Developments in the Cryopreservation of Marine Dinoflagellates

Abstract

<p><strong>Dinoflagellates are a taxonomically and functionally diverse groups of microalgae. Many strains have been isolated and cultured for scientific, ecological, industrial, pharmaceutical, and agricultural applications. Systematic maintenance of these culture collections may affect their long-term viability, often resulting in changes in their gene functions or contamination from other microorganisms. There is an urgent need to establish a cheap and effective way to store and protect these microalgal cultures for an extended period. In this study, I explored preservation protocols that can be used for the long-term maintenance of different dinoflagellate species especially those belonging to family Symbiodiniaceae. Cryopreservation allows long term storage, and the preserved isolates maintain their morphological, biochemical, and genetic properties, hence protecting their diversity. However, cryopreservation of dinoflagellates is challenging. I first explored the effects of different types of cryoprotectant agents (CPAs) and two freezing methods on three species: Vulcanodinium rugosum, Alexandrium pacificum and Breviolum sp. A total of 12 CPA treatments were assessed at concentrations between 5 - 15%, as well as in combination with dimethyl sulfoxide (DMSO) and other non-penetrating CPAs. Two freezing techniques were employed: rapid freezing and controlled-rate freezing. Breviolum sp. was successfully cryopreserved using 15% DMSO. Breviolum sp. cryopreserved with higher cell viability (45.4±2.2 (standard deviation) %) when using the controlled-rate freezing compared to the rapid freezing technique (10.0±2.8%). Despite exploring different CPAs and optimizing the freezing techniques, V. rugosum and A. pacificum could not be cryopreserved.</strong></p><p>After optimizing a cryopreservation protocol with dimethyl sulfoxide as the preferred CPA, 15 Symbiodiniaceae culture isolates from six genera were selected. Using 15% DMSO, 10 isolates were successfully cryopreserved using either rapid freezing or controlled-rate freezing. Cultures that failed to cryopreserve or had low survival post-thawing, were subjected to (i) a reduction of CPA to 10%, or (ii) increased salinity treatment before freezing. At 10% DMSO, three further isolates were successfully cryopreserved. At 15% DMSO there were high cell viabilities in Symbiodinium pilosum treated with 44 parts per thousand (ppt) and 54 ppt culture medium. Fugacium sp. successfully cryopreserved after salinity treatments of 54 ppt and 64 ppt. Descriptive fatty acid (FA) analyses of the increased salinity treated Symbiodiniaceae isolates showed changes in their FA production.</p><p>Symbiodinium pilosum was selected for further analysis due to its higher survival rates after pre-treatment in media with increased salinity. Investigations into FA production and the genes involved in FA biosynthesis and degradation pathways after both standard salinity (SS, 34 ppt) and increased salinity (IS, 54 ppt) treatments during freezing were carried out. There was a twofold increase in the mass of FAs produced by the isolate after treatment with IS media set at 54 ppt. Addition of DMSO and incubation before cryopreservation led to a further nine-fold increase in the mass of FAs produced in the SS treatment as compared to a five-fold increase in the IS treatment. Transcriptomic analyses of genes associated with FA biosynthesis showed the acyl carrier protein gene (acpP; K02078) was significantly upregulated after DMSO treatment in the SS cultures. The cytochrome P450 /NADPH-cytochrome P450 reductase gene (K14338) significantly downregulated after DMSO addition in the SS treatment, and this is likely to have prevented the degradation of FAs. These changes in the expression of FA biosynthesis and degradation genes contributed to the formation and increase of FAs in SS grown isolates as compared to IS treated isolates. The high amount of PUFAs in the IS treatments might have provided energy to the cells during high salinity and DMSO stress leading to higher cell survival during cryopreservation as compared to SS treated isolates.</p><p>The growth rates and photosynthetic efficiency of two isolates, Breviolum psygmophilum and Effrenium voratum before and after cryopreservation were also investigated. Rapid light curves (RLCs) produced using Pulse Amplitude Modulated Fluorometry (PAM) were used to characterise photosystem II (PSII). The maximum electron transport rate (ETRmax) and the quantum yield (Fv/Fm) of the control (non-cryopreserved) and cryopreserved culture isolates were assessed across the growth cycle. The non-cryopreserved isolate of B. psygmophilum had a higher quantum yield than the cryopreserved isolate from day 12 to day 24, but there were no differences from day 28 to the late stationary phase. No significant differences were observed in quantum yield or ETRmax between the control and cryopreserved E. voratum isolates. Overall, this thesis has developed and optimised cryopreservation protocol for Symbiodiniaceae isolates from a variety of different genera. Constant disruptions to major marine habitats through global climatic changes threatens the diversity of the Symbiodiniaceae and freezing these species will ensure they are kept alive indefinitely, protecting them for future ecological restoration of coral reef habitats.</p>