Novel Non-paralytic Shellfish Toxin and Non-spirolide Toxicity to Finfish, Brine Shrimp, and Rotifer Observed in a Culture of the Dinoflagellate Alexandrium insuetum Isolated From the Coastal Water of China

Xiaoying Song,Shuang Hao,Xinyu Zhai,Zhangxi Hu,Yunyan Deng,Junhui Chen,Zhaoyang Chai,Ying Zhong Tang,Lixia Shang

doi:10.3389/fmars.2021.735752

Abstract

The genus Alexandrium is one of the major harmful algal blooms (HABs)-forming dinoflagellate group and at least half of ~40 described species have been reported to produce paralytic shellfish toxins (PSTs). The potentially harmful species Alexandrium insuetum has been reported from many countries of Asia and Europe, and to have paralytic shellfish poisoning toxicity, but no mortality of marine animals was observed during its bloom. Therefore, it is ecologically important to characterize the possible toxicity and toxins of this organism. In this study, based on the establishment of two clonal cultures through cyst germination collected from the Yellow Sea, we identified A. insuetum from China as the first record via light microscopy (LM) and scanning electron microscopy (SEM) observations and phylogenetic analyses. The cultures of A. insuetum were further observed to be toxic to finfish and zooplankton and deleterious to rotifer eggs via laboratory bioassays. The exposure bioassays using rotifer (Brachionus plicatilis), brine shrimp (Artemia salina), and larval finfish (Oryzias melastigma) demonstrated that A. insuetum caused significant lethal effects on finfish and zooplankton species. Rotifer bioassays using cell-free culture medium, heat-treated cultures, and water, methanol, and trichloromethane extracts of algal cells revealed that A. insuetum produced heat-labile, water-soluble toxin(s) that could be excreted from A. insuetum cells and steadily accumulated in the medium during the growth phases. Hatching success of rotifer eggs was also found to be seriously affected by the exposure to A. insuetum. Importantly, ultra-high performance liquid chromatography-tandem mass spectrometry [UPLC (or LC)-MS-MS] analyses suggest the above-described toxicity of A. insuetum was caused by neither PSTs nor spiroimines (13-desmethyl spirolide C and gymnodimine). Collectively, our findings demonstrated the novel toxicity to finfish and zooplankton in A. insuetum, which is ecologically important in not only possibly contributing to population dynamics and even the formation of HABs of the species, but also affecting the on-the-spot survival and the reproduction potency of marine animals. The present work is believed to set a cornerstone for the monitoring and risk assessment of the species along the coastal waters of China and for understanding the general ecology of A. insuetum.

Highlights

Dinoflagellates are important primary producers and grazers of aquatic ecosystems, and responsible for the most harmful algal blooms (HABs) (Hallegraeff et al, 2021)
A volume of 6 mL culture of A. insuetum (4,000, 6,000, and 8,000 cells·mL−1, respectively) was added into the insert and the membrane with pore size of 8.0 μm would allow the cell-free culture medium to seep out the insert, but prevent A. insuetum cells (>10 μm) moving out the insert, we examined that no cells passed through the membrane during the experiment under a light microscope (IX73, Olympus, Japan)
The nucleus was located in the hypocone of cell (Figure 2B), and numerous rod-like chloroplasts were located almost throughout the cell (Figure 2E)

Summary

Introduction

Dinoflagellates are important primary producers and grazers of aquatic ecosystems, and responsible for the most harmful algal blooms (HABs) (Hallegraeff et al, 2021). The dinoflagellate genus Alexandrium Halim, one of the most important genera responsible for causing HABs, has negative impacts on marine ecosystems, economy, and human health worldwide (Anderson, 1998; Anderson et al, 2012). Many blooms that were attributed to A. tamarense (but possibly either A. catenella or A. pacificum; see John et al, 2014; Fraga et al, 2015), a PSTs-producing species, have been observed in the coastal waters of Dalian, Yantai, Zhoushan, and Xiamen, China, causing massive wild and caged fish kills (Zhou and Zhu, 2006; Zou et al, 2014; Yu and Liu, 2016). It is essential to identify Alexandrium species accurately, as this genus includes many hard-to-differentiate and toxic species that cause diverse impacts in aquaculture, human health, and marine ecosystems (Bravo et al, 2006)

Methods

Results

Conclusion