PCMHAB 2015: Project Summaries

Institutions:  Texas A&M University, Woods Hole Oceanographic Institution

Investigators:  Lisa Campbell, Heidi M. Sosik

The frequency of harmful algal blooms (HABs) is increasing worldwide. In the Gulf of Mexico, concern focuses on not only the nearly annual blooms of Karenia brevis, but also emerging HAB species such as Dinophysis and Pseudo-nitzschia. Early warning, provided by monitoring combined with rapid response, has been identified as one of the most effective ways to mitigate the impact of HABs; however, obtaining species-specific results with sufficient temporal resolution to provide early warning is challenging. Monitoring programs for HABs typically rely on toxin analysis of seafood samples or time-consuming manual enumeration of phytoplankton, which limit the frequency of observations and the potential for early warning.

Automated submersible microscopes such as Imaging FlowCytobot (IFCB) have changed this situation. Using a combination of flow cytometric and video technology to capture high resolution images of suspended particles and machine learning technology to classify the images, the IFCB can identify potentially toxic species within hours. Long term continuous plankton imaging by the IFCB deployed at Port Aransas, TX has provided early warnings of six HAB events since 2007. We now propose to expand the early warning capabilities in the Gulf of Mexico by advancing an IFCB network to increase the spatial coverage of HAB detection, training additional end-users, and developing an information support system to improve management applications of this technology.

The goals of this project are to increase the number of end-users and extend a network of IFCBs in the Gulf of Mexico along the Texas coast for improved detection and management of HAB events that threaten human and ecosystem health. The transfer of this mature technology will provide a model for scaling up the network and expanding it to other regions. One commercial IFCB will be purchased and added to the network of existing IFCBs in Port Aransas and Galveston. Through development of generalized training sets and improved automated classification to target a variety of HAB species, an outcome of this project will be a tool that is more useful to a wider scope of end-users. To facilitate use of the IFCB by managers and researchers outside of our laboratories, the project will include a number of partners from Texas A&M University at Galveston (TAMU-G), Texas Parks and Wildlife Department (TPWD), Department of State Health Services, University of California Santa Cruz, and Woods Hole Oceanographic Institution. Participation by potential users (Mission-Aransas National Estuarine Research Reserve, US Geological Survey, Texas Coastal Ocean Observing Network (TCOON) and members of the Interstate Shellfish Sanitation Conference (ISSC) and current IFCB owners will be welcomed. A Transition Advisory Committee (TAC), including Yuki Honjo, McLane Research Laboratories, Inc. (MRL), the commercial manufacturer of IFCB; Jonathon Deeds, Food and Drug Administration (FDA); Rebecca Hensley (TPWD), Kendra Daly, University of South Florida and Ocean Observing Initiative PI; and Matthew Howard, Gulf of Mexico Coastal Ocean Observing System (GCOOS), will provide feedback throughout the project, with the goal of improving the transfer of technology and knowledge to the state and federal managers who require early warning of HABs for effective mitigation. Outcomes include training additional end-users, which will facilitate a broader scale implementation of IFCB technology for mitigation of HABs, and production of a user manual for field deployment and analysis.

Institutions: University of South Florida, Fish and Wildlife Research Institute

Investigators: Robert H. Weisberg (lead), John J. Walsh, Jason M. Lenes, Yonggang Liu, Lianyuan Zheng, Alina Corcoran

Upwelling is a necessary condition for Karenia brevis HABs along the west Florida coastline and elsewhere, but too much upwelling may obviate such occurrences.  Upwelling results from local (wind-driven) and deep-ocean (Loop Current driven) forcing.  Local forcing is what generally accounts for K. brevis bloom onsets in fall months, and once a bloom is detected it may be tracked short-term via model simulations.  Anomalously persistent and intense upwelling of new inorganic nutrients across the shelf slope (as may occur when the Loop Current contacts the shelf slope near the Dry Tortugas) favors diatoms over dinoflagellates, thereby suppressing HAB development.  These findings were successfully employed to explain why there was no K. brevis bloom in 2010 and then subsequently to predict several months in advance that a major bloom would not occur 2013, but then would occur in 2014.  These accomplishments form the basis for our PCMHAB proposal, the objectives of which are to: (1) develop quantitative predictor and predictand relationships for seasonal prediction, transferrable to management agencies for their use and (2) further refine an existing short term prediction tool that is already in daily automated use (in collaboration with FWC) by adding biochemistry to the inert 3.5 day particle trajectories.  This work will allow management agencies (e.g., FWC resource managers, Florida Department of Health, Florida Department of Agriculture and Consumer Services and county agencies) to better mitigate the negative effects of blooms.  Improved understanding of bloom behavior and pathways may also provide a basis for prevention and control.  No such viable basis can exist without such understanding.

Institution: University of Delaware

Investigators: Kathryn J. Coyne (lead), Mark E. Warner, Jonathan H. Cohen, Timothy Targett

Abstract: Dinoflagellates make up about 75% of HAB species that are capable of producing toxins. These toxins can be transferred up the food web and represent a major threat to human health and marine life. Costs associated with monitoring, loss of revenue for local fisheries, and other human health-related impacts during dinoflagellate blooms can reach into the tens of millions of dollars for a single event. Previous PCMHAB-supported research to Coyne and Warner demonstrated that Shewanellasp. IRI-160 produces an algicidal compound, termed “IRI- 160AA” that specifically targets dinoflagellates while having no effect on a broad range of other phytoplankton. Results of small scale microcosm experiments during blooms of harmful dinoflagellates were consistent with laboratory culture experiments, and demonstrated a shift in community structure after application of the algicide, with a decrease in dinoflagellates and dose- dependent increases in ciliates and diatoms.  The proposed research builds on these previous results with the aim to provide management and end-users with information about the effectiveness of IRI-160AA in prevention and mitigation of harmful dinoflagellate blooms, and the potential risks to other species by application of the algicide.

Previous research was conducted with a bacteria-free filtrate of the culture medium, which could be partially purified by solid phase extraction techniques. Quantitative assessment of the impacts of IRI-160AA across a broad range of taxa for this study will require that the algicide be further purified. In preliminary work, HPLC analysis revealed the presence of four major peaks in the bioactive fraction. For this project, each of these peaks will be isolated to identify and characterize the bioactive compound. The minimum (EC5) and median (EC50) effective concentrations as well as the concentration required to cause 95% mortality (EC95) in dinoflagellates will be determined using protocols and guidelines developed by the EPA. These concentrations will then be used to assess dose response in natural community experiments conducted under bloom and non-bloom conditions. Effects on vertebrate and invertebrate species will then be determined.  In support of this line of research, preliminary data shows no decrease in viability of a fish gill cell line after 24 hours exposure to the algicide. Here, the lethal and sublethal impacts of the purified algicide will be determined for copepods and blue crab larvae and megalopae stages. Primary and tertiary stress responses across a range of locally important fish species will then be assessed with the algicide alone or in conjunction with diel-cycling of dissolved oxygen as a secondary stressor. Our overall goal will be to gain more information about the impacts of the algicide on dinoflagellates and non-dinoflagellate species so that we can accurately predict the overall effects on resident populations during field application. After careful review of the results by PCMHAB management and the Transition Advisory Committee, a field demonstration will be conducted to determine the effectiveness of the algicide and overall effects on water quality and biology. The demonstration will follow guidelines for site selection and monitoring requirements provided in the draft Programmatic Environmental Assessment (PEA) for PCMHAB. Successful completion of this work will provide management with knowledge of the potential risks and benefits associated with the application of IRI-160AA as a tool to prevent or mitigate harmful dinoflagellate blooms.

Institution: University of South Florida, Fish and Wildlife Research Institute

Investigators: John Paul (Lead), Katherine Hubbard

The marine dinoflagellate, Karenia brevis, blooms annually in the Gulf of Mexico (GOM) and negatively impacts human and ecosystem health through production of a suite of toxins known as brevetoxins. Blooms of K. brevis cause widespread fish kills, and negatively impact human

health when toxins become aerosolized along beaches, resulting in respiratory irritation. Brevetoxins also concentrate in shellfish during K. brevis blooms, resulting in Neurotoxic Shellfish Poisoning if consumed. Rapid, specific, and accurate quantification of K. brevis is needed to monitor waters in shellfish harvesting areas (SHAs) for certain cell thresholds, and to allow more timely warning of bloom conditions in coastal areas. Currently, samples are collected by an extensive phytoplankton monitoring network consisting of volunteers as well as local, county, and state partners. Cells of K. brevis are enumerated by phytoplankton analysts, in either fixed or live samples, using light microscopy. Expertise is required to discriminate K. brevis from non-toxic but morphologically similar taxa, and samples are processed individually. Samples are not easily enumerated at sea or in most field locations and are shipped to shore- based labs, causing delays in the public access to critical bloom information.

A rapid, sensitive, and specific assay for the detection of K. brevis based on nucleic acid amplification technology has been developed and successfully adapted for use with the QuadPyre, a handheld sensor that detects isothermal amplification of nucleic acids using thermoregulated fluorometry. The Overarching Goal of the proposed research is to develop, demonstrate, and transfer hand-held genetic sensors for K. brevis detection to end users that monitor the coastal and estuarine waters of the Gulf of Mexico. Accordingly, the proprosed PCMHAB research is broken into three specific phases: I-Enhancement of hand-held genetic sensors for K. brevis detection through research and development; II-Demonstration and validation of K. brevis sensors in field and lab trials; and III- Transfer of technology to end-users and integration of genetic data into HAB observing networks. To advance the utility of the assay, Phase I of the project has the specific aims: 1) Simplify the extraction and analysis of high quality RNA from a variety of samples to allow field-based detection and quantification of K. brevis cells; 2) Install a second fluorescence channel on the QuadPyre to allow the addition of an internal control (K. brevis calibrator molecule) for improved quantification and detection of inhibitors; and 3) Increase utility of QuadPyre through software developments. To demonstrate and validate the assay, Phase II aims to: 1) Train end users, primarily state and county monitoring agencies, in the use of QuadPyre; 2) Integrate the QuadPyre into pilot monitoring projects to allow quantitative validation with cell counts conducted by state monitoring agencies. The final phase of the project will enable the transfer of data and technology to end-users, including coastal managers and regional observing networks. Specific aims for Phase III include: 1) Provision of technology and/or synthesis of genetic data to end user groups and 2) Integration of genetic data into existing HAB reporting structures in the Gulf of Mexico (e.g. FWC HAB Monitoring; NOAA and GCOOS observing networks).