Karenia brevis is the dominant toxic red tide algal species in the Gulf of Mexico. It produces potent neurotoxins (brevetoxins [PbTxs]), which negatively impact human and animal health, local economies, and ecosystem function. Field measurements have shown that cellular brevetoxin contents vary from 1–68 pg/cell but the source of this variability is uncertain. Increases in cellular toxicity caused by nutrient-limitation and inter-strain differences have been observed in many algal species. This study examined the effect of P-limitation of growth rate on cellular toxin concentrations in five Karenia brevis strains from different geographic locations. Phosphorous was selected because of evidence for regional P-limitation of algal growth in the Gulf of Mexico. Depending on the isolate, P-limited cells had 2.3- to 7.3-fold higher PbTx per cell than P-replete cells. The percent of cellular carbon associated with brevetoxins (%C-PbTx) was ∼ 0.7 to 2.1% in P-replete cells, but increased to 1.6–5% under P-limitation. Because PbTxs are potent anti-grazing compounds, this increased investment in PbTxs should enhance cellular survival during periods of nutrient-limited growth. The %C-PbTx was inversely related to the specific growth rate in both the nutrient-replete and P-limited cultures of all strains. This inverse relationship is consistent with an evolutionary tradeoff between carbon investment in PbTxs and other grazing defenses, and C investment in growth and reproduction. In aquatic environments where nutrient supply and grazing pressure often vary on different temporal and spatial scales, this tradeoff would be selectively advantageous as it would result in increased net population growth rates. The variation in PbTx/cell values observed in this study can account for the range of values observed in the field, including the highest values, which are not observed under N-limitation. These results suggest P-limitation is an important factor regulating cellular toxicity and adverse impacts during at least some K. brevis blooms.

Ciguatera fish poisoning is an illness suffered by > 50,000 people yearly after consumption of fish containing ciguatoxins (CTXs). One of the current methodologies to detect ciguatoxins in fish is a radiolabeled receptor binding assay (RBA_(R)). However, the license requirements and regulations pertaining to radioisotope utilization can limit the applicability of the RBA_(R) in certain labs. A fluorescence based receptor binding assay (RBA_(F)) was developed to provide an alternative method of screening fish samples for CTXs in facilities not certified to use radioisotopes. The new assay is based on competition binding between CTXs and fluorescently labeled brevetoxin-2 (BODIPY^®- PbTx-2) for voltage-gated sodium channel receptors at site 5 instead of a radiolabeled brevetoxin. Responses were linear in fish tissues spiked from 0.1 to 1.0 ppb with Pacific ciguatoxin-3C (P-CTX-3C) with a detection limit of 0.075 ppb. Carribean ciguatoxins were confirmed in Caribbean fish by LC-MS/MS analysis of the regional biomarker (C-CTX-1). Fish (N = 61) of six different species were screened using the RBA_(F). Results for corresponding samples analyzed using the neuroblastoma cell-based assay (CBA-N2a) correlated well (R² = 0.71) with those of the RBA_(F), given the low levels of CTX present in positive fish. Data analyses also showed the resulting toxicity levels of P-CTX-3C equivalents determined by CBA-N2a were consistently lower than the RBA_(F) affinities expressed as % binding equivalents, indicating that a given amount of toxin bound to the site 5 receptors translates into corresponding lower cytotoxicity. Consequently, the RBA_(F), which takes approximately two hours to perform, provides a generous estimate relative to the widely used CBA-N2a which requires 2.5 days to complete. Other RBA_(F) advantages include the long-term (> 5 years) stability of the BODIPY^®- PbTx-2 and having similar results as the commonly used RBA_(R). The RBA_(F) is cost-effective, allows high sample throughput, and is well-suited for routine CTX monitoring programs.

Blooms of the toxic microalga Karenia brevis occur seasonally in Florida, Texas and other portions of the Gulf of Mexico. Brevetoxins produced during Karenia blooms can cause neurotoxic shellfish poisoning in humans, massive fish kills, and the death of marine mammals and birds. Brevetoxin-containing aerosols are an additional problem, having a severe impact on beachgoers, triggering coughing, eye and throat irritation in healthy individuals, and more serious respiratory distress in those with asthma or other breathing disorders. The blooms and associated aerosol impacts are patchy in nature, often affecting one beach but having no impact on an adjacent beach. To provide timely information to visitors about which beaches are low-risk, we developed HABscope; a low cost (~$400) microscope system that can be used in the field by citizen scientists with cell phones to enumerate K. brevis cell concentrations in the water along each beach. The HABscope system operates by capturing short videos of collected water samples and uploading them to a central server for rapid enumeration of K. brevis cells using calibrated recognition software. The HABscope has a detection threshold of about 100,000 cells, which is the point when respiratory risk becomes evident. Higher concentrations are reliably estimated up to 10 million cells L^-1. When deployed by volunteer citizen scientists, the HABscope consistently distinguished low, medium, and high concentrations of cells in the water. The volunteers were able to collect data on most days during a severe bloom. This indicates that the HABscope can provide an effective capability to significantly increase the sampling coverage during Karenia brevis blooms.

Paralytic shellfish poisoning (PSP) is precipitated by a family of toxins produced by harmful algae, which are consumed by filter-feeding and commercially popular shellfish. The toxins, including saxitoxin, neosaxitoxin, and gonyautoxins, accumulate in shellfish and cause intoxication when consumed by humans and animals. Symptoms can range from minor neurological dysfunction to respiratory distress and death. There are over 40 different chemical congeners of saxitoxin and its analogs, many of which are toxic and many of which have low toxicity or are non-toxic. This makes accurate toxicity assessment difficult and complicates decisions regarding whether or not shellfish are safe to consume. In this study, we describe a new antibody-based bioassay that is able to detect toxic congeners (saxitoxin, neosaxitoxin, and gonyautoxins) with little cross-reactivity with the low or non-toxic congeners (decarbamoylated or di-sulfated forms). The anti-saxitoxin antibody used in this assay detects saxitoxin and neosaxitoxin, the two most toxic congers equally well, but not the relatively highly toxic gonyautoxins. By incorporating an incubation step with L-cysteine, it is possible to convert a majority of the gonyautoxins present to saxitoxin and neosaxitoxin, which are readily detected. The assay is, therefore, capable of detecting the most toxic PSP congeners found in commercially relevant shellfish. The assay was validated against samples whose toxicity was determined using standard HPLC methods and yielded a strong linear agreement between the methods, with R2 values of 0.94–0.96. As ELISAs are rapid, inexpensive, and easy-to-use, this new commercially available PSP ELISA represents an advance in technology allowing better safety management of the seafood supply and the ability to screen large numbers of samples that can occur when monitoring is increased substantially in response to toxic bloom events

Climate change-related ocean warming and reduction in Arctic sea ice extent, duration and thickness increase the risk of toxic blooms of the dinoflagellate Alexandrium catenella in the Alaskan Arctic. This algal species produces neurotoxins that impact marine wildlife health and cause the human illness known as paralytic shellfish poisoning (PSP). This study reports Paralytic Shellfish Toxin (PST) concentrations quantified in Arctic food web samples that include phytoplankton, zooplankton, benthic clams, benthic worms, and pelagic fish collected throughout summer 2019 during anomalously warm ocean conditions. PSTs (saxitoxin equivalents, STX eq.) were detected in all trophic levels with concentrations above the seafood safety regulatory limit (80 μg STX eq. 100 g^-1) in benthic clams collected offshore on the continental shelf in the Beaufort, Chukchi, and Bering Seas. Most notably, toxic benthic clams (Macoma calcarea) were found north of Saint Lawrence Island where Pacific walruses (Odobenus rosmarus) are known to forage for a variety of benthic species, including Macoma. Additionally, fecal samples collected from 13 walruses harvested for subsistence purposes near Saint Lawrence Island during March to May 2019, all contained detectable levels of STX, with fecal samples from two animals (78 and 72 μg STX eq. 100 g^-1) near the seafood safety regulatory limit. In contrast, 64% of fecal samples from zooplankton-feeding bowhead whales (n = 9) harvested between March and September 2019 in coastal waters of the Beaufort Sea near Utqiaġvik (formerly Barrow) and Kaktovik were toxin-positive, and those levels were significantly lower than in walruses (max bowhead 8.5 μg STX eq. 100 g^-1). This was consistent with the lower concentrations of PSTs found in regional zooplankton prey. Maximum ecologically-relevant daily toxin doses to walruses feeding on clams and bowhead whales feeding on zooplankton were estimated to be 21.5 and 0.7 μg STX eq. kg body weight^-1 day^-1, respectively, suggesting that walruses had higher PST exposures than bowhead whales. Average and maximum STX doses in walruses were in the range reported previously to cause illness and/or death in humans and humpback whales, while bowhead whale doses were well below those levels. These findings raise concerns regarding potential increases in PST/STX exposure risks and health impacts to Arctic marine mammals as ocean warming and sea ice reduction continue.