present in a sample then qPCR would miss those probably due to lack of a developed assay. However, LM does require high levels of taxonomic skills and the precision in identification is only as accurate as the taxonomist allows. Different taxonomists trained in different ways using different identification literature can cause large person-person differences. The ease of identification is also species dependent. For example highly plastic species, or those with a variable life cycle are harder to identify and can often be easily misidentified. The naked form of Dictyocha speculum can easily be confused with the rounded cells of Pseudochattonella. Some species of the genus Alexandrium cannot be identified to species level due their very subtle morphological differences in their thecal plates. When dealing with toxic species, false positives are less problematic but can cause substantial financial losses if they result in the closure of a fishery, but when toxic or problematic organisms are missed completely this could have dire consequences. To reduce confusion, each taxonomist should be provided with a checklist of common species with up to date taxonomic names. Undertaking frequent inter-comparison exercises, e.g. the ring test or the International Phytoplankton Intercomparison (IPI) exercise, provide feedback on how individual laboratories and taxonomists perform. This forum also provides an opportunity to convene a discussion on nomenclatural changes and new technological advances in monitoring techniques. As error can be introduced in various different forms, in order to get the most accurate and reproducible results each individual step from: collection, storage, subsampling, homogenisation, filling the chamber, settling and counting strategies all require their own standardised protocols. All aspects of the protocol need to be considered from the storage containers to the type of fixative used. Many cells e.g. Pseudochatonella spp. are sticky and can adhere to plastic walls and as plastic bottles is often preferred over glass especially when transporting samples this can become problematic for accurate cell enumeration. The choice of preservative is important and often the optimal preservation methods 26 are taxa specific. It is not always easy to obtain reliable estimates from fixed material; preservatives can alter the sample in various ways creating a biased measurement. Lugols iodine [4] has long been the fixative of choice due to its relatively low toxicity and high stability. However, it is known to introduce artefacts such as changes to cell size, a reduction in cell number and in some instances it may fail to preserve certain taxa all together [5-6]. Each alternative fixative comes with its own issues [7]. Settling chambers themselves can be another source of variation. For reliable cell counts, specimens must be completely randomly distributed within the chamber. If cells do not follow a poisson distribution then it will bias the enumeration and any statistical analysis will be affected. To prevent uneven settling, the samples must be at a constant temperature during the settling period. For a higher chance of getting a well-mixed distribution then samples must first be homogenised. The best way to homogenise a sample is the Paul-Schatz figure of eight rotation method where samples are mixed 100 times in a rhythmic pulsating motion. Even when all precautions are taken, it is still almost impossible for cells to be randomly distributed due to issues such as cell clumping caused by polysaccharide fibrils or inconsistent settling conditions. Due to radial abundance gradients cell abundances at the periphery can be up to 50% lower than at the center, causing a settling bias. Uneven settling will affect the counting strategy. For any counting strategy a predetermined number of units must be observed. The number of units differs depending on the organism and the research objectives. Typically to reach an accuracy of 10% at least 400 cells must be counted [8]. Whole chamber cell counts should be carried out where possible but other counting strategies are often used such as transects and random fields. qPCR When carefully designed, with optimisation and validation, qPCR assays are highly accurate and sensitive, but without due care and optimisation, qPCR can be plagued by reproducibility and reliability problems. The quality of the starting material is one of the key determinants for obtaining reliable and reproducible data. As with microscopy fixatives and storage techniques play a large part in quality of the samples. Fortunately for short term storage Lugols iodine is the most ideal fixative and the same sample can be used for both microscopic and qPCR analysis [7]. Before amplification DNA must be extracted from the cellular material and commonly commercial extraction kits are applied. To get purified genomic DNA the sample must undergo a number of steps to lyse the cells, remove contaminants and purify the resulting DNA. In cases where the purification step is inefficient the resulting DNA may not be representative of the sample and/or contain compound(s) that will cause assay interference. In these cases the performance of the reaction will be sub-optimal, causing a reduction in the sensitivity and/or amplification efficiency. Inhibition of amplification can occur in different ways. Firstly, when high molecular weight compounds, e.g. humic acids or complex carbohydrates, combine with metal ions to sequester the nucleic acids away from the polymerases and prevent amplification. While some molecules block or inhibit the polymerase or alter the specificity of the primers, inhibitors which block or delay polymerase activity are highly problematic and they can lead to an underestimation of material in the sample or false negatives [9]. Typical approaches to combat inhibition include alternative DNA extraction kits, dilution, specialised polymerases, addition of adjuvants and internal controls. When designing a qPCR assay it is important to select an appropriate target and to design specific primers with no cross reactivity with other organisms. This study used previously validated species-specific hydrolysis probes in combination with primers to add an extra level of specificity. To ensure accuracy, time is required to optimise the efficiency of the assay and validate it multiple times. This sometimes means altering the constituents of the master-mix used, e.g. nucleotides, magnesium chloride or polymerase concentrations. This is extremely relevant when tackling issues arising from multiplexing assays [9-10]. Once optimised users can still face a number of precision related challenges. HARMFUL ALGAE NEWS NO. 59 / 2018 Harmful Algae News An IOC Newsletter on Toxic Algae and Algal Blooms No. 59 - February 2018 www.ioc-unesco.org/hab Content 25 years of HAN and IPHAB...... 1 25 YEARS Harmful Algae News was first published in early 1992 in response to requests from the participants at a number of IOC meetings and pacted by harmful algal events. Since Harmful Algae News turned 20 years old in 2012, it has been a web based e-newsletter which meant longer issues were possible and back issues easily accessible. We are currently working on a searchable index for all Harmful Algae News issues. The start of Harmful Intergovernmental Panel on Harmful Algal Blooms also turned 25! During 1992, the same Year as the IOC published the first issue of Harmful Algal News, it also established an Intergovernmental Panel on Harmful Algal Blooms (IPHAB) which has met every second year since it was formed. The Panel is com A retrospective look at the early days of HAB cyst research, and a look to the future On this occasion of the 25th anniversary of the publication of Harmful Algae News, several of us were asked to look backwards in time to some of the earlier days of HAB science. One area of study that has been a ma dormant stages and that these stages might be associated with certain bottom sediments. This then brings up the question, if benthic resting stages of certain dinoflagellates actually seed coastal red tides, are there localized areas of accumulation, or what we could call seedbeds?..........The poss day, we still do not know if there are other factors at work perhaps a density-dependent or quorum-sensing type of response, or even a response to the presence of grazers or parasites. Exploration of this response has long been limited by the constraints associated with laboratory cultures, but now approach did not stand up to data at my study locations. I raise this issue in this narrative because I want to correct what I feel are unjustified recommendations that may prevent those working on cysts from obtaining the type of biological data that can advance our understanding of certain types o number of cysts in subsurface layers unable to germinate or emerge, presumably due to lack of oxygen or to the tortuous pathway posed by sediment grains and detritus. Many might think that major storms and waves can erode significant layers of sediment and transport cysts long distances, but here ag How do algal blooms kill finfish and how can we mitigate their impacts? Algal blooms, water discolorations and their association with fish kills have been recorded since historic times, such as the description in the Bible (1000 years BC) all the waters that were in the river were turned to blood. A Table. 1. Economic losses from algal blooms for finfish aquaculture in different parts of the world HAB species Chattonella Heterosigma Cochlodinium polykrikoides Heterosigma Chaetoceros Heterosigma Karenia digitata Karenia mikimotoi Country Financial Losses Japan Korea, China Canada British Col emergency harvest operations. To prevent the buildup of histamines, fish should be kept alive as long as possible during harvesting. This can be achieved by diluting algal concentrations via airlift upwelling, or by targeted in-pen emergency application of clays [23] that mop up ichthyotoxins at cla As part of the 25th anniversary issue of Harmful Algal News I am providing an overview of the IOC-UNESCO Taxonomic Reference List of Harmful Algae (www. marinespecies.org/hab/) and will highlight some of the problems which have faced or are facing the Intergovernmental Panel on Harmful Algal Blooms Dinophyceae the authors intended the new genus to be described according to the botanical nomenclature. The lack of a Latin diagnosis therefore made it invalid. The confusion has actually still not been resolved, and the problem needs to be discussed and decided upon by the International Nomenclatur geographically widespread species being able to form fertile offspring, while other populations of the same species are not. Molecular techniques have contributed very significantly to solving many taxonomic problems, but they have not resulted in the emergence of a finite species concept. We have t Algal toxin discovery, management and regulation over the last 25 years Algal toxins in the dark ages (pre-1992) From a historic perspective, knowledge about algal toxins can be divided into truly prehistoric occurrences such as known from paleontological studies [1-2] and more recent historic recor lar rapid increase in known analogues has been observed for the azaspiracid (AZA) group, with the first analogue described in 1998 [64] and a review in 2014 reporting 30 analogues [65]. Only three years later, over 50 analogues are known for this group, including novel phosphate derivatives [66-71]. Butterflies in Brazil Abstracts are not always reliable guides to authors intentions. They are not expected to reveal a great deal about the evidence to be deployed in support of the science, evidence that may not even exist before deadlines for writing abstracts! Nevertheless, as examples of a mino on a decadal time scale, and identified palaeoclimatic oscillations are not necessarily a useful guide to its interpretation. An obvious obstacle to detecting climate signals in HAB data is posed by anthropogenic eutrophication. Another obstacle is the fact that phytoplankton respond directly to the trends, Karenia brevis might appear more often in the South Atlantic Bight of the US and Gymnodinium catenatum bloom more often in northwestern Iberian waters. There was also a warning by Barrie Dale germane to such speculations, that large scale climate models cannot predict local changes. Little m Red tides in Kamchatka coastal waters (Bering Sea, Russia) are a barrier for the salmon fishery and Pacific salmon Fig. 1. Map of Olyutorskiy Bay (Kamchatka, Bering Sea) where a red tide, reported by fishermen, occurred in July 2017. The stars denote fishery sites: red, affected by the bloom; green Fig. 2. Red tide in Olyutorskiy Bay on 15th July 2017 grounds of the Olyutorskiy Bay river basin performed during the second half of August showed an atypical distribution in the rivers. Maximal escapes were recorded in river basins located in the western and eastern parts of the Olyutorskiy Bay ar First report of Gambierdiscus in the Western Mediterranean Sea (Balearic Islands) Gambierdiscus (Dinophyceae) species are benthic dinoflagellates living in marine littoral zones of circumtropical areas and have recently been described in temperate waters [1]. Some species are producers of potent neu the SEASENSING (BIO2014-56024C2-2-R) project and the CERCA Programme/Generalitat de Catalunya. A. Tudó and A. Toldrà acknowledge IRTAURV-Banco Santander for their respective PhD grants (2016 PMF-PIPF-74 and 2015PMF-PIPF-67). The authors are grateful to Vanessa Castan and José Luis Costa for sampling Comparison by light microscopy and qPCR of potentially ichthyotoxic microalgae in Danish on-shore lagoons producing European flounder (Platichthys flesus): Pros and cons of microscopical and molecular methods Fig. 1. Lagoon used for production of European flounder at Fishlab, Denmark. Evaluation o Fig. 2. Comparison of phytoplankton species identification and cell densities (cells L-1) by qPCR (A and C) and light microscopy (target species and groups which potentially could comprise ichthyotoxic organisms) (B and D) in lagoon 1 (A and B) and lagoon 6 (C and D), respectively. The right Y-axis present in a sample then qPCR would miss those probably due to lack of a developed assay. However, LM does require high levels of taxonomic skills and the precision in identification is only as accurate as the taxonomist allows. Different taxonomists trained in different ways using different identif As qPCR measures genetic material rather than viable cells an over estimation of cell numbers can occur due to the inclusion of dead or dying cells. Problems may also occur when targeting multiple copy genes where the organism carries different numbers of the target depending on nutritional status, The Cawthron Institute Culture Collection of Micro-algae (CICCM) The CICCM is designated as a nationally significant database by the New Zealand government and so receives partial funding for its continued existence. Isolates from 13 classes of micro-algae are maintained either as live cultures or c The XVIII International Conference on Harmful Algae is approaching! It is time for nominations for achievement awards (Yasumoto Life Time and Patrick Gentien Young Scientist), registration to the conference as a student if you wish to participate in the Maureen Keller Award competition and fundraisi Forthcoming Events First announcement of the 11th International Conference on Toxic Cyanobacteria (ICTC) We are pleased to disseminate the first announcement of the 11th International Conference on Toxic Cyanobacteria (ICTC) that will be held in Krakow, Poland from May 5 10, 2019. The ICTC is a per International Coordination of Research on Harmful Algal Blooms From GEOHAB to GlobalHAB International cooperation is fundamental to advance understanding of HAB dynamics and to improve our ability to predict them. Fostering this international cooperation was the mission of GEOHAB (Global Ecology and 18th International Conference on Harmful Algae www.icha2018.com IMPORTANT DEADLINES Abstract submission deadline: 15 April 2018 Early bird registration: 15 July 2018 Get the 17 ICHA Proceedings at www.issha.org Eds-in-chief Beatriz Reguera, IEO, Vigo, Spain Eilen Bresnan, MARLAB, Scotland, UK Regi