The effect of temperature and salinity on Margalefidium polykrikoides group III VA, USA strain growth

M.polykrikoides is a cosmopolitan harmful dinoflagellate that blooms in coastal waters worldwide. Despite genomic evidence that it is in Group III and so closely related to isolates from Puerto Rico, Malaysia, North America, and Central America, M. polykrikoides group III VA, isolated from the lower Chesapeake Bay, bloom at warmer temperatures and lower salinities than in coastal ecosystems occupied by its closest relatives. In contrast, M. polykrikoides blooms in other regions initiate when salinities are higher (e.g., 30) and water temperatures are cooler, leading to the question of whether there are differences in salinity and temperature preferences for different groups and strains of M. polykrikoides, or whether the realized niche of M. polykrikoides varies based on local conditions and competitive interactions . In this study, the effect of temperature and salinity on the exponential growth rate and total cell yield of an M. polykrikoides VA culture isolate were examined. A clonal culture of M. polykrikoides (MP-LRCB-082419) was established by isolating a cell from the Lafayette River, a sub-tributary of the lower Chesapeake Bay, on August 24, 2019. Lafayette River water of 20 salinity was filtered through Thermo ScientificTM NalgeneTM Rapid-FlowTM sterile, 1,000 mL disposable filter units containing a 90 mm diameter and 0.1 µm pore size PES membrane. Filtered river water was augmented with L1 medium (Guillard & Hargraves, 1993) nutrient concentrations (excluding silicate) that was then used as the culture medium. The M. polykrikoides cell was isolated in Dr. Margaret Mulholland's lab via serial transfer into fresh medium using a 100-1000 µL Eppendorf® Research® pipette, Costar® 12-well clear TC-treated sterile well plates, and an OLYMPUS CKX41 inverted microscope. Then, the cell was inoculated into VWR® tissue culture flasks (25mL, non-treated, plug seal cap, and sterilized) containing L1 medium and incubated at 28 ̊ C, under 140µmol photons m-2 s-1 lights on a 14 h:10h light:dark cycle. M. polykrikoides cultures were acclimated to multiple temperature (16, 18, 20, 24, 28, 30, 32, and 34 ̊ C) and salinity (10, 15, 20, 25, and 30) treatments by stepwise transfer for 7 to 38 days (Yamaguchi & Honjo, 1989). Light was maintained under constant conditions at 140 µmol photons m-2 s-1 supplied over a 14h:10h light:dark cycle. […]

M.polykrikoides是一种世界性的有害甲藻（dinoflagellate），在全球沿海水域形成水华。尽管基因组证据表明其属于第三组，与来自波多黎各、马来西亚、北美和中美洲的分离株亲缘关系密切，但从切萨皮克湾下游分离出的M.polykrikoides第三组VA菌株，其水华形成的温度高于其近缘种所在的沿海生态系统，盐度则更低。相比之下，其他地区的M.polykrikoides水华在盐度较高（如30）且水温较低时启动，这引发了一个问题：不同组和菌株的M.polykrikoides在盐度和温度偏好上是否存在差异，或者其实际生态位（realized niche）是否因当地条件和竞争相互作用而变化。

在本研究中，我们检测了温度和盐度对M.polykrikoides VA培养分离株指数生长速率（exponential growth rate）和总细胞产量（total cell yield）的影响。2019年8月24日，从切萨皮克湾下游的拉法耶特河分离出单个细胞，建立了M.polykrikoides的克隆培养物（clonal culture）（MP-LRCB-082419）。将盐度为20的拉法耶特河河水通过Thermo Scientific™ Nalgene™ Rapid-Flow™无菌一次性过滤装置（容积1000 mL，含直径90 mm、孔径0.1 µm的PES膜）过滤。过滤后的河水添加L1培养基（Guillard & Hargraves, 1993）的营养浓度（不含硅酸盐），随后用作培养基。

M.polykrikoides细胞在Margaret Mulholland博士的实验室中通过连续转移建立培养：使用100-1000 µL的Eppendorf® Research®移液器、Costar® 12孔透明TC处理无菌孔板和OLYMPUS CKX41倒置显微镜，将细胞转移至新鲜培养基中。随后，将细胞接种到含L1培养基的VWR®组织培养瓶（25 mL，未处理，塞封盖，无菌）中，在28℃、140 µmol光子m⁻²s⁻¹光照强度、14h:10h光暗周期下培养。

M.polykrikoides培养物通过逐步转移（Yamaguchi & Honjo, 1989）适应多种温度（16、18、20、24、28、30、32和34℃）和盐度（10、15、20、25和30）处理，适应期为7至38天。光照条件保持恒定：140 µmol光子m⁻²s⁻¹光照强度，14h:10h光暗周期。[…]