15] examined differences in the 16S ribosomal RNA gene (16S) and reported 94% of non-clinical isolates were 16S type A whereas 76% of clinical strains were 16S type B, with 94% of clinical fatalities associated with 16S type B. Therefore, most clinical strains are identified as 16S type B, vcg type C [16]. Supporting this relationship, this clinical genotype was increased in oysters from Galveston Bay, TX in July through September 2010, which coincides well with the foodborne vibriosis trends for that year [17], whereas the environmental genotypes (16S type A, vcg type E) dominated the rest of the year [18].

V.p. and V.v. concentrations are correlated with temperature and salinity, with both species being more abundant in warm temperatures and brackish salinities; however abundances are influenced by numerous abiotic and biotic parameters which are not fully understood [16]. Studies report plankton as a reservoir of both vibrio species [13, 19, 20], and more specifically, increased abundances of diatoms are associated with higher vibrio counts [21, 22]. Of more importance from a human health risk perspective, strains of V.p. and V.v. with pathogenic genotypes were positively correlated with copepod abundance [23]. Additionally, bacterial interactions within the environment are likely to regulate vibrio populations due to the presence of bacteria that are obligate bacteriovores which directly ingest vibrio [24], and bacterial species that produce antimicrobials against these pathogens [25–27]. Despite several studies that have characterized the bacterial communities (prokaryotic microbiome) associated with bivalves [28, 29], to our knowledge only one study has correlated that community with vibrio counts [30]. Marcinkiewicz et al. [30] did not find a relationship between the overall community and grouped (i.e., medium and high) V.p. concentrations; however, this method of analysis likely missed specific bacterial taxa that are correlated with V.p. concentrations. In addition, there was no data relating microbiome relationships with pathogenic V.p. As planktonic communities (eukaryotic microbiomes) play an important role as a reservoir of these pathogens and bacterial interactions can alter vibrio populations, these biotic parameters are essential in understanding total and pathogenic vibrio dynamics in the marine environment.

Metals are often overlooked as abiotic parameters that may influence vibrio abundances. Iron is often a limiting nutrient in the marine environment, but it is vital for the persistence of vibrio species [31]; as such, vibrios have iron-sequestering systems to help them obtain this limited nutrient from the environment. Iron availability plays a large role in virulence, as patients with high serum iron have increased susceptibility to vibriosis [32, 33]. Iron participates in regulation of quorum-sensing systems that alter expression of toxins in V.v. [34] and impacts cytotoxicity in V.p. [35]. Calcium also seems to play an important role in regulating behaviors in these pathogens. Biofilm formation and virulence is controlled at least in part by calcium in both pathogens [35, 36]. This element may impact their ability to attach to plankton [37] and therefore determine the abundance of vibrio that are ingested by filter-feeding bivalves. It is unknown if iron and calcium influence environmental and clinical vibrio strains differently in the environment; thus, these metals will be investigated in the proposed work.

The priority of the proposal that addresses ISSC activities I.A.(1) “Evaluate environmental factors such as temperature, salinity, sediment type, etc. which could be controlling distribution,

6 occurrence, and prevalence of total and pathogenic strains of vibrios”, and I.A.(2) “Assess the environmental factors that influence growth and distribution of pathogenic strains of vibrios” is aimed at environmental monitoring of total and pathogenic V.p. and V.v. and often overlooked biotic and abiotic parameters in oysters, sediments, and water to increase understanding of pathogenic vibrio dynamics in the eastern Gulf of Mexico, a high vibriosis risk region of the United States.

Objectives for Priority #2:

1. Quantify differential abundances of total and pathogenic V.p. and V.v. in natural environments in the eastern Gulf of Mexico throughout a year. 2. Characterize the bacterial and eukaryotic microbiomes of oysters, sediment, and water in natural environments. 3. Record abiotic and biotic sediment and water parameters in oyster habitats. 4. Generate models relating total and pathogenic V.p. and V.p. to abiotic and biotic environmental parameters and bacterial and eukaryotic microbiomes.

Activities and techniques for Priority #2:

Activity 1. Sample collection. Samples will be collected from two locations in western Florida within 1-hr driving distance of Sarasota. The proximity of sampling locations is necessary as microbial composition can change in a matter of hours for copiotrophic microbes such as the Vibrio (class Gammaproteobacteria) [38], so samples must be processed immediately to prevent these microbial changes from influencing results. Collection sites will include “Conditionally Approved” areas in the Tampa Bay (Manatee County) and Charlotte Harbor (Lee County) regions (Fig. 3). These regions had relatively high rates of vibriosis infections in 2017 in comparison to the rest of the state [39], making them ideal locations for these examinations.

Tampa Bay Manatee County

Charlotte Harbor Lee County

Figure 3. Map of sampling areas in the proposed project.

Sampling will occur at each site every other week from mid-July 2020 until mid-June 2021 (22 sampling points per location). Water quality parameters (temperature, salinity, pH, dissolved oxygen, turbidity) will be measured using a multiparameter meter. Oysters (12 individuals per

7 sample), sediment, and water will be collected at each sampling point. Samples will be transported to the laboratory on ice and processed immediately upon arrival.

Activity 2. Total and pathogenic V.p. and V.v. enumeration and determination. Total and pathogenic V.p. and V.v. will be determined using the most probably number polymerase chain reaction (MPN-PCR) method as described within the FDA Bacteriological Analytical Manual Chapter 9 Vibrio [12]. Briefly, a 1:2 dilution for each sample type (oyster, sediment, water) will be prepared in sterile phosphate buffered saline (PBS). For oyster samples, 12 individuals will be pooled into one sample by thorough homogenization using a sterilized blender. Following homogenization, serial dilutions of each sample will be prepared in PBS. Alkaline peptone water (APW) will be inoculated with each dilution and incubated at 35°C overnight. The following day, a 3 mm loop from the top 1 cm of the three highest dilutions showing growth via turbidity will be plated onto thiosulfate citrate bile salts sucrose (TCBS) agar incubated at 35°C or modified cellobiose polymyxin colistin (mCPC) incubated at 39°C for isolation of V.p. and V.v., respectively. Presumptive colonies (round, opaque, and green-blue for V.p. and round, flat, opaque, and yellow for V.v.) will be collected in pure culture and tested for confirmation of identity using PCR. Total and pathogenic V.p. and V.v. will be determined using the genes and primers [14, 40– 43] indicated in Table 1. Upon confirmation, MPN/g of oyster and sediment and MPN/mL of water will be determined using previously prepared tables (Appendix II of FDA BAM: Vibrio). The potential pathogenicity of V.v. isolates will be further determined based on 1) presence of a capsule [12] using colony morphology and a negative stain, and 2) indole production (biotype 1) using the indole spot test.

Activity 3. Bacterial and eukaryotic microbiome characterization. A second water sample will be collected from each site at each sampling point and filtered through a 0.2 µm polycarbonate filter to collect microorganisms for microbiome analysis. A small (approximately 250 mg) subsample of the original oyster and sediment homogenate will also be processed for microbiome composition. Samples will be subjected to whole community DNA extraction using previously established protocols [44]. DNA will be sequenced using standard protocols by the Earth Microbiome Project (www.earthmicrobiome.org, Accessed 12 May 2020). For characterization of the prokaryotic microbiome, the 16S small subunit (SSU) rRNA is sequenced, whereas the 18S SSU rRNA is used for eukaryotic communities. Sequences will be processed using standard methods [44, 45].

Sequence analysis will be performed using Mothur [46] and R [47]. For measurements of alpha- diversity, microbe species richness (number of microbial species) and evenness (Shannon evenness index) will be determined for each community and compared using ANOVA, with data transformation where necessary to meet assumptions. Beta-diversity (microbial differences among samples) will be investigated using multiple statistical analyses. Relationships between water quality parameters and microbial taxa will be determined using partial least square (PLS) regression in R. The core microbiome for each sample type will be determined using the core function in the ‘microbiome’ R package [48]. The core microbiome is defined as the set of microbial species found in 50-100% of samples with a relative abundance of at least 0.01%.

Activity 4. Water and sediment chemistry. Water samples will be collected and analyzed for suspended particulate matter will be determined using previously described methods [49].

Table 1. Primers for isolate confirmation. Pathogen Gene Primers Reference

Total tdh tdh-1 5’-CCATCTGTCCCTTTTCCTGCC-3’ [40] V. tdh-4c 5’-CCACTACCACTCTCATATGC-3’ parahaemolyticus Pathogenic tlh L-tl 5’-AAAGCGGATTATGCAGAAGCACTG-3’ [42] V. R-tl 5’-GCTACTTTCTAGCATTTTCTCTGC-3’ parahaemolyticus

trh L-trh 5’-TTGGCTTCGATATTTTCAGTATCT-3’ [42] R-trh 5’-CATAACAAACATATGCCCATTTCCG- 3’ Total vvhA Vvh-785F 5’-CCGCGGTACAGGTTGGCGCA-3’ [41] V. vulnificus Vvh-1303R 5’-CGCCACCCACTTTCGGGCC-3’

Pathogenic vcgC P1 5’-AGCTGCCGATAGCGATCT- 3’ [14] V. vulnificus P3 ‘5-CGCTTAGGATGATCGGTG-3’

vcgE P2 5’-CTCAATTGACAATGATCT-3’ [14] P3 ‘5-CGCTTAGGATGATCGGTG-3

16S-A 16S-AF 5’-CATGATAGCTTCGGCTCAAA3’ [43] 16S-AR 5’-TAGTGCTATTAACACTACCAC-3’

16S-B 16S-BF 5’-CATGATGCCTACGGGCCAAA-3’ [43] 16S-BR 5’-TGCCGCTATTAACGACACCAC-3’

All sediment samples are analyzed in the laboratory for grain size, carbonate content, and total organic matter (TOM) by standard, accepted methods. Grain size is determined by initially wet sieving the sample through a 63 µm screen. The sand- and gravel-size (>63 µm) fraction is then analyzed by settling tube [50], and the percentage of each size interval within the sand/gravel fraction is calculated using the settling rate (Stoke’s Law). The fine-size (<63 µm) fraction is determined by pipette [51], which is also based upon settling rates, and the percentages of silt- and clay-size sediments are calculated. Data from both analyses are combined, calculations made, and results are expressed as mean grain size, %gravel, %sand, %silt ,%clay and %mud (silt+clay). Note, mean grain size is expressed in phi (ø ) units, where ø=-log2 (grain dia. in mm). Hence, the larger the ø value the finer the grain size. For reference, the sand/mud boundary is 4ø.

Calcium carbonate content is determined by the acid leaching method according to the procedure described in Milliman [52]. A 10% hydrochloric acid (HCL) solution is added to a pre-weighed sample. After the reaction reaches completion (i.e. all calcium carbonate is dissolved) each sample is washed four times, dried and weighed again. The difference is weight represents the calcium carbonate fraction.

Total organic carbon (TOC) is determined by loss on ignition (LOI) according to Dean [53]. Approximately 1 g of insoluble residue (i.e. the sediment remaining after acid leaching) is ashed in a muffle furnace at 550˚ C for at least 2.5 hours. The sample is weighed again and the difference in weight represents the total organic matter in the sample.

Water samples will be analyzed for total organic carbon (TOC), total nitrogen (TN), lipid class composition and fatty acid analyses. Total nitrogen (TN) will be determined in water samples using pyrolysis gas chromatography coupled to a quadrupole mass spectrometer [54]. Lipid class and fatty acid composition will be characterized after liquid-liquid solvent extraction of water subsamples for each analysis. For lipid class composition, water extracts are analyzed, by thin layer chromatography/flame ionization detection using an Iatrascan [55]. Fatty acids will be identified by first derivatizing to picolinyl esters then analyzed for saturated and unsaturated fatty acid compounds with gas chromatography-mass spectrometry [56].

Activity 5. Relating total and pathogenic V.p. and V.v. to environmental parameters. Vibrio groups from MPN-PCR results will be characterized as in Table 2.

Table 2. Vibrio groups analyzed in this study. Vibrio Characterization Results V.p. Total tlh+ Pathogenic tdh+ trh+ V.v. Total vvhA+ Pathogenic vcg type C 16S type B Biotype 1 (indole-positive)

A data matrix will be formed for V.p. and V.v. groups described in the table above at each sampling point for comparison against bacterial and eukaryotic microbiomes as well as environmental measurements (temperature, salinity, pH, dissolved oxygen, turbidity, suspended particulate matter, iron, calcium, sediment size, lipid class, and fatty acid composition). Relationships between vibrio groups and measured variables will be determined using partial least square (PLS) regression and visualized using clustered image maps (CIM; ‘heat maps’) in R. The microbial taxa that are most positively and negatively correlated with each vibrio species will be identified at various taxonomic levels. To confirm relationships with environmental parameters, generalized linear models (GLM) will be performed as previously described [57]. Briefly, initial correlations between each factor and each bacterial group will be performed. If a parameter is correlated (r > 0.3, P < 0.05) with any of the groups, it will be included in GLMs. GLMs will be performed for all bacterial groups using the same group of factors for consistency, and interactions between parameters will also be examined. The final results from Activity 5 will be correlations of microbial taxa and environmental parameters with each vibrio group listed above and estimated coefficients for the model of each vibrio group and measured environmental parameters, identifying a subset of factors that are important for predicting total and pathogenic vibrio levels in the natural environment.

References: 1. Saerens D, Huang L, Bonroy K, Muyldermans S (2008) Antibody Fragments as Probe in Biosensor Development. Sensors 8:4669–4686. doi: 10.3390/s8084669 2. Holt LJ, Enever C, de Wildt RM, Tomlinson IM (2000) The use of recombinant antibodies in proteomics. Curr Opin Biotechnol 11:445–449. 3. Nelson PN, Reynolds GM, Waldron EE, et al. (2000) Monoclonal antibodies. Mol Pathol 53:111–117. 4. Thiaville PC, Bourdage KL, Wright AC, et al. (2011) Genotype is correlated with but does not predict virulence of Vibrio vulnificus biotype 1 in subcutaneously inoculated, iron dextran-treated mice. Infect Immun 79:1194–1207. doi: 10.1128/IAI.01031-10 5. Vongxay K, Wang S, Zhang X, et al. (2008) Pathogenetic characterization of Vibrio parahaemolyticus isolates from clinical and seafood sources. Int J Food Microbiol 126:71– 75. doi: 10.1016/j.ijfoodmicro.2008.04.032 6. Yeung PSM, Hayes MC, DePaola A, et al. (2002) Comparative phenotypic, molecular, and virulence characterization of Vibrio parahaemolyticus O3:K6 isolates. Appl Environ Microb 68:2901–2909. doi: 10.1128/AEM.68.6.2901-2909.2002 7. Kawatsu K, Ishibashi M, Tsukamoto T (2006) Development and evaluation of a rapid, simple, and sensitive immunochromatographic assay to detect thermostable direct hemolysin produced by Vibrio parahaemolyticus in enrichment cultures of stool specimens. J Clin Microbiol 44:1821–1827. doi: 10.1128/JCM.44.5.1821-1827.2006 8. Jadeja R, Janes ME, Simonson JG (2015) Development of rapid and sensitive antiflagellar monoclonal antibody based lateral flow device for the detection of Vibrio vulnificus fromoyster homogenate. Food Control 56:110–113. doi: 10.1016/j.foodcont.2015.03.019 9. Zhao Y, Wang H, Zhang P, et al. (2016) Rapid multiplex detection of 10 foodborne pathogens with an up-converting phosphor technology-based 10-channel lateral flow assay. Sci Rep 6:1–8. doi: 10.1038/srep21342 10. Yonekita T, Morishita N, Arakawa E, Matsumoto T (2020) Development of a monoclonal antibody for specific detection of Vibrio parahaemolyticus and analysis of its antigen. J Microbiol Methods 173:105919. doi: https://doi.org/10.1016/j.mimet.2020.105919 11. Crowther JR (1995) ELISA. Theory and Practice. Methods Mol Biol Vol 42. Humana Press, Totowa, NJ, p 223 12. Kaysner CA, DePaola Jr. A, Jones J (2004) Vibrio. Bacteriol Anal Man. p Chapter 9 13. Maugeri TL, Carbone M, Fera MT, Gugliandolo C (2006) Detection and differentiation of Vibrio vulnificus in seawater and plankton of a coastal zone of the Mediterranean Sea. Res Microbiol 157:194–200. doi: 10.1016/j.resmic.2005.06.007 14. Rosche TM, Yano Y, Oliver JD (2005) A rapid and simple PCR analysis indicates there are two subgroups of Vibrio vulnificus which correlate with clinical or environmental isolation. Microbiol Immunol 49:381–389. doi: 10.1111/j.1348-0421.2005.tb03731.x 15. Nilsson WB, Paranjype RN, DePaola A, Strom MS (2003) Sequence polymorphism of the 16S rRNA gene of Vibrio vulnificus is a possible indicator of strain virulence. J Clin Microbiol 41:442–446. doi: 10.1128/JCM.41.1.442-446.2003 16. FAO/WHO (2020) Risk assessment tools for Vibrio parahaemolyticus and Vibrio vulnificus associated with seafood. 17. CDC (2013) National Enteric Vibrio Cases Reported to CDC, 2010. 18. Cam S (2016) Environmental influences on virulence factors in Vibrio vulnificus. doi: 10.1017/CBO9781107415324.004

19. Wright AC, Hill RT, Johnson JA, et al. (1996) Distribution of Vibrio vulnificus in the Chesapeake Bay. Appl Environ Microb 62:717–724. doi: 10.1128/aem.62.2.717-724.1996 20. Sarkar BL, Nair GB, Sircar BK, Pal SC (1983) Incidence and level of Vibrio parahaemolyticus associated with freshwater plankton. Appl Environ Microb 46:288–290. doi: 10.1128/aem.46.1.288-290.1983 21. Vezzulli L, Brettar I, Pezzati E, et al. (2012) Long-term effects of ocean warming on the prokaryotic community: evidence from the vibrios. ISME J 6:21–30. doi: 10.1038/ismej.2011.89 22. Main CR, Salvitti LR, Whereat EB, Coyne KJ (2015) Community-Level and species- specific associations between phytoplankton and particle-associated Vibrio species in delaware’s inland bays. Appl Environ Microb 81:5703–5713. doi: 10.1128/AEM.00580- 15 23. Turner JW, Malayil L, Guadagnoli D, et al. (2014) Detection of Vibrio parahaemolyticus, Vibrio vulnificus and Vibrio cholerae with respect to seasonal fluctuations in temperature and plankton abundance. Environ Microbiol 16:1019–1028. doi: 10.1111/1462- 2920.12246 24. Chen H, Athar R, Zheng G, Williams HN (2011) Prey bacteria shape the community structure of their predators. ISME J 5:1314–1322. doi: 10.1038/ismej.2011.4 25. Priyaja P, Jayesh P, Correya NS, et al. (2014) Antagonistic effect of Pseudomonas aeruginosa isolates from various ecological niches on Vibrio species pathogenic to crustaceans. J Coast Life Med 2:76–84. doi: 10.12980/jclm.2.2014j30 26. Kamarudheen N, George CS, Pathak S, et al. (2015) Antagonistic activity of marine Streptomyces sp. on fish pathogenic vibrio species isolated from aquatic environment. Res J Pharm Technol 8:1529–1533. doi: 10.5958/0974-360X.2015.00273.5 27. Wu HJ, Sun L Bin, Li CB, et al. (2014) Enhancement of the immune response and protection against Vibrio parahaemolyticus by indigenous probiotic Bacillus strains in mud crab (Scylla paramamosain). Fish Shellfish Immun 41:156–162. doi: 10.1016/j.fsi.2014.08.027 28. Pierce ML, Ward JE (2018) Microbial Ecology of the Bivalvia, with an Emphasis on the Family Ostreidae. J Shellfish Res 37:793–806. doi: 10.2983/035.037.0410 29. Rubiolo JA, Botana LM, Martínez P (2019) Insights into Mussel Microbiome. Microb Communities Aquac Ecosyst. pp 95–120 30. Marcinkiewicz AL, Schuster BM, Jones SH, et al. (2017) Bacterial community profiles and Vibrio parahaemolyticus abundance in individual oysters and their association with estuarine ecology. BioRxiv 156851. 31. Payne SM, Mey AR, Wyckoff EE (2016) Vibrio Iron Transport: Evolutionary Adaptation to Life in Multiple Environments. Microbiol Mol Biol R 80:69–90. doi: 10.1128/mmbr.00046-15 32. Thompson FL, Iida T, Swings J (2004) Biodiversity of vibrios. Microbiol Mol Biol R 68:403–431. doi: 10.1128/MMBR.68.3.403 33. Li G, Wang MY (2020) The role of Vibrio vulnificus virulence factors and regulators in its infection-induced sepsis. Folia Microbiol (Praha) 65:265–274. doi: 10.1007/s12223- 019-00763-7 34. Hwang Kim I, Wen Y, Son JS, et al. (2013) The fur-iron complex modulates expression of the quorum-sensing master regulator, smcr, to control expression of virulence factors in vibrio vulnificus. Infect Immun 81:2888–2898. doi: 10.1128/IAI.00375-13

35. Gode-Potratz CJ, Chodur DM, McCarter LL (2010) Calcium and iron regulate swarming and type III secretion in vibrio parahaemolyticus. J Bacteriol 192:6025–6038. doi: 10.1128/JB.00654-10 36. Garrison-Schilling KL, Grau BL, McCarter KS, et al. (2011) Calcium promotes exopolysaccharide phase variation and biofilm formation of the resulting phase variants in the human pathogen Vibrio vulnificus. Environ Microbiol 13:643–654. doi: 10.1111/j.1462-2920.2010.02369.x 37. Williams TC, Ayrapetyan M, Oliver JD (2015) Molecular and physical factors that influence attachment of vibrio vulnificus to Chitin. Appl Environ Microb 81:6158–6165. doi: 10.1128/AEM.00753-15 38. Fuhrman JA, Cram JA, Needham DM (2015) Marine microbial community dynamics and their ecological interpretation. Nat Rev Microbiol 13:133–146. doi: 10.1038/nrmicro3417 39. Florida Department of Health (2017) Section 1 : Data Summaries for Common Reportable Diseases / Conditions. Florida Annu Morb Stat Rep. pp 15–94 40. Nishibuchi M, Hill WE, Zon G, et al. (1986) Synthetic oligodeoxyribonucleotide probes to detect Kanagawa phenomenon-positive Vibrio parahaemolyticus. J Clin Microbiol 23:1091–1095. doi: 10.1128/jcm.23.6.1091-1095.1986 41. Hill WE, Keasler SP, Trucksess MW, et al. (1991) Polymerase chain reaction identification of Vibrio vulnificus in artificially contaminated oysters. Appl Environ Microb 57:707–711. doi: 10.1128/aem.57.3.707-711.1991 42. Bej AK, Patterson DP, Brasher CW, et al. (1999) Methods Detection of total and hemolysin-producing Vibrio parahaemolyticus in shellfish using multiplex PCR amplification of tl , tdh and trh. 36:215–225. 43. Senoh M, Miyoshi SI, Okamoto K, et al. (2005) The cytotoxin-hemolysin genes of human and eel pathogenic Vibrio vulnificus strains: Comparison of nucleotide sequences and application to the genetic grouping. Microbiol Immunol 49:513–519. doi: 10.1111/j.1348- 0421.2005.tb03756.x 44. Tarnecki AM, Brennan NP, Schloesser RW, Rhody NR (2018) Shifts in the skin- associated microbiota of hatchery-reared common snook Centropomus undecimalis during acclimation to the wild. Microb Ecol 77:770–781. 45. Tarnecki AM, Wafapoor M, Phillips RN, Rhody NR (2019) Benefits of a Bacillus probiotic to larval fish survival and transport stress resistance. Sci Rep 9:4892. doi: 10.1038/s41598-019-39316-w 46. Schloss PD, Westcott SL, Ryabin T, et al. (2009) Introducing mothur: Open-source, platform-independent, community-supported software for describing and comparing microbial communities. Appl Environ Microb 75:7537–7541. doi: 10.1128/AEM.01541- 09 47. R Core Team (2016) R: A language and environment for statistical computing. 48. Leo L, Sudarshan S (2017) Tools for microbiome analysis in R. Microbiome package version 2.1.1. URL: http://microbiome.github.com/microbiome. 49. Johnson CN, Bowers JC, Griffitt KJ, et al. (2012) Ecology of vibrio parahaemolyticus and vibrio vulnificus in the coastal and estuarine waters of Louisiana, Maryland, Mississippi, and Washington (United States). Appl Environ Microb 78:7249–7257. doi: 10.1128/AEM.01296-12 50. Gibbs RJ (1974) A settling tube system for sand-size analysis. J Sediment Petrol 44:583– 588.

51. Folk RL (1965) Petrology of sedimentary rocks. 52. Milliman JD (1974) Marine carbonates. 53. Dean WE (1974) Determination of carbonate and organic matter in calcareous sediments and sedimentary rocks by loss on ignition; comparison with other methods. J Sediment Res 44:242–248. 54. Nakajima K (1986) Simultaneous determination of total organic carbon and total nitrogen in waters by pyrolysis-gas chromatography-mass spectrometry. Water Res 20:233–235. 55. Sloan, C.A., B.F. Anulacion, K.A. Baugh, J.L. Bolton, D. Boyd, R.H. Boyer, D.G. Burrows, D.P. Herman, R.W. Pearce and GMY (2014) Northwest Fisheries Science Center’s Analyses of Tissue, Sediment, and Water Samples for Organic Contaminants by Gas Chromatography/Mass Spectrometry and Analyses of Tissue for Lipid Classes by Thin Layer Chromatography/Flame Ionization Detection. 56. Dubois N, Barnathan G, Gouygou JP, Bergé JP (2009) Gas chromatographic behavior of fatty acid derivatives for mass spectrometry on low-polarity capillary columns. Eur J Lipid Sci Technol 111:688–697. doi: 10.1002/ejlt.200800148 57. Black RA, Taraba JL, Day GB, et al. (2014) The relationship between compost bedded pack performance, management, and bacterial counts. J Dairy Sci 97:2669–2679. doi: 10.3168/jds.2013-6779

Project Deliverables

• Two anti-V. parahaemolyticus mAbs along with their hybridoma cell lines that can be used as a perpetual source of the developed mAbs for future use in an RDT. • Two anti-V. vulnificus mAbs along with their hybridoma cell lines that can be used as a perpetual source of the developed mAbs for future use in an RDT. • Environmental monitoring metadata including: total and pathogenic V.v. and V.p. abundances, temperature, salinity, pH, dissolved oxygen, turbidity, suspended particulate matter, total nitrogen, total organic carbon, total lipid class composition, fatty acid composition, and bacterial microbiomes and eukaryotic microbiomes. • Statistical models indicating significant relationships between total and pathogenic V.p. and V.v. and measured environmental parameters. • Final report to ISSC.

Project Management Approach

The following outlines the Mote Marine Laboratory project management approach for completion of deliverables specified in this proposal with support from ISSC.

Definition of Roles. Table 2 includes assigned work groups, the phases and activities for which they will be responsible, and their deliverables.

Mote Marine Laboratory Work Group Tasks & Deliverables

Target Deliverable Work Group Phases/Activities Major Deliverables Completion Date Sponsored Programs Phase 1 • Project contract • 06-30-2020 Office Environmental Laboratory Phase 2, Priority • 2 anti-V.p mAbs • 06-30-2021 for Forensics #1, Activities 1-3 • V.p. hybridoma cell lines • 06-30-2021 • 2 anti-V.v. mAbs • 06-30-2021 • V.v. hybridoma cell lines • 06-30-2021 Phase 2, Priority • Water chemistry data • 07-01-2021 #2, Activity 4 Marine Microbiology Phase 2, Priority • Environmental metadata • 07-01-2021 #2, Activities 1-3, 5 • Statistical models Phase 3 • Final report • 08-01-2021 • 08-20-2021

Sponsored Programs Office: Mote Marine Laboratory’s Sponsored Programs Office is responsible for stewardship of funding contracts and funds. Environmental Laboratory for Forensics. The most fundamental research done in the ELF lab is the quantification of environmental levels of organic contamination from petroleum, PCBs, flame retardants, pesticides, and other industrial contaminants; however it is also strongly engaged in developing novel techniques for understanding cause and effects of stressors on species, populations, and overall environmental health. Towards this end, they develop and employ a variety of cutting-edge approaches based on human health models. The lab holds a patent for MML which uses a biomarker to determine the sex of a fish using blood and is currently working on other quick test methodologies. The ELF team includes: • Dr. Dana Wetzel, Senior Scientist, Program Manager: Dr. Wetzel will oversee all operations in ELF. • Dr. Tracy Sherwood, Staff Scientist: Dr. Sherwood will lead Phase 2 Priority #1 Activities, including development, optimization, and validation of anti-V.p. and anti-V.v. mAbs. • Christelle Miller, Senior Chemist: Mrs. Miller will perform chemical analyses on sediment and water samples, including total nitrogen, total organic carbon, lipid class composition, and fatty acid analysis. Marine Immunology: Research in the Marine Immunology Program is focused on basic and applied research on the health and immune systems of marine vertebrates. The program is providing new knowledge about how certain environmental stressors affect the health of marine species. Within this program, Dr. Andrea Tarnecki’s research focuses on the relationship between microbiomes, the environment, and host animals. She has previously published on post-harvest processing methods in oysters for V.p. and V.v. depuration. Current research with Vibrio includes

15 description of Vibrio species composition of marine aquaculture, antagonism studies with probiotic bacteria, and discovery of secondary metabolites. The Marine Immunology team includes: • Dr. Andrea Tarnecki, Staff Scientist: Dr. Tarnecki will act as Principal Investigator (PI) and oversee all aspects of project management, including experimental design, sample collection, Vibrio quantification and isolate description, isolate maintenance, microbiome data, data analysis, and the final report. She has 10 years’ experience in marine microbiology, including culture-based and culture-independent methodologies and field collection. The entire team will be responsible for contributing to and reviewing deliverables to ensure high quality and accuracy.

Data Management: PI Tarnecki will be responsible for ensuring that the data collection is carried out in accordance with the data management plan described herein. The Mote Marine Laboratory librarian will be responsible for archiving project reports and publications on behalf of the project team.

Samples to be collected include oysters, sediment, and water and filters for Vibrio quantification and pure culture maintenance and microbiome analysis, as well as DNA isolated from these sample types. Data collected will include environmental measurements (temperature, salinity, pH, dissolved oxygen, turbidity, suspended particulate matter, total nitrogen, total organic carbon, lipid class composition, fatty acids), total and pathogenic V.p. and V.v. counts, sequencing data (raw and processed), photographs, and a final report. Samples will be collected from oysters, sediment, and water in natural environments. Environmental parameters will be measured using multiparameter multimeters. Bacterial counts will be determined from MPN-PCR couple with plating on selective and differential culture media. Sequencing data will be collected using the Illumina MiSeq sequencing platform. Results from collected data will be collected into a metadata file and compiled into a final report.

All environmental measurements and Vibrio counts will be recorded in laboratory notebooks documenting experimental design, methodology, and results. Notebooks will be labeled by date and be detailed enough as to allow future researchers to duplicate experiments. Collected environmental data, bacterial counts, and processed sequencing data (Operational Taxonomic Unit (OTU) and/or Amplicon Sequencing Variant (ASV) tables), will be stored digitally in spreadsheet tables. Raw sequencing data will be available in BAM format and uploaded to the Sequence Read Archive (https://www.ncbi.nlm.nih.gov/sra) upon completion of the project. Processed microbiome OTUs/ASVs will be uploaded into GenBank. Images will be stored in standard forms such as JPEG, PNG, or TIFF. Reports will be available in PDF through Mote Marine Laboratory’s technical report repository. Other data collected will be shared with researchers upon request following the completion of the project.

Data and metadata collected during the project will be made available to the public immediately upon completion of the final report, with additional data available upon approval from the project team. Re-use and re-distribution requests will be considered on a case-to-case basis and with guidance from ISSC. Mote Marine Laboratory recognizes that the legitimate proprietary concerns of research, sponsors, and the effective protection and commercial application of inventions and

16 works may require confidentiality and limited delays in the publication of certain information or the sharing of data.

Extracted nucleic acids and collected Vibrio isolates will be maintained onsite and be available upon request. All electronic data will be stored on a minimum of two external hard drives and continually backed up to such drives. One drive will be kept onsite, and the other offsite. The final report will be retained by the Mote Marine Laboratory library for an indefinite period not less than 5 years. Hard copies of measurement data described above will be retained in laboratory notebooks onsite. Sequencing data will be additionally maintained in the Sequence Read Archive and GenBank. Links to this data will be included in resulting publications. Additional data will be available to other researchers upon request.

Monitoring and Reporting: There will be a shared metadata file available in Google Docs to which all members of the team will have access. As data is generated, it will be entered into the file. The PI will ensure timely update of this file.

The PI will maintain an Excel workbook with all required Phases and Activities linked the project schedule shown above. On a bi-weekly basis, the PI will collect information from the ELF and Marine Immunology programs regarding costs, schedules, and progress towards each specified piece of the project. Progress will be updated in work schedule. A second spreadsheet will be used to monitor costs associated with the project and ensure the team stays on budget. This constant communication is to ensure early identification of any issues that may impact the schedule or success of the project and quickly apply corrective actions to remediate these issues.

Project Timeline: 2020 2021

Activity Jun Jul Aug Sep Oct Nov Dec Jan Feb Mar Apr May Jun Jul Aug Sep PHASE 1 Notification of Award Project Contract Planning and Supplies Protocol Finalization PHASE 2 P R Priority #1: mAb Development O Activity 1 J Activity 2 E Activity 3 C Priority #2: Environmental Monitoring T Activity 1 E Activity 2 N Activity 3 D Activity 4 Activity 5 PHASE 3 Final Report Preparation Submission of Deliverables

Detailed and Itemized Pricing

Budget Table

ISSC Match Total Salaries PI Tarnecki $18,900 $0 $18,900 Co-PI Wetzel $12,523 $11,927 $24,450 Co-PI Sherwood $13,869 $0 $13,869 Chemist Miller $11,981 $0 $11,981 A. Total Salaries $57,273 $11,927 $69,200 B. Fringe $20,475 $4,264 $24,739 C. Travel $2,530 $0 $2,530 D. Supplies $25,610 $0 $24,610 E. Testing & Analysis $33,680 $0 $33,680 F. Other $0 $8,000 $8,000 G. Total Direct $139,568 $24,191 $163,759 H. Indirect Charges $0 $70,445 $70,445 I. Matching funds $0 $94,636 $94,636 J. Totals $139,568 $94,636 $234,204 Percent Match 67.8%

Budget Justification

A. Salaries: $57,273 • Dr. Andrea Tarnecki, Staff Scientist, 4.5 mos: Dr. Tarnecki will act as Principal Investigator (PI) and oversee all aspects of project management, including experimental design, sample collection, Vibrio quantification and isolate description, isolate maintenance, microbiome data, data analysis, data management, and the final report. • Dr. Dana Wetzel, Senior Scientist, 0.5 mos: Dr. Wetzel (Co-PI) will oversee all operations in the Environmental Lab for Forensics. She will also provide 0.5 mos as match (see below). • Dr. Tracy Sherwood, Staff Scientist, 3 mos: Dr. Sherwood (Co-PI) will lead Phase 2 Priority #1 Activities, including development, optimization, and validation of anti-V.p. and anti-V.v. mAbs. She will be responsible for all communication and collaboration with Raybiotech, Inc. She will be responsible for finding optimal mAb pairs and validating them against other bacterial species. • Christelle Miller, Staff chemist, 3 mos: Mrs. Miller will perform all chemical analyses and compile all resulting data from total nitrogen, total organic carbon, lipid classes, and fatty acid analyses.

B. Fringe benefits: $20,475 Mote Marine Laboratory’s fringe rate is 35.75% of salaries and covers sick leave, vacation, and holiday pay; partial support of health insurance, workers compensation, etc.

C. Travel: $2,530 Travel for sample collection, calculated at 44 trips, 100 miles per trip, at $0.575 per mile.

D. Research Supplies: $25,610 • Sampling and sample processing: Bottles, microcentrifuge tubes, DNA extraction kits, polycarbonate filters, GF/F filters, standard and filter pipet tips, filtration apparati for water filtration, sterilizable blenders • Essential laboratory maintenance: Pipettor calibrations, biosafety cabinet hood check, HEPA filters for DNA extraction clean room, DI water filters • Culturing supplies: Petri dishes, media components (TCBS, yeast extract, peptone, NaCl, beef extract, agar, bromothymol blue, cresol red, ethanol, cellobiose, CPC antibiotics), 96- well plates and sealers, culture loops, Nigrosin stain, indole spot test supplies • PCR and electrophoresis: PCR master mix, molecular grade water, primers, agarose, DNA ladder, ethidium bromide • Virulent Vibrio isolates • ELISA supplies: well plates, plate covers, buffers, mAbs, chromogenic substrate • Office: telephone, printing, photocopying • Postage/shipping: for sending samples for microbiome sequencing, sediment analysis, mAb development

E. Testing & Analysis: $33,680 • $6,600 microbiome sequence: 132 samples at $50 per sample • $24,000 mAbs: 2 pairs at $12,000 per pair • $3,080 sediment analysis: 44 samples of each, metals at $20 per sample and grain size at $50 per sample

F. Other: $0

G. Total Direct Charges: $139,568

H. Indirect Charges: $0

I. Matching funds: $94,636 (67.8%) • $6,000 Mote REU program. A $6,000 student stipend to support a student that will be working within the Marine Immunology program on the proposed project. • $2,000 Instrument use fee. • $16,191 Dr. Wetzel salary/fringe: For 0.5 months dedicated to this project. • $70,445 Indirect costs. Mote’s negotiated indirect rate is 74.99% of salary and fringe.

J. Total Project Cost: $234,204

Appendix: References

Charles Sidman, Associate Director for Research Florida Sea Grant PO Box 110400 Gainesville, FL 32611-0400 (352) 392-5870 [email protected] Completed Project: Manipulating microbes to improve aquaculture efficiency. Andrea Tarnecki, Mote Marine Laboratory. Bacterial management issues cause mass mortality in fish aquaculture creating economic losses for the seafood and aquarium trade and affecting the success of stock enhancement efforts. This project will test naturally occurring probiotics for the aquaculture industry to reduce current survival bottlenecks with guidance from an advisory board of industry partners. R/LR-A-54.

Lisa Manning, Ph.D. National Listing Coordinator Endangered Species Division (PR3) Office of Protected Resources NOAA Fisheries [email protected] Project: Development of hand held testers for gender identification in Siberian sturgeon.

Robert Suydam, Ph.D. Senior Wildlife Biologist North Slope Borough Department of Wildlife Management Box 69 Barrow, AK 99723 Projects: Health biomarkers and contaminants in whales, seals, polar bears, fish, etc.

Appendix: Project Team Staffing No employees working on this engagement have ever been convicted of a felony.

Andrea Tarnecki, PhD. Staff Scientist in the Marine Immunology program at Mote Marine Laboratory. Earned her Ph.D. in the School of Fisheries, Aquaculture and Aquatic Sciences at Auburn University, with her discipline focused in Microbiology. She has over 10 years of experience in culturing and maintaining microorganisms as well as characterizing microbiomes using high-throughput sequencing methods. During her dissertation work, she explored post- harvest processing in the eastern oyster for depuration of V.p. and V.v., quantifying these pathogens using the FDA-approved MPN methodology. Recently she has formed collaborations with local shellfish farmers to investigate mortality events that occur in aquaculture of these animals from a microbiological perspective. She has also investigated Vibrio populations in recirculating aquaculture systems and tested antagonistic activity of probiotic bacteria against these microorganisms. She currently maintains a library of over 1,000 bacterial isolates and continues to investigate microbial ecology using standard culture-based techniques in combination with sequencing technologies.

Dana Wetzel, Ph.D. Program Manager for the Environmental Laboratory for Forensics at Mote Marine Laboratory, Wetzel has over 20 years of toxin-exposure organismal health, biomarker and antibody discovery experience. She has been successful in identifying and validating targeted antibodies of specialized biomarkers for in-situ biosensors. In addition to the gender identification biomarker antibody discovery and validation work for a future rapid biosensor, Wetzel and Sherwood have completed research that conclusively identifies a biomarker reflecting eminent breeding status in equines, and are progressing to the antibody development phase to create a stall- side in-situ biosensor for horse breeding/reproduction diagnostics applications. Wetzel and Sherwood have recently been selected to develop a rapid in-situ biosensor for brevetoxin detection in shellfish. Most importantly, Mote Marine Laboratory has worked with the aquaculture industry for decades on a range of important research issues and maintains a strong partnership with aquaculture farmers.

Tracy Sherwood Ph.D. Earned her Ph.D. in Medical Science from the University of South Florida, College of Medicine, Department of Molecular Medicine, discipline in Medical Microbiology and Immunology. Current appointment is at Mote Marine Laboratory in the Department of Environmental Laboratory for Forensics. She has over 15 years’ experience in immunoassay implementation and development. Recently along with Co-PI Wetzel, she has developed and validated monoclonal antibodies for a gender identification biomarker previously isolated from an important commercial aquaculture species, to create in-situ biosensors testers. In addition, along with Co-PI Wetzel holds two provisional patents; U.S. Provisional Application No. 62/934,351: Brevetoxin Detection Device and Method, and U.S. Provisional Application No. 63/017,540: Velvet Disease Detection Device, System and Method. Her role for this project will be the source of contact and oversight for monoclonal antibody development and validation, as well as lead all ELISA evaluations including; optimization, specificity and selectivity, and reproducibility.

Christelle Miller, Senior Chemist for the Mote’s Environmental Laboratory for Forensics, has over 10 years of experience with qualitative and quantitative analyses. She excels in method

21 development and implementing and maintaining quality assurance/ quality control criteria. She is certified in both Agilent and Thermo Fisher Scientific software and instrumentation. She is highly proficient with GCMS, LCMS, HPLC, Iatrascan, GPC and ACE instrumentation for analyses.

Appendix: Company Overview Company Information Mote Marine Laboratory Inc. DUNS 0791940800000 SIC 87,873 1600 Ken Thompson Parkway Sarasota, FL 34236-1006 Phone: (941) 388-4441 Fax: (941) 388-1986

Key Contact Andrea M. Tarnecki, Ph.D. Staff Scientist 12300 Fruitville Road Sarasota, FL 34240-8933 Phone: (941) 388-4541 ext124 Fax: (941) 371-5946 1. Authorized Person: Michael P. Crosby, Ph.D. 1600 Ken Thompson Parkway Sarasota, FL 34236-1006 Phone: (941) 388-4441 Fax: (941) 388-1986

Brief History of Mote Marine Laboratory Mote Marine Laboratory is one of the oldest marine research laboratories in Florida. It opened its doors as the Cape Haze Marine Laboratory in Placida, Florida, in 1955. The lab was founded on three basic principles: passion, philanthropy, and partnership. The lab was later renamed to honor a major benefactor, William R. Mote. Mote currently has more than 200 staff members, including more than 30 Ph.D. scientists. Mote’s vision is to be a leader in nationally and internationally respected research programs that are relevant to the conservation and sustainable use of marine biodiversity, healthy habitats, and natural resources. Mote is one of the world’s few remaining private research laboratories and, as a nonprofit organization, is funded through federal, state, and local grants. Mote has been handling research contracts for over 60 years. In 2019, it handled nearly $4M in federal and state contracts and grants from agencies such as NOAA, EPA, NSF, U.S. Department of Defense, and Fish and Wildlife Services (FWS).

Conflicts of Interest No members of the project team have any personal, professional, or financial conflicts of interest associated with the proposed work.