Cryoprotectant treatment tests on three morphologically diverse marine dinoflagellates and the cryopreservation of Breviolum sp. (Symbiodiniaceae)

Joseph Kanyi Kihika,Ken G Ryan,Kirsty F Smith,Susanna A Wood,Lucy Thompson,Sarah Challenger,Lesley Rhodes

doi:10.1038/s41598-021-04227-2

Joseph Kanyi Kihika, Ken G Ryan + Show 5 more

Open Access

https://doi.org/10.1038/s41598-021-04227-2

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Abstract

Dinoflagellates are among the most diverse group of microalgae. Many dinoflagellate species have been isolated and cultured, and these are used for scientific, industrial, pharmaceutical, and agricultural applications. Maintaining cultures is time-consuming, expensive, and there is a risk of contamination or genetic drift. Cryopreservation offers an efficient means for their long-term preservation. Cryopreservation of larger dinoflagellate species is challenging and to date there has been only limited success. In this study, we explored the effect of cryoprotectant agents (CPAs) and freezing methods on three species: Vulcanodinium rugosum, Alexandrium pacificum and Breviolum sp. A total of 12 CPAs were assessed at concentrations between 5 and 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. Despite exploring different CPAs and optimizing the freezing techniques, we were unable to successfully cryopreserve V. rugosum and A. pacificum. For Breviolum sp. there was higher cell viability (45.4 ± 2.2%) when using the controlled-rate freezing compared to the rapid freezing technique (10.0 ± 2.8%). This optimized cryopreservation protocol will be of benefit for the cryopreservation of other species from the family Symbiodiniaceae.

Highlights

Dinoflagellates are among the most diverse group of microalgae
Vulcanodinium rugosum and Breviolum sp. were both grown at 25 °C and A. pacificum and A. carterae at 18 °C5,46
When the cryoprotectant agents (CPAs) concentration was increased to 8%, there was a 100% cell survival in glycerol while > 90% of the cells survived in dimethyl sulfoxide (DMSO) and DEG

Summary

Introduction

Dinoflagellates are among the most diverse group of microalgae. Many dinoflagellate species have been isolated and cultured, and these are used for scientific, industrial, pharmaceutical, and agricultural applications. Cultures of dinoflagellates are used in research programmes worldwide including in the development of certified reference standards for phytoplankton monitoring, development and testing of molecular DNA assays, and the production of a diverse array of bioactive secondary compounds that include toxins with pharmaceutical potential[5]. Some species, such as those from the family Symbiodiniaceae, are currently being used globally to understand a multitude of stressors, including ocean warming, ocean acidification, and eutrophication, all of which have caused a decline in coral health and cover in recent decades[6]. It produces saxitoxins that accumulate in shellfish and can cause Paralytic Shellfish Poisoning in humans if contaminated shellfish are consumed[19,21]

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