Quantity of the dinoflagellate sxtA4 gene and cell density correlates with paralytic shellfish toxin production in Alexandrium ostenfeldii blooms Henna Savela a, *, Kirsi Harju d , Lisa Spoof b , Elin Lindehoff e , Jussi Meriluoto b , Markus Vehnia¨ inen a , Anke Kremp c a Biotechnology, Department of Biochemistry, University of Turku, Tykisto¨katu 6 A 6th Floor, FI-20520 Turku, Finland b Biochemistry, Faculty of Natural Science and Engineering, A ˚ bo Akademi University, Tykisto¨katu 6A 3rd Floor, FI-20520 Turku, Finland c Marine Research Centre, Finnish Environment Institute, Erik Palme´nin aukio 1, FI-00560 Helsinki, Finland d VERIFIN – Finnish Institute for Verification of the Chemical Weapons Convention, Department of Chemistry, P.O. Box 55, FI-00014 University of Helsinki, Finland e Ecology and Evolution in Microbial model System (EEMiS), Department of Biology and Environmental Science (BoM), Linnæus University, Kalmar 39182, Sweden 1. Introduction Marine dinoflagellates are known for their ability to produce numerous highly toxic compounds. The best-known are saxitox- ins, a group of potent neurotoxins including saxitoxin itself and its closely related structural analogues. These small hydrophilic alkaloids are also known as paralytic shellfish toxins (PST) due to their tendency to accumulate in the food chain, mainly to filter- feeding bivalves which can then act as vectors of PST poisoning. In mammals the toxins act by reversibly blocking voltage-gated sodium channels (Catterall, 1980), thus inhibiting the transmis- sion of neuronal signals. Symptoms are dose-dependent, and range from slight numbness and tingling of face and extremities to total neuromuscular paralysis, respiratory arrest and death (Llewellyn, 2006). The estimated mortality rate of PST poisoning can be as high as 15% (Hallegraeff, 2003), and treatment is limited to supportive care. Most developed countries with commercial shellfish farming have set monitoring programs in place to prevent food-related PST poisoning. Major economic losses are suffered due to the combined public health costs and forced harvesting closures of fish and mussel farms (Hoagland and Scatasta, 2006). Harmful Algae 52 (2016) 1–10 A R T I C L E I N F O Article history: Received 23 June 2015 Received in revised form 29 October 2015 Accepted 29 October 2015 Keywords: Dinoflagellate qPCR paralytic shellfish toxin Alexandrium sxtA4 A B S T R A C T Many marine dinoflagellates, including several species of the genus Alexandrium, Gymnodinium catenatum, and Pyrodinium bahamense are known for their capability to produce paralytic shellfish toxins (PST), which can cause severe, most often food-related poisoning. The recent discovery of the first PST biosynthesis genes has laid the foundation for the development of molecular detection methods for monitoring and study of PST-producing dinoflagellates. In this study, a probe-based qPCR method for the detection and quantification of the sxtA4 gene present in Alexandrium spp. and Gymnodinium catenatum was designed. The focus was on Alexandrium ostenfeldii, a species which recurrently forms dense toxic blooms in areas within the Baltic Sea. A consistent, positive correlation between the presence of sxtA4 and PST biosynthesis was observed, and the species was found to maintain PST production with an average of 6 genomic copies of sxtA4. In August 2014, A. ostenfeldii populations were studied for cell densities, PST production, as well as sxtA4 and species-specific LSU copy numbers in Fo¨ glo¨, A ˚ land, Finland, where an exceptionally dense bloom, consisting of 6.3  10 6 cells L 1 , was observed. Cell concentrations, and copy numbers of both of the target genes were positively correlated with total STX, GTX2, and GTX3 concentrations in the environment, the cell density predicting toxin concentrations with the best accuracy (Spearman’s r = 0.93, p < 0.01). The results indicated that all A. ostenfeldii cells in the blooms harbored the genetic capability of PST production, making the detection of sxtA4 a good indicator of toxicity. ß 2015 Elsevier B.V. All rights reserved. * Corresponding author. E-mail address: henna.savela@utu.fi (H. Savela). Contents lists available at ScienceDirect Harmful Algae jo u rn al h om epag e: ww w.els evier.c o m/lo cat e/hal http://dx.doi.org/10.1016/j.hal.2015.10.018 1568-9883/ß 2015 Elsevier B.V. All rights reserved.