Overall objective: To characterize the genetic and environmental basis for toxin production, to determine the mode of action of selected toxins, and to address several limitations in toxin analysis and field detection.
Rationale. Toxins are a cross-cutting subject and a common concern through all the GlobalHAB themes. In consideration of their importance, GlobalHAB includes a specific topic on toxins to facilitate addressing fundamental and applied objectives that would be beneficial to the scientific community, and to management and regulatory authorities. Research should concern some of the aspects already indicated in the Biogeography and Biodiversity, Adaptive Strategies, Nutrients and Eutrophication themes and the ones specified here. The research objectives are focused on known phycotoxins as well as “rare” or “emerging” toxins such as gonyodomines and gymnodomines since the causative organisms may be expanding (see related references in Berdalet et al. 2016).
A first challenge is to obtain comprehensive understanding of toxin biosynthesis pathways and the genes involved. This knowledge is necessary to better predict the probable impact of changes in the global marine environment on the toxicity of HAB species. Knowledge of the genetic basis of toxin production could be revealed if there are suitable target genes for the discrimination of toxic species and their detection in situ (e.g., Murray et al. 2011; Farrell et al. 2016). Additionally, such knowledge will lead to better understanding of environmental controls of toxin production. Significant progress has been made in this regard for certain known and emerging phycotoxins, although knowledge gaps still exist (see e.g., Kellmann et al. 2008 for saxitoxin; Krüger et al. 2010 for ß-methylamino-L-alanine [BMAA]; Savela et al. 2014 for microcystin; Jeffery et al. 2004 for domoic acid; Kohli et al 2015 for polyketides), while no studies have addressed okadaic acid so far. The development of metabolomics from both intra- and extracellular compounds produced by HABs would bring great advances in that field (Pisapia et al. 2017). The role of different toxin analogues in species that produce multiple toxin families, and their biostability for overall toxicity, are also aspects that require further attention (e.g., cyanobacteria that produce combinations of paralytic shellfish toxins (PST) and hepatotoxins; Pearson et al. 2016).
A second challenge is identification of the mode of action of microalgae-produced toxins. After decades of research, it is now fairly well understood that many of these toxins are neurotoxic to mammals, operating through the blockage or activation of sodium or calcium channels (e.g., Durán-Riveroll et al. 2016). Many algal species are known to be cytotoxic, allelopathic (Legrand et al. 2003) or hemolytic; however, the nature and mode of actions in other organisms remain unclear. Along with identification of unknown emerging toxins, toxicity tests should be performed on different target cells, as some toxins may have different targets in nature.
Above: Razor clams harvested from the Washington State, USA, Pacific coast beaches. These clams can retain the toxin, domoic acid, for many months. Photo credit: Vera Trainer, NOAA.
Right: Fish kills caused by Karlodinium australe bloom. Photo: P.T. Lim, University of Malaya.
In the case of fish-killing species, such as Margalefidinium (=Cochlodinium) polykrikoides, Chattonella spp., Pseudochattonella spp., Heterosigma akashiwo, and Karlodinium australe (e.g., Lim et al. 2014, Wagoner et al. 2014) the toxins produced could act through physical damage, hypoxia or anoxia, ichtyotoxicity, or synergistic effects through known or unknown direct biological effects (Black et al. 1991). The mechanism of fish-kill events associated with the colonial diatom species, Leptocylindrus minimus (Clément and Lembeye 1993), Skeletonema costatum, and Thalassiosira spp. (Kent et al. 1995) required further clarification. This information is crucial in the development of countermeasures by the aquaculture industry in dealing with massive fish-kill events due to different microalgal blooms. However, establishing general mitigation strategies is difficult because the impacts can vary among regions. For example, in 2002, Prorocentrum minimum blooms in the Philippines caused massive fish kills (Azanza et al. 2005), but similar blooms in Johor Strait, Malaysia did not cause any losses to local aquaculture (Usup et al. 2003).
A third need is the availability of sensitive, accurate, and cost-effective means of microalgal toxin analysis for the protection of public health and food security, and sustainability of aquaculture. Progress has been made in the detection and characterization of several microalgal toxins due to the advancement in analytical techniques, cell-based and functional assays. However, official methods are not yet available for some toxins, with ciguatoxin being the most notable.
Over the past two decades, live animal bioassays for microalgal toxins have gradually been replaced by analytical methods and in vitro assays, such as the neuroblastoma (N2A) assay, and sodium and calcium channel receptor-binding assay-RBA (e.g., PSTs, Oshima 1995, Turner et al. 2015; ciguatoxins, Yogi et al. 2011; karlotoxin, Wagoner et al. 2008). While several of these analytical and in vitro methods still require further development and validation in order to be accepted as official methods, they do offer the sensitivity, accuracy and high throughput needed for regulatory purposes. More studies are needed to get these methods accepted as official methods, and improve their robustness and user-friendliness. Similar efforts are required for the extraction of problematic toxins, such as CTX. A very important aspect of detecting and quantifying toxins is the availability of pure toxins, certified reference materials and secondary standards in the case of RBA. The need is most urgent for the two arguably most important toxin groups, namely PSP and CFP toxins.
A major problem faced by many toxin-testing laboratories, particularly in developing countries, is the inability to cope with the financial and staffing demands of testing large numbers of fish and shellfish samples in order to produce results in a timely manner. Under normal conditions most of those samples could be negative to toxin presence in seafood and do not require detailed testing. Thus, it would be highly desirable to make available reliable and affordable toxin screening kits that could be used on site or in laboratories with minimal facilities.
Specific objectives
- Determine the genetic basis of, and environmental influence on, toxin production and gene expression of toxin-producing algae.
- Establish the modes of action of the known and unknown emerging toxins and their activities (cytotoxic, allelopathic or hemolytic) on different aquatic organisms (fish, shellfish).
- Encourage production of certified standards and reference material for all major microalgal toxins.
- Promote and facilitate inter-laboratory validation studies that lead to acceptance of toxin analysis methods as official methods.
- Establish working linkages with different agencies in the development of CFP toxin detection and monitoring methods, including production of toxin reference material.
Example tasks
- Revision of the current state of knowledge and gaps on the genetic basis of selected microalgae toxicity, and its modulation by environmental factors; identification of the technology and methods availability and needs for progress in this area including toxin gene detection in the field.
- Revision of the current knowledge and priority research areas on the mode of action of toxins of fish-killing HABs and genetic data relevant to HAB toxicity.
- Endorsement of research on new instrumental methods of toxin analysis and support of inter-laboratory trials and validation studies to prove their efficacy and enable their acceptance as accredited methods by international food safety authorities.
- GlobalHAB will encourage the organization of training and inter-laboratory validation of rapid methods of analysis using toxin test kits.
Outcomes
- An updated list with inclusion of toxin-producing and ichthyotoxic harmful algal species in Algae Base and IOC Taxonomic Reference list of Harmful Algae.
- A document on the genetic basis of and environmental influence on selected microalgae toxicity, summarizing priority research areas, best practice guidelines for research, and including a list of Task Team experts.
- Publication of the results of the validation studies on toxin kits or other analytical approaches in appropriate journals (e.g., Journal of the Association of Official Analytical Chemists).
- INFORMATION FOR POLICY MAKERS and aquatic products industry on the impacts of HABs in marine fish and shellfish aquaculture and fisheries activities.
References
The complete list of references can be found here.
