Table of Contents
- Cysts and Life Cycles of Harmful Dinoflagellates
- Introduction to Microalgal Culturing Techniques
- Identification and Taxonomy of Unarmored Dinoflagellates
- Identifying Harmful Diatoms
- Introduction to Interactive Key to Species
The workshop opened with an introduction covering the significance of HAB research, given the socioeconomic and environmental impact of HAB events, with special focus on the Western Pacific region, in which South East Asian countries are located.
With growing global population and demand and the decrease in fisheries output, mariculture has become a vital industry. However, the nature of aquaculture leads to the enrichment of coastal waters with unused nutrients, which are then taken advantage of by existing aquatic communities, leading to harmful algal blooms. HABs, in turn, may lead to massive losses through fish kills as well and can adversely affect human (consumer) health. It is therefore important to have well-documented and organized systems in place to monitor and disseminate information regarding these events.
In line with this, it is necessary to build the capacity to conduct these monitoring, management, and research activities within the region and to strengthen the sharing of information between the many countries involved, particularly since HABs issues may cross borders.
It is also important to improve understanding of bloom dynamics of harmful species in order to better tailor measures to mitigate the effects of their occurrence. A paper published in 2017, Life-history stages of natural bloom populations and the bloom dynamics of a tropical Asian ribotype of Alexandrium minutum (Lau WLS, et.al.), was also discussed in the presentation. The paper goes into detail regarding the mechanism of encystment and excystment via a study of a bloom event involving A. minutum in 2015.
A. minutum is of a genus known to produce a number of saxitoxins (STXs), a potent neurotoxin known to cause paralytic shellfish poisoning (PSP) upon consumption of contaminated shellfish by humans. The species has been noted to be particularly capable of adapting to adverse conditions via a number of mechanisms throughout its life cycle and stages of reproduction. Through carefully-planned sampling and analysis via microscopy and flow cytometry, the different life stages of A. minutum populations were studied and, together with environmental data and factors affecting or triggering stages such as cyst production and sexual reproduction led to understanding how algal blooms develop, persist, and are terminated. It was found that excystment contributes to the initiation and prolongation of bloom periods while fluctuating levels of salinity, temperatures, and available macronutrients were crucial to ending the bloom events.
Cysts and Life Cycles of Harmful Dinoflagellates
Certain species of dinoflagellates are known to produce cysts as part of their life cycle and reproduction. These cysts can accumulate and lie dormant until favourable conditions allow them to germinate and are thus capable of providing the initial population boost leading to algal blooms.
Historic studies were described, primarily focused on the discovery of general morphological features of cysts and the myriad biological functions they serve, including as a means of survival under unfavourable conditions, bloom initiation and termination, the ability of a species of a specific genetic make-up to spread over time and space, and how they can serve as seed banks preserving DNA. These lead to cysts having significant contributions to population, genetic diversity, and the distribution of dinoflagellate species.
Current studies have further explored the morphology and taxonomy of known cysts, identifying important and distinctive structures that can lead to identifying species and life stages while using various microscopic techniques.
It was also discussed that some of the challenges that come with their study is that dinocysts can have common morphological types for several species and that cyst-theca relationship is not completely known. As such identification/ taxonomy may require the use of genetic/ molecular techniques.
Introduction to Microalgal Culturing Techniques
Due to the nature of algae, one of the challenges in their study is obtaining and maintaining sufficient quantities of a species for purposes such as taxonomic confirmation and toxin analysis. Microalgal culturing then becomes a valuable technique to address this problem, providing an artificial environment approximating conditions in the wild as closely as possible that will allow algae to grow. The culture process can take from weeks upto months, beginning with single cell-isolation, culture establishment, and culture maintenance, until enough cells are obtained.
Prior to beginning culturing, it is important to consider the facilities available, the purpose for culturing, approach to culturing, and knowledge of the species to be cultured in order to achieve optimum conditions and ensure the favoured/ required nutrients are present. The source and ambient conditions where the target species thrives are very important in determining what culture conditions should be maintained and how culturing should be approached (e.g. what media to use, the temperature and salinity, etc.)
The lecture further describes the differences between different culture conditions, which are namely:
- Batch culture conditions, where cells are allowed to grow exponentially until the limited supply of nutrients are consumed and the growth cycle is completed;
- Semi-continuous culture, which is kept at exponential growth by sub-culturing (taking an aliquot and adding fresh media for further culture) within every few generations to reduce cell density; and
- Continuous cultures, where cultures are kept at constant conditions and are more complicated to maintain.
The lecture also covered the necessary preparations to be carried out prior to culture as well as the different cell isolation techniques such as dilution, agar streaking, centrifugation, sieving, and micropipetting (among others).
Lab Practical 1&2: Exposure to basic techniques in microalgal culturing
Sterilization and sterile techniques/ Preparation of Culture Media
The participants were able to observe demonstrations and participate in limited hands-on exercises in the following activities:
- Cleaning culture vessels: Culture vessels and plugs/covers must be sterilized and autoclaved prior to use. Specific techniques and conditions were discussed (wet autoclaving, preparation of plugs). The choice of culture vessels was also discussed (must be chemically inert and non-toxic, reasonably transparent, easy to clean, and with a large surface area to volume ratio;
- Seawater preparation: Natural seawater is used as the base of all culture media and as such is aged in darkness for several months to allow living microorganisms to die, filtered via cartridge (0.2 microns) and/or via membrane filters (typically nitrocellulose though type may vary depending on purpose and source) or glass fiber filters. Filtration is done in order to remove unwanted organisms to ensure pure cultures. The seawater is also tested for salinity and adjusted in accordance with the optimum salinity for its intended purpose. The seawater is also sterilized and allowed to equilibrate prior to use to allow the diffusion of gas.
- Media Preparation: Different kinds of media are utilized for different species of algae, based on their individual needs in terms of nutrients. Formulas for several kinds of media (ES-DK, f/2, L1, etc.) were given, along with the treatment, handling and storage of each and all their stock components. Instructions for the above are detailed in the handouts given to each participant. Sensitive components such as vitamins must be sterilized via filtering (not autoclaved).
- Culture facility and conditions: Cultures are kept in chambers observing light/dark cycles (ratio of L:D dependent on species) and temperature-controlled. Transferring cultures/subcultures must also observe specific conditions (preferably under laminar flow and with sterile surfaces and implements, with an open flame).
Identification and Taxonomy of Unarmored Dinoflagellates
A number of microalgae causing red tide and toxicity belong to the unarmored class of dinoflagellates which are species of naked dinoflagellates without the so-called thecal plates.
Their lack of thecal plates allows for identification of living samples using light microscopy by observing the position, size, shape, presence, and torsion of organelles and cellular features such as the cingulum, sulcus, nucleus, and chloroplast. The cingulum, which accommodates the transverse flagellum, and its properties are notable as the main diagnostic character upon which previous taxonomy of unarmored dinoflagellates were based. Scanning electron microscopy can also allow identification via the apical groove. Other identifying traits include whether the species forms cell chains or not.
Common species (e.g. Karlodinium, Cochlodinium, Takayama, etc.) and their identifying characteristics were presented during the lecture.
Identifying Harmful Diatoms
Similar to the previous lecture, this covered and discussed the various characteristics of diatoms observable via microscopy, using common species as examples. It also gave examples of harmful diatoms which can cause fish kills through interfering with biological processes solely by their physical properties. Examples of toxin-producing diatoms were also given, with particular attention to toxic species of Pseudo-nitzschia and Nitzschia, a group of diatoms producing domoic acid and responsible for Amnesic Shellfish Poisoning in humans. Extensive details about important morphological characteristics useful in identifying species of Pseudo-nitzschia and Nitzschia confirmed to be toxic were presented. The action limits (20 mg/kg wet weight) and symptoms of domoic acid toxicity (mostly affecting neurological functions) were also discussed.
Introduction to Interactive Key to Species
With the growing number of plankton species discovered as well as the amount of resources available, it has become not only convenient but also sensible to take advantage of computer-based programs, resources, and databases. Such programs are known as Interactive Keys to Species (IKS), as opposed to traditional means of identifying the taxonomy of biological entities.
Lab Practical 3: Interactive key in species identification: Identifying species using the 3I key (Pseudo-nitzschia sp.)
The 3I key, a free, open access platform, was chosen as the Interactive Key to Species to be discussed for the purpose of the lab practical and which the participants worked with during the hands-on session.
Participants were given files containing micrographs of unidentified 12 plankton samples determined to be of the genus Pseudo-nitzschia and tasked to identify their species. The 3I key to species makes use of a taxonomical database which includes such information as species profiles, micrographs of species, auto-generated descriptions, distribution maps, study materials, and references which can be searched and used to compare to available data on hand for the species to be identified.