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University of Southern California Sea Grant Proposal PROJECT TITLE: DOCUMENTING MULTIPLE PHYCOTOXINS IN COASTAL ECOSYSTEMS OF THE CALIFORNIA COAST PRINCIPAL INVESTIGATORS: David A. Caron, Professor, University of Southern California Avery O. Tatters, Postdoctoral Investigator, University of Southern California Eric A. Webb, Associate Professor, University of Southern California FUNDING REQUESTED: 2016-2017 $60,183 Federal/State $40,836 Match 2017-2018 $61,232 Federal/State $42,060 Match STATEMENT OF THE PROBLEM The widespread occurrence and distribution of marine Harmful Algal Blooms (HABs) and their toxins in coastal waters of California have been documented during the last 15 years (Scholin et al., 2000; Anderson et al., 2006; Schnetzer et al., 2007; Kudela et al., 2008; Schnetzer et al., 2013). In contrast to these now-well-characterized ‘showcase’ marine phycotoxins such as domoic acid and saxitoxins, there is a paucity of research relating to many other algal and cyanobacterial-derived toxins that can occur in coastal environments. Specifically, discharges into estuaries and along the open coast from freshwater sources (e.g. rivers, creeks, coastal lagoons) can also contribute harmful freshwater cyanobacteria or algae and their toxins to the ocean, posing significant health risks to marine animals in close proximity to these sources (Miller et al., 2010) and also potentially threatening human seafood safety. We hypothesize that these freshwater species, together with marine-sourced toxins, result in elevated levels of diverse phycotoxins in coastal marine ecosystems in close proximity to freshwater discharges, creating matrices of toxins in estuaries that are much more complex than presently recognized. Our lack of knowledge regarding this important problem in coastal marine ecosystems stems from the fact that previous studies of harmful algal bloom-forming species and their toxins in these ecosystems have focused almost exclusively on marine taxa, with very little consideration that fresh or brackish water phycotoxins may be influencing marine mammals, birds, shellfish, finfish, and ecosystem health in general. In large part, this lack of attention is related to state and federal agencies that typically provide support for studying exclusively oceanic or freshwater ecosystems, but not their convergence. Consequently, the occurrence of ‘freshwater’ toxic species and/or toxins in regional estuaries has only rarely been considered or investigated (Lehman et al., 2005; Miller et al., 2010; Gibble and Kudela, 2014).

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During the Fall of 2014, ad-hoc sampling of brackish/estuarine and coastal interface waters along the coast of southern California revealed the consistent and simultaneous presence of several toxin-producing species of algae and cyanobacteria, and/or multiple toxins in discrete water samples (see MOTIVATION for details). The identities of these species and the toxins they produce indicate a freshwater source even though they were detected in estuarine and coastal waters. These toxins, together with well-characterized marine algal toxins occurring in coastal waters, create the potential for multiple stressors for estuarine and near-coast marine communities. The existence of numerous, undocumented phycotoxins is highly concerning, given that these toxins have rarely been recognized or characterized within coastal ecosystems. This proposal will assess known and presently understudied algal and cyanobacterial toxins, and their producers, in coastal ecosystems in close proximity to freshwater discharges throughout southern California. Our work will focus on the land-sea interface, and encompass both highly urbanized regions as well as relatively undeveloped regions along the coast of the Southern California Bight. This is largely a discovery-based project because, while our preliminary data indicate that the accumulation of multiple phycotoxins may be a common situation in brackish/estuarine habitats of Southern California, the diversity of toxins and the species that produce some of them are unknown at this time. The primary goal of this project is therefore to establish baseline information on the number and identity of the toxins, and relate them to the species producing them. As part of this study, we will build on our existing culture collection of toxin-producing species of algae and cyanobacteria in order to create a community resource for studying these species. We will identify the cultured species by both traditional morphological criteria and genetic means. Through experimental studies that will be a part of this project, we will begin to understand the physiology and ecology of these species, and thereby determine if these organisms are ‘transients’ in brackish and marine ecosystems (i.e. produced in fresh water and merely transported into marine ecosystems), or capable of growth and toxin production in marine waters. We will perform experimental studies with cultured species to investigate their response to environmental stimuli (temperature, salinity), as we believe these are fundamental factors controlling their ability to growth in fresh, brackish and marine waters. We hypothesize that the current extreme drought throughout the region, as well as large-scale climate change forecasted to persist throughout the southwestern U.S., may be producing environmental conditions conducive to blooms of toxic species and subsequent ecosystem level toxin accumulations in fresh and estuarine ecosystems. Our experimental studies will help test this possibility. The information resulting from this study will begin to assess the significance of these species and their toxins for ecosystem health and sustainability. INVESTIGATORY QUESTIONS Our investigatory questions revolve around studies to address three basic aspects of cyanobacterial and algal toxins along the coast of the Southern California Bight: i. establishing the diversity and concentrations of previously undocumented phycotoxins present in estuarine and coastal regions, ii. identifying the source of these toxins (i.e. marine or freshwater) and the

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species that are producing them, and iii. experimental studies aimed at understanding if these species are growing and producing toxins in these estuaries, or if their presence is a result of transport from freshwater or marine ecosystems. Specifically, we will address the following major questions, and aspects related to them: What is the extent of cyanobacterial and algal-derived toxins at the land-sea interface?

• What is the geographical distribution and diversity of ‘freshwater’ cyanobacterial or algal toxins along the coast of the Southern California Bight?

• What is their proximity to sources/inputs of fresh and coastal water? • Do toxins co-occur at a single site, and what concentration of toxins are present?

Can we link toxins observed in estuarine and coastal waters along the coast of the Southern California Bight to putative toxin producers?

• Which species of cyanobacteria or algae are producing the observed toxins? • Can individual species biosynthesize complex mixtures of toxins? • What toxins are being produced by cultured species? • What are the salinity and temperature optima and tolerances of toxic algae and

cyanobacteria in our study area? Are near-coast freshwater environments along urbanized regions of the southern California coast major sources of high abundances and/or diversity of toxic cyanobacteria and algae to the coastal ecosystem?

• Is estuarine or coastal contamination with HAB species and toxins related to urbanization along the drainage system?

• What are the monitoring needs, related to the findings of this study, that must be developed and where should they be enacted to track potentially dangerous situations in our coastal ecosystems?

MOTIVATION The coastline of Southern California is arguably one of the most beautiful and valuable coastlines in the United States but in specific regions it is also one of the most impacted, in part due to extreme population density. The Greater Los Angeles area is the second largest metropolitan region in the U.S. (determined either by total population, or population density per unit area). The highly urbanized region of its coastline extends from Santa Monica Bay to the northern shores of Orange County, a coastline of only ≈50 miles that supports a massive population that is presently estimated at 18.5 million people and continues to grow at a rapid rate (U.S. Census Bureau. 2015. Annual Estimates of the Resident Population: April 1, 2010 to July 1, 2014 – Combined Statistical Area). The region is economically vibrant, and vital to the state because of its ports/harbors, fisheries, agriculture, recreational opportunities and tourism. However, high population density and its many consequent land uses (e.g. industry, agriculture, sewage and waste disposal) have created significant negative impacts on downstream estuarine and coastal environments. These impacts include negative feedback for humans through the microbiological contamination of estuaries and beaches, and for marine animals through the accumulation of various chemical contaminants (e.g. PCBs and DDT) or endocrine disruptors

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that alter behavior and development (McDermott-Ehrlich et al., 1978; Jiang et al., 2001; Baker et al., 2009; McQuaig et al., 2012). Another well documented issue for both human and marine animal health is the contamination of marine food resources via the stimulation of toxic marine algae as a consequence of human-sourced nutrient enrichment of the coastal ocean (Glibert et al., 2005; Reifel et al., 2013; Schnetzer et al., 2013). Most notably in our region, domoic acid (the cause of Amnesic Shellfish Poisoning: ASP) and saxitoxins (the cause of Paralytic Shellfish Poisoning: PSP) are known to be common constituents in the coastal ocean. Both the species that produce these phycotoxins and the chemicals themselves are regularly monitored in the plankton and in sentinel marine animals (e.g. mussels), as well as seafood resources, by the California Department of Public Health. These powerful neurotoxins are also the topics of monitoring and research to understand the environmental factors leading to the success of the toxin-producing species in plankton communities, and their effects on marine and human food chains. Marine algal toxins produced along the California coast are presently recognized as a significant, episodic source of mortality for our ‘charismatic macrofauna’ (Scholin et al., 2000; Gulland and Hall, 2007), and algal blooms and their toxins are an important consideration for seafood safety (Etheridge, 2010) and a variety of water uses (Caron et al., 2010). It has recently been determined that human mediated discharges (e.g. sewage effluent, storm drains) in urbanized regions of southern California result in nutrient loading of our coastal waters that are now similar in magnitude to natural sources of nutrients such as upwelling (Howard et al., 2014). That is, anthropogenic sources of nutrients along the urbanized coast of southern California may be contributing to the growth and toxin production of marine algae. In Greater Los Angeles alone, nearly one billion gallons of treated sewage effluent is released into the coastal ocean on a daily basis. The issues noted above relating to HABs in marine ecosystems are now well documented, and constitute an area of active scientific research and environmental monitoring. On the freshwater side of the equation, cyanobacterial and algal toxins have also become a major concern for drinking water quality, recreational exposure, and animal mortality (Backer et al., 2008; Roelke et al., 2011; Wilhelm et al., 2011), and these events appear to be increasing as a consequence of climate change and human-induced eutrophication (Davis et al., 2009; Paerl and Huisman, 2009; Paerl and Paul, 2011; Paerl, 2014). One important aspect concerning the health of coastal ecosystems related to HABs and their toxins that has gone completely ‘under the radar’ until very recently, however, is the potential contribution of cyanobacterial and algal toxins produced in freshwater ecosystems that are subsequently transported to the coast via creeks, rivers or breaches of coastal lagoons. This issue garnered considerable public attention with the recent documentation of sea otter deaths in Monterey Bay that were attributed to cyanobacterial toxins (microcystins) produced in rivers flowing into the Monterey Bay National Marine Sanctuary, where the otters were exposed to the toxins via accumulation in the marine food web (Miller et al., 2010). Cyanobacterial toxins were extraordinarily high in some local lakes and rivers, presumably due to agricultural activity in the area that contributed to nutrient loading and cyanobacterial growth. Movement of this water into the coastal ecosystem exposed the marine organisms in the estuaries and coastal ocean to these

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chemicals, creating trophic links between the freshwater toxin and marine animals. The incident described above raised awareness of the potential for a ‘freshwater’ class of toxins as a significant concern in coastal California, but microcystins are certainly not the whole story with respect to cyanotoxins (or algal toxins) in marine ecosystems along the coast. Nodularins have been documented in food webs of the Baltic Sea (Sivonen et al., 1989; Repka et al., 2004), and threats to seafood safety by cyanobacterial toxins have been documented from estuaries in other parts of the world (Falconer et al., 1992; Mulvenna et al., 2012). We recently obtained very preliminary findings that highlight the co-occurrence of multiple toxin-producing cyanobacteria/algae, and mixtures of phycotoxin classes, in a variety of locations along the southern California coastline (Table 1). Table 1. Preliminary results (only a single sample was analyzed from each location) of toxin screening from estuarine and coastal locations within the Southern California Bight, showing widespread occurrence of algal/cyanobacterial toxins. An asterisk (*) indicates a toxin detected in an environmental sample and in a cultured organism from the same location.

Our results clearly indicate a presently overlooked, potentially significant environmental and public health concern in estuarine/coastal ecosystems that span the geographic breadth of the Southern California Bight. A wide variety of previously undocumented phycotoxins were

Toxin/class detected

Mode of action in mammals

Estuary (1-33ppt) Locality detected likely producer

Marine ( > 33ppt) Locality detected likely producer

Domoic Acid KA/GLU receptor binding antagonist ? Pseudo-nitzschia spp. ubiquitous Pseudo-nitzschia

spp.

Paralytic Shellfish Poisoning Toxins

(PSPs)

Na+ channel antagonist, neurotoxins

San Elijo Lagoon, Batiquitos Lagoon

Alexandrium sp.*, Aphanizomenon sp.,

Cylindrospermopsis sp.*, Lyngbya sp.*,

Phormidium sp.*

Newport Point, Ventura Harbor, Jalama

Alexandrium spp.*,

Lyngbya sp.,

Diarrhetic Shellfish Poisoning Toxins

(DSPs)

Protein Phosphatase inhibitor (PPi) ? Dinophysis spp. ? Dinophysis spp.

Microcystins hepatotoxins, PPi San Elijo Lagoon Microcystis spp.* ? -

Anatoxins nAChR agonists Lagoon at Pt.

Mugu, Santa Clara River estuary

Anabaena spp., Dolichospermum sp.,

Oscillatoria spp.? ? Oscillatoria spp.

Cylindrospermopsins Potent hepatotoxins, multiple effects

upstream from lagoon at Pt. Mugu, Santa Clara River

Cylindrospermopsis raciborskii ? -

Nodularin potent PPi Santa Clara River Nodularia sp. * ? -

Cycloimines nAChR and mAChR

antagonists, neurotoxins

? ? Newport Point,

Point Dume, Ventura Harbor

Gymnodimium sp., Alexandrium

spp. *

Lyngbyatoxin A Dermatitis, tumor promoters, other

Ventura County, Los Penasquitos

Lagoon Lyngbya spp.* Ventura

County Lyngbya spp.*

Phormidolide unknown Santa Clara River estuary, Jalama

creek Phormidium spp.* ? Phormidium spp.

Microginins metalloprotease inhibitor San Elijo Lagoon Microcystis sp. ? ?

Microviridins PPi, elastase inhibitor San Elijo Lagoon Microcystis sp. ? ?

Karlotoxins cytotoxins ? Karlodinium veneficum ? Karlodinium

veneficum

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observed across the estuaries sampled. Moreover, some of the locations had multiple toxins. These data indicate that the accumulation of multiple phycotoxins may be a common situation in brackish/estuarine habitats of Southern California, but the number of co-occurring species and toxins within the estuaries in the region, their concentrations, seasonality, and their relationship to human activities on land, are poorly understood at this time. Additionally, we currently have only circumstantial information linking putative toxin-producing species (which can be numerous in a given ecosystem) to actual toxin production. Without that information, a monitoring plan that specifically targets toxic cyanobacteria and algae cannot be developed. In order to generate an accurate risk assessment related to phycotoxins, we propose a coordinated sampling and research effort to establish baseline information on the occurrence of various toxins and their producers in a wide array of estuarine environments in the Southern California Bight. We feel that characterizing the extent of toxic algae and associated chemicals within estuarine ecosystems throughout the region is an important first step in establishing the extent of this problem, and expanding our awareness of the presence of multiple stressors in urbanized regions of the coastal ocean. Consequently our primary motivation in this proposal is the discovery and characterization of a diversity of compounds/organisms in the coastal ecosystems of southern California. The proposed work is cutting edge in that these species and compounds have thus far gone undocumented. We propose to survey all estuarine/lagoonal/river/creek entry points to the coastline across the length of the Southern California Bight in order to assess which freshwater ecosystems are contributing phycotoxins, and to identify the major producers of these toxins. Co-occurrence of multiple marine- or freshwater-derived toxins in estuaries creates the potential for multiple stressors of the organisms living in these environments. This project will bring fuller awareness of the connections between freshwater, estuarine and marine habitats by improving our understanding of the cyanobacterial/algal toxins at the land-sea interface. It is highly likely that the contribution of freshwater phycotoxins to estuaries and along the coast is strongly influenced by rain events (i.e. flushing), land use of the areas drained by freshwater creeks/lagoons, seasonality, climate, and potentially other factors. A number of freshwater discharges into the ocean in southern California are seasonal. Accumulations of algal biomass and toxins in lagoons and creeks therefore can occur over several months, with massive ‘dosing’ occurring when rainfall is sufficient to allow discharge, or breaching of coastal land barriers to the ocean. We are therefore motivated to synchronize our sampling with major weather/seasonal events such as a ‘first flush’, the first major rain event in the region which brings large amounts of rainwater into local waterways and for many creeks and lagoons results in the reestablishment of connectivity to the ocean. Additionally, there is little to no baseline information on the role of human activities in influencing cyanobacterial/algal communities in water discharging to coastal ecosystems in the region. We will establish baseline information for southern Californian estuaries so that future assessments can identify changes in response to land use, climate change, etc. Our expectation is that fresh waters in highly developed areas will accumulate large amounts of anthropogenically-derived substances including nutrient-rich runoff and wastes that could dramatically stimulate the development of freshwater cyanobacterial/algal abundances relative to undeveloped areas.

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Connectivity of those freshwater bodies to the coast (location and timing) will be fundamental determinants of the areas of the coastline that experience multiple freshwater phycotoxins. Water quality concerns stemming from rapid human population growth along the coast in southern California may be exacerbated by the large-scale drought presently taking place in the region as well as erratic annual weather patterns. Therefore, we hypothesize that urbanization (and the attendant eutrophication that can accompany it), possibly enhanced by present climatic conditions in the region, are major factors affecting the number and concentrations of toxins in freshwater entering marine ecosystems. Ultimately, we want to understand not only the presence of previously undocumented phycotoxins in the environment, but which cyanobacterial or algal species are producing them. Populations of the same species in disparate areas may biosynthesize different compounds due to site-specific environmental drivers, and specific classes of toxins may be synthesized by very different species. Due to this anticipated inter- and intra-specific variability, we are motivated to establish a well-curated culture collection of putative toxin producers that can be assessed for their ability to produce toxins, and ultimately studied to better understand their physiology and ecology. It is generally thought that the mere presence of some of these organisms within a system is cause for concern. One of the goals of this proposal is establishing a biological reference collection that will facilitate toxin discovery, and provide an essential source of reference material for the community that can be used to design and building region- and site-specific monitoring programs as well as support the development of research into the molecular taxonomy of important toxin-producing species identified in this work. Thus, the culture collection is fundamental for defining candidate species for future HAB monitoring programs. An indirect motivation for this proposed Sea Grant project relates to our desire to characterize ‘what’s in the water’. In this case, the focus is on the fresh and brackish waterbodies impinging on coastal ecosystems. This water is now and will be used/considered for public/private use in the future (e.g. irrigation), and thus this topic should be of interest to all consumers. Our preliminary findings highlight a variety of previously undocumented toxins in estuarine waters, and many of these are almost certainly sourced from nearby freshwaters. GOALS AND OBJECTIVES The overarching goals of this research project are to build awareness of the presence of undocumented phycotoxins in estuaries and coastal regions adjacent to freshwater discharge along the coast of the Southern California Bight, to provide insight into the causative species for these toxins, and to perform experimentation aimed at understanding if these species are growing and producing toxins in these estuaries or if their presence is a result of transport from freshwater or marine ecosystems. Ultimately, this work will contribute to establishment of guidelines for monitoring potentially toxic species and their products in the environment. As noted above and supported by our preliminary results (Table 1), the confluence of freshwater and marine ecosystems along the coast of southern California can be affected by multiple toxins of both freshwater and marine origin. The geographic distribution and types of these chemical threats is presently unknown. Given these overarching goals, the specific objectives of the project are:

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(1) Determine the extent of known or presently-undocumented phycotoxins in southern Californian coastal waters and estuarine ecosystems: This objective will be accomplished by conducting an extensive spatial survey of toxic algae and cyanobacteria along the coast of the Southern California Bight during Year 1 of the project. The survey will also provide live material for visually identifying potentially toxic species of algae and cyanobacteria, which will then be targeted for isolation and culture. (2) Isolate, culture and identify the causative cyanobacteria/algae that are the origin of toxins observed in the Year 1 survey: A culture collection consisting of both marine and freshwater toxic cyanobacteria and algae will be initiated from the survey samples, and used to directly link toxins to putative producers. Toxin-producing species will be identified by morphological and molecular approaches, and made publicly available to the scientific community. The biological collection and databases will be melded in future with other regional collections and databases now emerging within the state to provide a source of reference organisms and toxins in support of statewide monitoring and management practices of toxic algae and cyanobacteria. (3) Conduct field work (Year 2) to obtain greater temporal resolution on the presence of phycotoxins at 3-5 ‘hot spots’ within the region: Study sites exhibiting multiple phycotoxins or high concentrations during the Year 1 field survey will be sampled at higher frequency (monthly) during the second year to provide information on the seasonality of toxins appearing at the sites, with concomitant characterization of the putative producers at the sites. (4) Establish the basic physiological tolerances of these species and their effects on toxin production: Experimental work will be conducted to examine the temperature and salinity optima of the cultured species, which will help identify the source of toxic species (marine or freshwater) and help establish if these species are likely growing within estuarine environments along the coast, or being transported there from marine or freshwater ecosystems. 5) Provide information for the development of future monitoring practices: Information resulting from this study will identify regional hot spots, the phycotoxins appearing in those areas, and the seasonality of their appearances. This information will be conveyed to the Regional Water Quality Control Boards and other agencies charged with water quality in the region. METHODS AND APPROACH A spatial survey will be conducted that encompasses major estuaries within the middle and northern Southern California Bight, dovetailing with a complementary program sampling by colleagues in the southern region of the Bight (Howard et al, SCCWRP). The survey will constitute a baseline determination of presently-overlooked phycotoxins along the coast (preliminary data in Table 1), conducted three times between early summer and late fall (when cyanobacterial abundances in freshwaters typically reach maximal abundances), and also repeated once after the first significant rainfall of the year (‘first flush’) when freshwater discharge into the coastal ocean is most likely to contribute high concentrations of phycotoxins

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to estuarine and coastal ecosystems. The first year’s field work will provide material for culturing and identifying potentially toxic cyanobacteria and algae, and also identify locations that exhibit multiple phycotoxins or particularly high concentrations. The second year of field work will focus on 3-5 of these latter locations. Spatial survey collection sites: The collection sites, shown in Figure 1 and listed below, represent virtually all connections (permanent or seasonal) between freshwater ecosystems and the ocean, along the coast of southern California from Santa Barbara in the north to Torrey Pines in the south. This geographical range extends from relatively undeveloped coastline north of the Greater Los Angeles region, through the highly urbanized coastline of LA, and then from southern Orange County to the south through relatively undeveloped regions. Note, three major river systems will specifically not be sampled (Los Angeles, San Gabriel and Santa Ana Rivers). Those rivers will be sampled as part of a new NOAA-funded program beginning in Fall 2015 (see RELATED RESEARCH below), and thus our sampling program is completely complementary to that study.

Figure 1. Site of specific sampling locations throughout the Southern California Bight (from north to south): (1) Atascadero Creek/San Jose Creek/San Pedro Creek/Tecolotito Creek confluences to Goleta Slough; (2) Ventura Harbor; (3) Santa Clara River Estuary/Lagoon; (4) Oxnard-Channel Islands Harbor; (5) Mugu Lagoon; (6) Zuma Lagoon; (7) Malibu Creek/Lagoon; (8) Topanga Creek/Lagoon; (9) Rustic Creek; (10) Ballona Lagoon; (11) Marina Del Rey; (12) Ballona Creek; (13) Del Rey Lagoon; (14) King Harbor, City of Redondo Beach; (15) Malaga Creek; (16) Colorado Lagoon Park/Alamitos Bay; (17) Anaheim Bay; (18) Bolsa Chica Channel/Basin; (19) Seal Beach National Wildlife Refuge; (20) Huntington Beach

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Channel/Harbour; (21) Upper Newport Beach Nature Preserve; (22) Aliso Creek; (23) Salt Creek; (24) Dana Point Harbor; (25) San Juan Creek ; (26) San Mateo Creek; (27) Santa Margarita River; (28) Oceanside Harbor; (29) San Luis Rey River; (30) Buena Vista Creek/Lagoon; (31) Agua Hendionda Carlsbad; (32) Batiquitos Lagoon State Marine Conservation Area; (33) San Eljjo Lagoon; (34) San Dieguito River/Lagoon; (35) Los Penasquitos Creek/Lagoon. Whole water and benthic grab samples will be collected at all localities. Dominant algae and cyanobacteria will be evaluated by microscopy and samples will be screened for toxins using HPLC and/or ELISA (see below). We will also deploy Solid Phase Adsorption Toxin Tracking (SPATT)(Lane et al., 2010), that will be retrieved for analysis after one week of exposure, in order to characterize phycotoxins that might be present at concentrations too low to be detected by direct sample analysis. SPATT technology allows for highly sensitive toxin determinations by adsorbing toxins from the water throughout the period of deployment. In this way, SPATT also provides a temporal integration of the presence of toxins at the site. Optimization of resins used for the application of SPATT for various toxins will be accomplished as part of the recently funded NOAA project in which Caron and Tatters are participants (see RELATED RESEARCH). Focused field work in second year: Based on findings from Year 1, we will narrow our field efforts to a more extensive study of 3-5 of the original sites. This strategy will permit increased sampling frequency and resolution of the most ‘active or interesting’ locations. We propose a monthly scheme (Feb-Nov 2017) that involves whole water and benthic sampling flanking deployment and retrieval of SPATT bags. The specific number of stations will be determined by the distance (and therefore travel time) between the most interesting sites (i.e. those site that show either high toxin concentrations, multiple co-occurring toxins, or both). Small scale spatial heterogeneity in the various source waters will be characterized at sites that have more than one freshwater input (e.g. the confluence of the Atascadero, San Jose, San Pedro and Tecolotito Creeks at Goleta Slough). Toxin analyses: For both field samples and samples of established cultures, filtered samples and SPATT bag elutions will be screened and analyzed for a suite of toxins using HPLC and/or ELISA (Table 2). HPLC will serve as our primary screening tool in addition to immunoassay and a receptor binding assay. A synthesis of literature for the toxins listed in Table 2 allowed us to devise HPLC methods that are able to detect, not quantify, several of these compounds in a single run (Astrachan and Archer, 1981; Ohtani et al., 1992; Lawton et al., 1994; Lawton et al., 1995; Hawkins et al., 1997; Kodania et al., 1999; Gugger et al., 2005; Meriluoto and Spoof, 2005; Tonk et al., 2009). We will screen for domoic acid, microcystins (LR, RR, YR), anatoxin-a, nodularin, cylindrospermopsin, lyngbyatoxin a, microginins, microviridins, karlotoxins, gymnodimines and various cyclic imines using an Agilent 1290 UHPLC equipped with UV-vis/DAD/FL detection (Table 2). Putative identification of toxins will be accomplished by comparison with reference standards, predicted retention times and absorbance spectra. Screening for the other toxins, okadaic acid and PSPs, will be performed using commercially available ELISA kits from Abraxis. Quantification of domoic acid, microcystins, nodularin, cylindrospermopsin and gymnodimine will be performed by HPLC with minor modifications to

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published methods (Harada et al., 1994; Lawton et al., 1994; Lawton et al., 1995; Marrouchi et al., 2010; Tatters et al., 2012). Measurements of okadaic acid and PSPs will be made with ELISA (Sassolas et al., 2013; Casero et al., 2014). Lastly, anatoxin-a and cyclic imine concentrations will be determined using colormetric receptor binding assays also available from Abraxis (Rubio et al., 2014). Table 2. Overview of the toxins that will be analyzed from field samples. Putative producers obtained in culture will also be assayed for the toxins they may produce.

Compound(s) Screening method

Quantification method

Domoic Acid HPLC/ELISA HPLC/ELISA Okadaic Acid ELISA ELISA

PSPs ELISA ELISA Microcystins HPLC/ELISA HPLC/ELISA Anatoxin-a HPLC RBA Nodularin HPLC HPLC

Cylindrospermopsin HPLC HPLC Lyngbyatoxin A HPLC -

Microginins HPLC - Microviridins HPLC -

Gymnodimines HPLC HPLC other Cyclic imines HPLC RBA

Karlotoxins HPLC - Establishment of cultures: Potential toxin-producing cyanobacterial and algal species (identities based on morphological identifications) will be collected and isolated from the field samples, which will be subsequently analyzed for phycotoxins. Algae/cyanobacteria will be brought into culture in an attempt to relate toxins at the sites to specific microbial taxa. These strains will be used to confirm toxin production, and to conduct simple experimental studies to examine growth conditions and toxin production (see immediately below). Cultured algae and cyanobacterial species will be identified based on morphological details (i.e. classical taxonomic criteria). In addition, small subunit ribosomal RNA genes (16S genes) of the cyanobacterial strains will be sequenced using ‘cyanobacterial specific’ PCR primers (Nübel et al., 1997) as recently described (Momper et al., 2015). If the 16S molecule does not provide enough information to positively identify the isolate, the internal transcribed spacer (ITS) region will also be sequenced (Orcutt et al., 2002; Webb et al., 2009). This effort will provide molecular identifications and comparison with national and international databases of known toxic and nontoxic cyanobacteria. As mentioned above, toxic species of cyanobacteria and algae cultured during this study will be made available to the scientific community, to serve as source material for the development of methodology for monitoring important species and studies of specific toxins and their production. Experimental studies with cultured algae and cyanobacteria: Lab studies will be conducted with toxin-producing cyanobacteria or algae to determine the ability of these species to grow and produce toxins across a range of salinities and temperatures.

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Where possible and where time permits, we will clean cultures of co-occurring heterotrophic bacteria, as growth or toxin production may be affected by these microbial associates (Shen et al., 2011). We anticipate that many of the toxins (and toxic cells) that we will encounter in the estuaries will be freshwater species transported from upstream locations, rather than endemic to saline environments. We will conduct experimental studies to determine optimal salinity, as well as salt tolerance of selected isolates. Growth across a range of temperatures will also be measured in order to determine the responsiveness of growth and toxin production to environmental temperature. Since it has been shown that cyanobacteria become a more significant fraction of algal blooms in shallow freshwater lakes as temperature increases (Kosten et al., 2012), these empirical data will be valuable for predicting local outcomes as climate change increases the average annual temperature in southern California. RELATED RESEARCH This project strongly leverages two research and monitoring efforts presently coming on-line in California. One effort (funded through the Regional Water Quality Control Boards) will begin this summer, and will investigate the production of cyanobacterial toxins and the causative species of those toxins in two freshwater, inland lakes in southern California (Lake Elsinore, Canyon Lake). This project is the beginning of what is planned to be a statewide effort to expand assessment of the occurrence of potentially toxic cyanobacteria (and a few eukaryotic algae) in the state’s freshwater waterways. Second, a NOAA based program is presently recommended for funding (start date Fall 2015; NOAA Monitoring and Event Response for Harmful Algal Blooms, MERHAB; Title: “Improving tools for monitoring multiple HAB toxins at the land-sea interface in Coastal California, HAB-SICC”). The latter program will focus on understanding the occurrence and impact of cyanobacterial toxins produced in freshwater ecosystems along the coastline of California as physiological stressors, at a few selected coastal locations. The study will examine locations in central and northern California beyond the geographical range of this Sea Grant project, as well as freshwater cyanobacteria and their toxins in three large river systems in southern California (Los Angeles, Santa Ana and San Gabriel rivers) and two estuaries in San Diego County (San Onofre, San Diego). The MERHAB locations will not be sampled as a part of this Sea Grant project in order to avoid overlap between the projects and maximize geographical coverage between them. Because the sites chosen for the Sea Grant work proposed here will complement the MERHAB sites, this project will expand and greatly improve the spatial resolution with which to assess potential links between urbanization and cyanobacterial occurrences in waterways leading to the coast. The Caron lab is involved in both of the projects described above, so coordination and cooperation among the three programs will greatly increase the chances of accomplishig the goals set forth in this Sea Grant project (see Letter of Support from Meredith Howard, Southern California Coastal Water Research Project, who is lead PI on both projects). The first component of the MERHAB project to be undertaken is an evaluation of the effectiveness of various resins for binding various cyanotoxins (for use in SPATT), and

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validation with field/culture samples of the SPATT approach. That information will be applied in the present Sea Grant project. SPATT validation is slated for the first year of the MERHAB project, and therefore the SPATT method for cyanobacterial toxins will be vetted by the time the first field season of this project would be conducted. SPATT technology for binding and analyzing marine toxins is well worked out, and has been applied in field studies for several years by the Caron lab. BUDGET-RELATED INFORMATION Personnel support for this project is requested for Avery O. Tatters (4 mos./year in both years), who is a Postdoctoral Investigator in Caron’s lab and who is partially supported on other grants. Tatters has extensive expertise in algal and cyanobacterial culturing and taxonomy and is an essential component of the culture establishment and morphological identifications. Funds ($1,000) are requested to support a part-time undergraduate student on the project to assist with the culture work and photographic documentation of the field samples and laboratory cultures established in the project. A Sea Grant Traineeship is also requested (For Joshua Kling), who is a Ph.D. graduate student in Webb’s lab, and who will be instrumental in the molecular identification of cyanobacterial cultures established in the project. Funds requested for materials and supplies ($11,500 in Years 1 and 2) will provide sampling containers, preservatives, culture vessels (flasks, tissue culture dishes, etc.), culture media and vessels, PCR primers and supplies, sequencing costs, as well as the large amounts of disposal tubes, gloves, pipettes, and miscellaneous reagents required for the work. Travel funds ($2,788/yr) are requested to cover substantial costs (mileage charges) for conducting the extensive field sampling programs that will be conducted in both years. No permanent equipment is requested, all facilities necessary to conduct the research are present in the PIs labs (incubators, microscopes, plate readers for ELISAs, a departmental HPLC, and various instruments for genetic analyses,). Matching Funds for this project will be provided by USC for 0.5 months/year academic salary (and associated fringe) for Caron and Webb. ANTICIPATED BENEFITS In a general sense, the outcomes of the study will be directly applicable to Sea Grant’s sustainable seafood and aquaculture initiatives. Characterizing the extent of toxic algae and their phycotoxins within estuarine ecosystems in the Southern California Bight is an important step in our growing awareness of the presence of multiple toxins in urbanized regions of the coastal ocean. Our very preliminary findings regarding the co-occurrence of multiple toxin producers

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and mixtures of toxin classes (Table 1) suggest a presently overlooked but potentially important environmental and public health concern in estuarine/coastal ecosystems. In order to enable an accurate risk assessment, a coordinated sampling effort is needed to establish the presence of these toxins, and their producers. Our present lack of understanding regarding the prevalence, diversity and geographical distribution of these toxins thwarts Sea Grant’s goal of achieving sustainable coastal ecosystems and the services they provide. The culture collection and toxin discovery provided by the study will be an essential source of reference material for designing and building region- and site-specific monitoring programs, support the development of a molecular taxonomy for species with difficult or ambiguous morphologies (many cyanobacteria), and research that might explain the distributions of these species. Several wetlands and estuarine preservation or conservation groups managing various sites (see list of sampling locations) will directly benefit from a knowledge of the toxins present in their ecosystems, the species of cyanobacteria and/or algae producing those substances, and their putative sources (i.e. upstream growth of freshwater species, introduced from the coastal ocean, or produced in-situ in the estuarine environment). This work also has direct implications for the location of proposed aquaculture operations in the region as well as seafood safety monitoring. The proposed physiological studies to understand the temperature and salinity limits of the species, and the responsiveness of toxin production to these variables, will help identify possible management strategies for these groups and agencies. This work will also contribute to an ongoing effort to generate a Southern California regionally-specific cyanobacteria guide. This identification manual will include photomicrographs of natural communities and culture material as well as morphological, physiological and phylogenetic information for select, ecologically important strains. This identification and overall ecological resource will immediately be useful to educators, HAB monitoring groups, water quality personnel; individuals and entities that routinely have difficulties identifying these cryptic organisms. COMMUNICATION OF RESULTS We have a variety of venues through which we have communicated our scientific results in the past, and which we will continue to use in the future. Our findings will be communicated to the scientific community through the normal venues of publication and scientific meetings in which we participate. An example of the latter is the upcoming meeting of the 8th Symposium on Harmful Algae in the U.S., Long Beach, CA, November 15-19, 2015, which Caron is co-hosting (with Meredith Howard; Southern California Coastal Water Research Project). That symposium takes place every 2 years, and we anticipate that our participation in 2017 will focus on the results of this study. The information resulting from our research will also be made available to water managers and regulators. Caron serves on the Technical Advisory Board of the Surface Water Ambient

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Monitoring Program (SWAMP) of the State Water Resources Control Board of California, formed to review and contribute to a statewide strategy for monitoring, assessing and reporting on cyanotoxins in California waterbodies. His presence on that Board provides direct interactions with many of the state groups and individuals developing criteria for assessing and managing freshwater toxic cyanobacteria/algae (e.g. California Cyanotoxin Harmful Algal Bloom network (CCHABs) and coastal water quality. His participation in a local workshop, Offshore Aquaculture in the Southern California Bight, Aquarium of the Pacific, Long Beach, CA, April 2829, 2015 also provided a means of informing state and federal agencies of the issues surrounding harmful algae and their toxins in California coastal waters. Our outreach and education activities make extensive use of USC Sea Grant’s outreach activities through a citizen-based harmful algal bloom monitoring program (HABWatch) that we have conducted during the past several years. This program is designed to incorporate the public, and personnel at public aquaria and informal learning centers into HAB-related monitoring of coastal waters. Participating groups (we have 11 groups that have or are participating) receive simple equipment and training in sample collection, processing and HAB species identification. Each group carries out weekly sampling and HAB monitoring at a locale near them, and the results are submitted and made available through a web portal. In addition to increasing public awareness and understanding of HABs, some of these groups have employed the program to develop teaching/exhibition modules for the public. Our results are made available to the general public through a number of activities. For example, recent public presentations of HAB-related information by Caron included a Discovery Lecture Series presentation at Cabrillo Marine Aquarium, San Pedro, CA, April 3, 2015 (Harmful Algal Blooms Along the California Coast: Their Ecosystem Impacts and Our Present Understanding). REFERENCES Anderson, C.R., Brzezinski, M.A., Washburn, L., Kudela, R., 2006. Circulation and

environmental conditions during a toxigenic Pseudo-nitzschia australis bloom in the Santa Barbara Channel, California. Mar. Ecol. Prog. Ser. 327, 119-133.

Astrachan, N.B., Archer, B.G. 1981. Simplified monitoring of anatoxin- a by reverse phase HPLC and sub-acute effects of anatoxin-a in rats. In: Carmichael, W.W. (ed.) The Water Environment--Algal Toxins and Health. Plenum Press, New York and London. 437-447.

Backer, L., Carmichael, W., Kirkpatrick, B., Williams, C., Irvin, M., Zhou, Y., Johnson, T., Nierenberg, K., Hill, V., Kieszak, S., Cheng, Y.-S., 2008. Recreational exposure to low concentrations of microcystins during an algal bloom in a small lake. Marine Drugs 6, 389.

Baker, M., Ruggeri, B., Sprague, L., Eckhardt-Ludka, C., Lapira, J., Wick, I., Soverchia, L., baldi, M., Polzonetti-Magni, A., Vidal-Dorsch, D., Bay, S., Gully, J., Reyes, J., Kelley, K., Schlenk, D., Breen, E., Šášik, R., Hardiman, G., 2009. Analysis of Endocrine Disruption in Southern California Coastal Fish Using an Aquatic Multispecies Microarray. Enviornmental Health Perspective 117, 223-230.

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Caron, D.A., Garneau, M.-v., Seubert, E., Howard, M.D.A., Darjany, L., Schnetzer, A., Cetinic, I., Filteau, G., Lauri, P., Jones, B., Trussell, S., 2010. Harmful algae and their potential impacts on desalination operations off southern California. Water Res. 44, 385-416.

Casero, M.C., Ballot, A., Agha, R., Quesada, A., Cires, S., 2014. Characterization of saxitoxin production and release and phylogeny of sxt genes in paralytic shellfish poisoning toxin-producing Aphanizomenon gracile. Harmful Algae 37, 28-37.

Davis, T.W., Berry, D.L., Boyer, G.L., Gobler, C.J., 2009. The effects of temperature and nutrients on the growth and dynamics of toxic and non-toxic strains of Microcystis during cyanobacteria blooms. Harmful Algae 8, 715-725.

Etheridge, S.M., 2010. Paralytic shellfish poisoning: Seafood safety and human health perspectives. Toxicon 56, 108-122.

Falconer, I.R., Choice, A., Hosja, W., 1992. Toxicity of edible mussels (Mytilus edulis) growing naturally in an estuary during a water bloom of the blue-green alga Nodularia spumigena. Environ. Toxicol. Water Qual. 7, 119-123.

Gibble, C.M., Kudela, R.M., 2014. Detection of persistent microcystin toxins at the land–sea interface in Monterey Bay, California. Harmful Algae 39, 146-153.

Glibert, P.M., Seitzinger, S., Heil, C.A., Burkholder, J.M., Parrow, M.W., Codispoti, L.A., Kelly, V., 2005. The role of eutrophication in the global proliferation of harmful algal blooms. Oceanography 18, 198-209.

Gugger, M., Lenoir, S., Berger, C., Ledreux, A., Druart, J.C., Humbert, J.F., Guette, C., Bernard, C., 2005. First report in a river in France of the benthic cyanobacterium Phormidium favosum producing anatoxin-a associated with dog neurotoxicosis. Toxicon 45, 919-928.

Gulland, F.M.D., Hall, A.J., 2007. Is Marine Mammal Health Deteriorating? Trends in the Global Reporting of Marine Mammal Disease. EcoHealth 4, 135-150.

Harada, K.I., Ohtani, I., Iwamoto, K., Suzuki, M., Watanabe, M.F., Watanabe, M., Terao, K., 1994. Isolation of cylindrospermopsin from a cyanobacterium Umezakia natans and its screening method. Toxicon 32, 73-84.

Hawkins, P.R., Chandrasena, N.R., Jones, G.J., Humpage, A.R., Falconer, I.R., 1997. Isolation and toxicity of Cylindrospermopsis raciborskii from an ornamental lake. Toxicon 35, 341-346.

Howard, M.D.A., Sutula, M., Caron, D.A., Chao, Y., Farrara, J.D., Frenzel, H., Jones, B., Robertson, G., McLaughlin, K., Sengupta, A., 2014. Anthropogenic nutrient sources rival natural sources on small scales in the coastal waters of the Southern California Bight. Limnol. Oceanogr. 59, 285-297.

Jiang, S., Noble, R., Chu, W., 2001. Human adenoviruses and coliphages in urban runoff-impacted coastal waters of southern California. Appl. Environ. Microbiol. 67, 179-184.

Kodania, S., Suzuukia, S., Ishidaa, K., Murakamia, M., 1999. Five new cyanobacterial peptides from water bloom materials of lake Teganuma (Japan). FEMS Microbiol. Lett. 178, 343-348.

Kosten, S., Huszar, V.L.M., Bécares, E., Costa, L.S., van Donk, E., Hansson, L.-A., Jeppesen, E., Kruk, C., Lacerot, G., Mazzeo, N., De Meester, L., Moss, B., Lürling, M., Nõges, T., Romo, S., Scheffer, M., 2012. Warmer climates boost cyanobacterial dominance in shallow lakes. Global Change Biol. 18, 118-126.

Kudela, R.M., Lane, J.Q., Cochlan, W.P., 2008. The potential role of anthropogenically derived nitrogen in the growth of harmful algae in California, USA. Harmful Algae 8, 103-110.

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Lane, J.Q., Roddam, C.M., Langlois, G.W., Kudela, R.M., 2010. Application of Solid Phase Adsorption Toxin Tracking (SPATT) for field detection of the hydrophilic phycotoxins domoic acid and saxitoxin in coastal California. Limnology and Oceanography, Methods 8, 645-660.

Lawton, L.A., Edwards, C., Codd, G.A., 1994. Extraction and high-performance liquid-chromatographic method for the determination of microcystins in raw and treated waters. Analyst 119, 1525-1530.

Lawton, L.A., Edwards, C., Beattie, K.A., Pleasance, S., Dear, G.J., Codd, G.A., 1995. Isolation and characterization of microcystins from laboratory cultures and environmental samples of Microcystis aeruginosa and from an associated animal toxicosis. Natural Toxins 3, 50-57.

Lehman, P.W., Boyer, G., Hall, C., Waller, S., Gehrts, K., 2005. Distribution and toxicity of a new colonial Microcystis aeruginosa bloom in the San Francisco Bay Estuary, California. Hydrobiologia 541, 87-99.

Marrouchi, R., Dziri, F., Belayouni, N., Hamza, A., Benoit, E., Molgo, J., Kharrat, R., 2010. Quantitative determination of gymnodimine-A by high performance liquid chromatography in contaminated clams from Tunisia coastline. Mar. Biotechnol. 12, 579-585.

McDermott-Ehrlich, D., Young, D.R., Heesen, T.C., 1978. DDT and PCB in flatfish around southern California municipal outfalls. Chemosphere 7, 453-461.

McQuaig, S., Griffith, J., Harwood, V.J., 2012. Association of fecal indicator bacteria with human viruses and microbial source tracking markers at coastal beaches impacted by nonpoint source pollution. Appl. Environ. Microbiol. 78, 6423-6432.

Meriluoto, J., Spoof, L. 2005. SOP: Analysis of microcystins by high-performance liquid chromatography with photodiode-array detection. In: Meriluoto, J., Codd, G.A. (eds.) Toxic cyanobacterial monitoring and cyanotoxinanalysis. Abo Akademi University Press, Finland. 77-84.

Miller, M.A., Kudela, R.M., Mekebri, A., Crane, D., Oates, S.C., Tinker, M.T., Staedler, M., Miller, W.A., Toy-Choutka, S., Dominik, C., Hardin, D., Langlois, G., Murray, M., Ward, K., Jessup, D.A., 2010. Evidence for a Novel Marine Harmful Algal Bloom: Cyanotoxin (Microcystin) Transfer from Land to Sea Otters. PLoS One 5, e12576.

Momper, L.M., Reese, B.K., Carvalho, G., Lee, P., Webb, E.A., 2015. A novel cohabitation between two diazotrophic cyanobacteria in the oligotrophic ocean. ISME J. 9, 882-893.

Mulvenna, V., Dale, K., Priestly, B., Mueller, U., Humpage, A., Shaw, G., Allinson, G., Falconer, I., 2012. Health risk assessment for cyanobacterial toxins in seafood. J. Environ. Res. Public Health 9, 807-820.

Nübel, U., Garcia-Pichel, F., Muyzer, G., 1997. PCR primers to amplify 16S rRNA genes from cyanobacteria. Appl. Environ. Microbiol. 63, 3327-3332.

Ohtani, I., Moore, R.E., Runnegar, M.T.C., 1992. Cylindrospermopsin: a potent hepatotoxin from the blue-green alga Cylindrospermopsis raciborskii. J. Am. Chem. Soc. 114, 7941-7942.

Orcutt, K.M., Rasmussen, U., Webb, E.A., Waterbury, J.B., Gundersen, K., Bergman, B., 2002. Characterization of Trichodesmium spp. by genetic techniques. Appl. Environ. Microbiol. 68, 2236-2245.

Paerl, H.W., 2014. Mitigating harmful cyanobacterial blooms in a human- and climatically-impacted world. Life 4, 988-1012.

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Paerl, H.W., Paul, V.J., 2011. Climate change: Links to global expansion of harmful cyanobacteria. Water Res. 46, 1349-1363.

Paerl, H.W., Huisman, J., 2009. Climate change: a catalyst for global expansion of harmful cyanobacterial blooms. Environ. Microbiol. Rep. 1, 27-37.

Reifel, K.M., Corcoran, A.A., Cash, C., Shipe, R., Jones, B.H., 2013. Effects of a surfacing effluent plume on a coastal phytoplankton community. Cont. Shelf Res. 60, 38-50.

Repka, S., Meyerhöfer, M., von Bröckel, K., Sivonen, K., 2004. Associations of cyanobacterial toxin, nodularin, with environmental factors and zooplankton in the Baltic Sea. Microb. Ecol. 47, 350-358.

Roelke, D.L., Grover, J.P., Brooks, B.W., Glass, J., Buzan, D., Southard, G.M., Fries, L., Gable, G.M., Schwierzke-Wade, L., Byrd, M., Nelson, J., 2011. A decade of fish-killing Prymnesium parvum blooms in Texas: roles of inflow and salinity. J. Plankton Res. 33, 243-253.

Rubio, F., Kamp, L., Carpino, J., Faltin, E., Loftin, K., Molgo, J., Araoz, R., 2014. Colorimetric microtiter plate receptor-binding assay for the detection of freshwater and marine neurotoxins targeting the nicotinic acetylcholine receptors. Toxicon 91, 45-56.

Sassolas, A., Catananta, G., Hayat, A., Stewart, L., Elliott, C., Marty, J., 2013. Improvement of the efficiency and simplification of ELISA tests for rapid and ultrasensitive detection of okadaic acid in shellfish. Food Control 30, 144-149.

Schnetzer, A., Jones, B.H., Schaffner, R.A., Cetinic, I., Fitzpatrick, E., Miller, P.E., Seubert, E.L., Caron, D.A., 2013. Coastal upwelling linked to toxic Pseudo-nitzschia australis blooms in Los Angeles coastal waters, 2005-2007. J. Plankton Res.

Schnetzer, A., Miller, P.E., Schnaffner, R.A., Stauffer, B.A., Jones, B.H., Weisberg, S.B., DiGiacomo, P.M., Berelson, W.M., Caron, D.A., 2007. Blooms of Pseudo-nitzschia and domoic acid in the San Pedro Channel and Los Angeles harbor areas of the Southern California Bight, 2003-2004. Harmful Algae 6, 372-387.

Scholin, C.A., Gulland, F., Doucette, G.J., Benson, S., Busman, M., Chavez, F.P., Cordaro, J., DeLong, R., De Vogelaere, A., Harvey, J., Haulena, M., Lefebvre, K., Lipscomb, T., Loscutoff, S., Lowenstine, L.J., Marin, R., Miller, P.E., McLellan, W.A., Moeller, P.D.R., Powell, C.L., Rowles, T., Silvagni, P., Silver, M., Spraker, T., Trainer, V., Van Dolah, F.M., 2000. Mortality of sea lions along the central California coast linked to a toxic diatom bloom. Nature 403, 80-84.

Shen, H., Niu, Y., Xie, P., Tao, M., Yang, X.I., 2011. Morphological and physiological changes in Microcystis aeruginosa as a result of interactions with heterotrophic bacteria. Freshwater Biol. 56, 1065-1080.

Sivonen, K., Kononen, K., Carmichael, W.W., Dahlem, A.M., Rinehart, K.L., Kiviranta, J., Niemela, S.I., 1989. Occurrence of the hepatotoxic cyanobacterium Nodularia spumigena in the Baltic Sea and the structure of the toxin. Appl. Environ. Microbiol. 55, 1990-1995.

Tatters, A.O., Fu, F.-X., Hutchins, D.A., 2012. High CO2 and silicate limitation synergistically increase the toxicity of Pseudo-nitzschia fraudulenta. PLoS One 7, e32116.

Tonk, L., Welker, M., Huisman, J., Visser, P.M., 2009. A production of cyanopeptolins, anabaenopetins, and microcystins by the harmful cyanobacteria Anabaena 90 and Microcystis PCC 786. Harmful Algae 8, 219-224.

Webb, E.A., Ehrenreich, I.M., Brown, S.L., Valois, F.W., Waterbury, J.B., 2009. Phenotypic and genotypic characterization of multiple strains of the diazotrophic cyanobacterium, Crocosphaera watsonii, isolated from the open ocean. Environ. Microbiol. 11, 338-348.

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Wilhelm, S.W., Farnsley, S.E., LeCleir, G.R., Layton, A.C., Satchwell, M.F., DeBruyn, J.M., Boyer, G.L., Zhu, G., Paerl, H.W., 2011. The relationship between nutrients, cyanobacterial toxin and the microbial community in Lake Tai (Taihu),China. Harmful Algae 10, 207-215.

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PROJECTED WORK SCHEDULE TITLE: DOCUMENTING MULTIPLE PHYCOTOXINS IN COASTAL ECOSYSTEMS OF THE CALIFORNIA COAST

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BUDGET (Year 1)

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BUDGET (Year 2)

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BRIEF CURRICULUM VITA (Caron) NAME DAVID A. CARON Address Dept of Biological Sciences, University of Southern California 3616 Trousdale Parkway, Los Angeles, CA 90089-0371 Phone (work) 213-740-0203 (home) 310-455-1659 Email dcaron@usc.edu EDUCATION 1975 B.S. University of Rhode Island, Microbiology 1977 M.S. University of Rhode Island, Oceanography 1984 Ph.D. Massachusetts Inst. of Technology and Woods Hole Oceanographic Inst., Joint Program in Biol. Oceanography POSITIONS HELD 1984 - 1985 Associate Research Scientist, LDEO of Columbia University. 1985 - 1989 Assistant Scientist, Woods Hole Oceanographic Institution. 1989 - 1993 Associate Scientist, Woods Hole Oceanographic Institution. 1993 - 1997 Associate Scientist with tenure, WHOI. 1997 - 1999 Senior Scientist, WHOI. 1999 - present Professor, University of Southern California 2010 - 2011 Interim Director, Wrigley Institute for Environmental Studies 2000 - 2003 Section Head, Marine Environmental Biology Section 2003 - 2006 Chair of the Department of Biological Sciences, USC SELECTED PUBLICATIONS (out of 207 published and in-press articles & book chapters) Das, J., F. Py, J. Harvey, J. Ryan, A. Gellene, R. Grahamk, D.A. Caron, K. Rajan and G,S. Sukhatme. 2015. Data-driven robotic sampling for marine ecosystem monitoring. International Journal of Robotics Research. In press. Liu, Z., A.E. Koid, R. Terrado, V. Campbell, D.A. Caron and K.B. Heidelberg. 2015. Changes in gene expression of Prymnesium parvum due to nitrogen and phosphorus limitation. Frontiers in Microbiology, Aquatic Microbiology. DOI: 10.3389/fmicb.2015.00319. Caron, D.A., P.D. Countway, A.C. Jones, D.Y. Kim, A. Schnetzer. Marine Protistan Diversity. 2012. Marine Protistan Diversity. Annual Review of Marine Science 4: 467-493. Liu, Z., A.C. Jones, V. Campbell, K.D. Hambright, K.B. Heidelberg and D.A. Caron. Gene expression in the mixotrophic prymnesiophyte, Prymnesium parvum, responds to prey availability. Frontiers in Microbiology 6 (2015). DOI: 10.3389/fmicb.2015.00319.

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Seegers, B.N., J.M. Birch, R. Marin III, C.A. Scholin, D.A. Caron, E.L. Seubert, M.D.A. Howard, G.L. Roberston and B.H. Jones. 2015. Subsurface seeding of surface harmful algal blooms observed through the integration of autonomous gliders, moored Environmental Sample Processors, and satellite remote sensing in Southern California. Limnology and Oceanography. 60: 754-764. Hu, S., Z. Liu, A.A. Y. Lie, P.D. Countway, D.Y. Kim, A.C. Jones, R.J. Gast, E.B. Sherr, B.F. Sherr, S.C. Cary and D.A. Caron. 2015. Marine microbial eukaryote diversity and biogeography inferred from three different approaches for processing DNA information. Journal of Eukaryotic Microbiology. DOI: 10.1111/jeu.12217. Howard, M.D.A., M. Sutula, D.A. Caron, Y. Chao, J.D. Farrara, H. Frenzel, B. Jones, G. Robertson, K. McLaughlin and A. Sengupta. 2014. Anthropogenic nutrient sources rival natural sources on small scales in the coastal waters of the Southern California Bight. Limnology and Oceanography. 59: 285-297. Seubert, E.L., M.D.A. Howard, R.M. Kudela, T.N. Stewart, R.W. Litaker, R. Evans and D.A. Caron. 2014. Development, comparison and validation using ELISAs for the analysis of domoic acid in California sea lion body fluids. Journal of AOAC International. 97: 345-355. Caron, D.A. 2013. Towards a molecular taxonomy for protists: benefits, risks and applications in plankton ecology. J. Euk. Microbiol. 60: 407-413. Jones, A.C., T.S. Vivian Liao, F.Z. Najar, B.A. Roe, K.D. Hambright and D.A. Caron. 2013. Seasonality and disturbance: annual pattern and response of the bacterial and microbial eukaryotic assemblages in a freshwater ecosystem. Environ. Microbiol. 15: 2557–2572. Seubert, E.L., A.G. Gellene, M.D.A. Howard, P. Connell, M. Ragan, B.H. Jones, J. Runyan, D.A. Caron. 2013. Seasonal and annual dynamics of harmful algae and algal toxins revealed through weekly monitoring at two coastal ocean sites off southern California, USA. Environ. Sci. Poll. Res. 20: 6878–6895. Lewitus A.J., R.A. Horner, D.A. Caron, E. Garcia-Mendoza, B.M. Hickey, M. Hunter, D.D. Huppert, D. Kelly, R.M. Kudela, G.W. Langlois, J.L. Largier, E.J. Lessard, R. RaLonde, J.E. Rensell, P.G. Strutton, V.L. Trainer, J.F. Tweddle. 2012. Harmful algal blooms in the North American west coast region: history, trends, causes, and impacts. Harmful Algae. 19: 133-159. Seubert, E.L., S. Trussell, J. Eagleton, A. Schnetzer, I. Cetinić, P. Lauri, B.H. Jones and D.A. Caron. 2012. Algal toxins and reverse osmosis desalination operations: laboratory bench testing and field monitoring of domoic acid, saxitoxin, brevetoxin and okadaic acid. Water Research. 46: 6563-6573. Caron, D.A. and D.A. Hutchins. 2013. The effects of changing climate on microzooplankton grazing and community structure: drivers, predictions and knowledge gaps. Journal of Plankton Research. 35: 235-252.

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BRIEF CURRICULUM VITA (Tatters) NAME AVERY O. TATTERS Address Dept. of Biological Sciences, University of Southern California 3616 Trousdale Parkway, Los Angeles, CA 90089 Phone (work) 910-393-7078 (home) 910-393-7078 Email tatters@usc.edu EDUCATION

2004 B.S. Biology, University of North Carolina-Wilmington 2007 Emerging Infectious Disease/Pathology, University of Texas Medical Branch 2009 M.S. Marine Science, University of North Carolina-Wilmington 2014 Ph.D. Marine Environmental Biology/Marine Biology and Biological

Oceanography, University of Southern California POSITIONS HELD

2003 - 2004 Research Assistant, University of North Carolina-Wilmington 2004 - 2005 Research Associate, University of North Carolina-Wilmington 2005 - 2007 Graduate Research Assistant, University of Texas Medical Branch 2007 - 2009 Graduate Research Assistant, University of North Carolina- Wilmington 2009 - 2014 Ph.D. Student- University of Southern California 2014 - present Postdoctoral Research Associate, University of Southern California

SELECTED PUBLICATIONS Bermudez, R., Feng, Y., Roleda, M.Y., Tatters, A.O., Hutchins, D.A., Larsen, T., Boyd, P.W., Hurd, C.L., Riebesell, U., Winder, M. 2015. Long-Term Conditioning to Elevated pCO2 and Warming Influences the Fatty and Amino Acid Composition of the Diatom Cylindrotheca fusiformis. PLoS ONE, 10 (5): e0123945. doi: 10.1371/journal.pone.0123945 Tatters, A.O., Flewelling, L.J., Fu, F., Granholm, A.A, Hutchins, D.A. 2013. High CO2 Promotes the Production of Paralytic Shellfish Poisoning Toxins by Alexandrium catenella from Southern California waters. 30: 37-43. doi: 10.1016/j.hal.2013.08.007 Tatters, A.O., Roleda, M.R., Schnetzer, A.., Fu, F., Hurd, C.L., Boyd, P.W., Caron, D.A., Lie, A.A.Y., Hoffmann, L.J., Hutchins, D.A. 2013. Short- and Long-Term Conditioning of a

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Temperate Marine Diatom Community to Acidification and Warming. Phil. Trans. Royal Soc. B. vol. 368, no. 1627. doi: 10.1098/rstb.2012.0437 Tatters, A.O., Schnetzer, A., Fu, F., Lie, A.Y.A., Caron, D.A., Hutchins, D.A. 2013. Short- versus Long-Term Responses to Changing CO2 in a Coastal Dinoflagellate Bloom: Implications for Interspecific Competitive Interactions and Community Structure. Evolution. 67(7) 1879-1891. doi: 10.1111/evo.12029 Fu, F., Tatters, A.O., Hutchins, D.A. 2012. Global change and the future of harmful algal blooms in the ocean. Mar. Ecol. Prog. Ser. 470:207-233 Tatters, A.O., Van Wagoner, R.M., Tomas, C.R., Wright, J.L.C. 2012. Regulation of spiroimine neurotoxins and hemolytic activity in laboratory cultures of the dinoflagellate Alexandrium peruvianum (Balech & Mendiola) Balech & Tangen Harmful Algae. 19:160-168 Tomas, C.R., Van Wagoner, R.M., Tatters, A.O., White, K.D., Hall, S., Wright, J.L.C. 2012. Alexandrium peruvianum (Balech and Mendiola) Balech and Tangen a new toxic species for coastal North Carolina. Harmful Algae. 17:54-63 Tatters, A.O., Fu, F., Hutchins, D.A. 2012. High CO2 and Silicate Limitation Synergistically Increase the Toxicity of Pseudo-nitzschia fraudulenta. PLoS ONE. 7(2):e32116. doi: 10.1371/journal.pone.0032116 Van Wagoner, R.M., Deeds, J.R., Tatters, A.O., Place, A.R., Tomas, C.R., Wright, J.L.C. 2010. Structure and Relative Potency of Several Karlotoxins from Karlodinium veneficum. Journal of Natural Products. 73(8) 1360-1365 Tatters, A.O., Muhlstein, H.I., Tomas, C.R. 2010. The hemolytic activity of Karenia selliformis and two clones of K. brevis throughout a growth cycle. J. Appl. Phycol. 22:435-442 Tomas, C.R., Peterson, J., Tatters, A.O. 2007. Harmful Algal Species from Wilson Bay, New River, North Carolina: Composition, Nutrient Bioassay, and HPLC Pigment Analysis. Water Resources. Research Institute Project 50337. Report 369

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BRIEF CURRICULUM VITA (Webb) Name: Eric A. Webb Address Dept. of Biological Sciences, University of Southern California 3616 Trousdale Parkway, Los Angeles, CA 90089 Phone (work) 213-740-7954 (home) 774-836-6778 Email eawebb@usc.edu EDUCATION 1994 B.Sc., The Ohio State University, Microbiology 1999 Ph.D., The University of Wisconsin-Madison, Bacteriology 1999-2001 Postdoctoral, The Woods Hole Oceanographic Institution (WHOI) POSITIONS HELD

2001- 2005 Assistant Scientist, Biology Department WHOI 2005-2006 Associate Scientist without tenure 2006-2011 Assistant Professor, USC, Biology Depart., MEB Section 2011-2015 Associate Professor with Tenure, USC, Biology Depart., MEB SELECTED PUBLICATIONS (WOS August 2014 h-index = 20)

Sohm, J.A., N. Ahlgren, Z. Thomson, C. Williams, J. W. Moffett, M. A. Saito, E. A. Webb, and G. Rocap. Synechococcus Diversity And Distribution in the Tropical and Subtropical Oceans. –in press at ISMEJ N.G. Walworth, U. Pfreundt, W.C. Nelson, T. Mincer, J. F. Heidelberg, F. Fu, J.B. Waterbury, T. Glavina del Rio, L. Goodwin, N. Kyrpides, M. Land, T. Woyke, D.A. Hutchins, W.R. Hess, and E.A. Webb. 2015 Trichodesmium genome maintains abundant, widespread noncoding DNA in situ, despite oligotrophic lifestyle. PNAS 112:4251–4256. Momper LM, Reese BK, Carvalho G, Lee P, and Webb EA. (2015). A novel cohabitation between two diazotrophic cyanobacteria in the oligotrophic ocean. ISME J 9:882–893. Fu FX, Yu E, Garcia NS, Gale J, Luo Y, Webb EA, Hutchins DA. 2014. Differing responses of marine N2 fixers to warming and consequences for future diazotroph community structure. Aquatic Microbial Ecol 72:33–46. S.A. Sañudo-Wilhelmy, L Gómez-Consarnau, C. Suffridge, and E.A. Webb. 2014. The Role of B Vitamins in Marine Biogeochemistry. Annu. Rev. Mar. Sci. 2014. Vol. 6: 339-367 doi: 10.1146/annurev-marine-120710-100912 Hutchins DA, Fu F-X, Webb EA, Walworth N, Tagliabue A. 2013. Taxon-specific response of marine nitrogen fixers to elevated carbon dioxide concentrations. Nature Geosci 6:790–795.

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Chappell, P.D., J.W. Moffett, A.H. Hynes, and E.A. Webb. 2012 Molecular evidence of iron limitation and availability for the global diazotroph Trichodesmium ISME J 1-12 doi:10.1038/ismej.2012.13 Hynes, A.M., E.A. Webb, S.C. Doney, and J.B. Waterbury. 2012. Comparison of cultured Trichodesmium (Cyanophyceae) with species characterized from the field. J Phyc 48: 196–210. doi: 10.1111/j.1529-8817.2011.01096.x Sohm, J. A., J. A. Hilton, A. E. Noble, J. P. Zehr, M. A. Saito, and E. A. Webb. 2011. Nitrogen fixation in the South Atlantic Gyre and the Benguela Upwelling System. Geophys Res Lett 38: Chappell, P.D. and E.A. Webb. 2010. A molecular assessment of the iron stress response in the two phylogenetic clades of Trichodesmium Environ Microbiol 12:13-27 Rivers, A.R., R. W. Jakuba, and E.A. Webb. 2009. Iron stress genes in marine Synechococcus and the development of a flow cytometric iron stress assay. Environ. Microbiol 11:382 - 396 Van Mooy, B. A., Fredricks, H. F., Pedler, B. E., Dyhrman, S. T., Karl, D. M., Koblizek, M., Lomas, M. W., Mincer, T. J., Moore, L. R., Moutin, T., Rappe, M. S., and E.A. Webb. 2009. Phytoplankton in the ocean use non-phosphorus lipids in response to phosphorus scarcity. Nature, 458(7234), 69-72. Webb E.A., I.M. Ehrenreich, S. Brown, F.W. Valois, and J.B. Waterbury. 2009. Phenotypic and Genotypic Characterization of Multiple Strains of the Diazotrophic Cyanobacterium, Crocosphaera watsonii, isolated from the Open Ocean. Environ Microbiol 11: 338-348. Webb, E. A., R. W. Jakuba, J. W. Moffett, and S. T. Dyhrman. 2007. Molecular assessment of phosphorus and iron physiology in Trichodesmium populations from the western Central and western South Atlantic. Limnol. Oceanogr. 52:2221-2232. (Impact factor 3.545, citations 21) Dyhrman, S.T., P. D. Chappell, S. T. Haley, J. W. Moffett, E. D. Orchard, J. B. Waterbury, and E. A. Webb. 2006. Phosphonate utilization by the globally important marine diazotroph Trichodesmium. Nature. 439:68-71 Ehrenreich, I.M., J.B. Waterbury, and E.A. Webb. 2005. The Distribution and Diversity of Natural Product Genes in Marine and Freshwater Cyanobacterial Cultures and Genomes Appl. Environ. Microbiol. 71:7401-7413 Webb, E. A., Moffett, J. W., and J. B. Waterbury. 2001. Iron Stress in Open Ocean Cyanobacteria (Synechococcus, Trichodesmium, and Crocosphaera): Identification of the IdiA protein. Appl. Environ. Microbiol. 67:5444-5452

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SUPPORTING DOCUMENTATION Letter of support: Dr. Meredith Howard, Southern California Coastal Water Research Project, Costa Mesa, CA

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SUMMARY PROPOSAL FORM PROJECT TITLE: DOCUMENTING MULTIPLE PHYCOTOXINS IN COASTAL

ECOSYSTEMS OF THE CALIFORNIA COAST OBJECTIVE: The overarching objectives of this research project are to build awareness of the presence of undocumented phycotoxins in estuaries and coastal regions adjacent to freshwater discharge along the coast of the Southern California Bight, and to provide insight into the causative species for these toxins through isolation and culture of toxin-producing species. The longer-term goal is to contribute to the establishment of guidelines for monitoring potentially toxic species and their harmful products in estuarine environments throughout the region. METHODOLOGY: A spatial and temporal survey to obtain baseline information on the presence of potentially harmful cyanobacteria and algae, and their phycotoxins, will be conducted at numerous sites along the southern California coastline. We will obtain water and grab samples and employ passive toxin sampling methodology to provide a temporal aspect to the analysis. Algae and cyanobacteria will be examined via microscopy, and toxins will be assessed by HPLC and/or ELISA. Species of interest will be isolated and cultured, and studied experimentally in order to provide further physiological and genetic characterization. RATIONALE: The widespread occurrence of marine harmful algal blooms and their toxins (in particular domoic acid and saxitoxins) in coastal waters of California has been documented during the last 15 years. In contrast to these ‘showcase’ marine phycotoxins, there is a paucity of research on many other algal and cyanobacterial toxins that occur in our coastal environment. During fall 2014, ad-hoc sampling of estuarine and coastal interface waters along the coast of southern California revealed the consistent and simultaneous presence of several toxin-producing species of algae and cyanobacteria, and/or multiple toxins. The potential human and environmental health risks posed by acute or chronic exposure to mixtures of these previously undocumented phycotoxins is unknown. We propose a combination of field and laboratory studies to characterize the extent and distribution of these toxins, to identify the causative species, and assess their environmental tolerances. We hypothesize that freshwater habitats of urbanized and developed land stimulate the growth of diverse and potentially toxic cyanobacterial/algal assemblages, creating ‘hot spots’ of complex matrices of toxins in estuaries and along coastlines. DATA SHARING: All sequence data obtained in the study will be deposited in public databases. Cultures of toxic cyanobacteria and algae will be made publicly available. Scientific data resulting from the study will be shared through publication in the primary literature, presentation at scientific meetings, and through interactions with various state agency charged with water quality management.