改良高通量测序技术揭示除藻剂对藻类群落的影响

doi:10.13560/j.cnki.biotech.bull.1985.2022-0193

摘要/Abstract

摘要：

研究藻华水体中浮游植物群落对除藻剂的响应，可为在实际应用中选择合适的除藻剂提供理论依据。近年来，高通量测序技术已逐步用于表征藻类群落结构，然而它无法区分群落中的“死亡”和“存活”个体。本研究利用经叠氮溴化丙锭（PMA）前处理和未经PMA前处理的高通量测序技术，探究Ca(ClO)₂、CuSO₄和一种市售生物除藻剂对某一存在藻华的景观水体中藻类群落组成、多样性和标志物种的影响。结果显示，3种除藻剂均能有效降低水样的藻密度；初始水样中绿藻门的单针藻属（Monoraphidium）相对丰度最大（74.34%），是造成藻华的优势藻；与非PMA处理组相比，经PMA处理后的测序结果更能体现不同除藻剂处理后活藻群落组成和多样性差异：在除藻剂作用下，绿藻门相对丰度下降至1.50%-24.61%，蓝藻门优势种Leptolyngbya boryana相对丰度上升至27.85%-41.52%；50 mg/L生物除藻剂能显著提升群落的Chao1指数、observed species指数、Pielou's evenness指数和Shannon指数，增加了群落的多样性、均匀性和稳定性；主坐标分析表明投加量增大不会显著改变Ca(ClO)₂和生物除藻剂处理组的活藻群落结构；物种差异分析显示，Chroococcidiopsis sp.这一典型的逆境环境优势藻种可在1.0 mg/LCuSO₄和50 mg/L生物除藻剂处理组中富集。基于PMA前处理的高通量测序技术，为研究不同类型除藻剂处理后藻华水体中活藻群落结构的变化提供了一种有效的技术手段。

关键词: 除藻剂, 碘化丙啶, 高通量测序, 藻类群落, 多样性

Abstract:

It is significant to study the response of algal community towards algicide in water body that suffers from algal bloom，because it can provide theoretical evidence for choosing appropriate algicide in practical application. Recently，high-throughput sequencing technology（HTST）has been gradually utilized in characterizing algal community structure. However，it can not distinguish ‘dead’ and ‘alive’ individuals in the community. In this study，a method of combing propidium monoazide（PMA）pretreatment with HTST and normal HTST were used to investigate the effects of Ca(ClO)₂，CuSO₄ and a commercially available bio-algicide towards the algal community composition，diversity and marked species in a landscape pond containing algal bloom. The results showed that three algicides were all able to efficiently decrease algal density. The results of high-throughput sequencing presented that Monoraphidium genus in Chlorophyta phylum dominated the original water sample，with a relative abundance of 74.34%. Compared with normal sequencing，the sequencing after pre-PMA treatment efficiently revealed the differences of living algal community composition and diversity among different algicides-treated samples. After algicide treatment，the relative abundances of Chlorophyta decreased to 1.50%-24.61%，while the relative abundances of Leptolyngbya boryana（bel-onging to Cyanobacteria phylum）increased to 27.85%-41.52%. 50 mg/L bio-algicide significantly increased Chao1 index，observed species index，Pielou's eveness index and Shannon index，i.e.，increased the diversity，evenness and stability of the alive algal community. Principal coordinate analysis showed that increasing dosages of Ca(ClO)₂ and bio-algicide did not significantly change the community structure of alive algae. Species differences analysis revealed that Chroococcidiopsis sp.，typical adversity environmental dominant algal species，enriched in the alive algal communities of 1.0 mg/L CuSO₄ group and 50 mg/L bio-algicide group. This study proved that PMA pretreatment coupled with HTST is an efficient technology to investigate the influences of different algicides on the alive algal community structures in water body that suffers from algal bloom.

Key words: algicide, PMA, high-throughput sequencing, algal community, diversity

陈宇捷, 郑华宝, 周昕彦. 改良高通量测序技术揭示除藻剂对藻类群落的影响[J]. 生物技术通报, 2022, 38(11): 70-79.

CHEN Yu-jie, ZHENG Hua-bao, ZHOU Xin-yan. Modified High-throughput Sequencing Reveals the Effects of Different Algicides towards Algal Community[J]. Biotechnology Bulletin, 2022, 38(11): 70-79.

图/表 9

表1 景观池塘水基本水质参数

Table 1 Basic water quality parameters of the landscape pond

氨氮Ammonia nitrogen/(mg·L^-1)	总氮Total nitrogen/(mg·L^-1)	总磷Total phosphorus/(mg·L^-1)	化学需氧量Chemical oxidation demand/(mg·L^-1)	浊度 Turbidity/(mg·L^-1)	藻细胞密度 Algal cell density/(10⁶ cell·mL^-1)	叶绿素a Chlorophyll a/(μg·L^-1)
0.60	0.79	0.05	35	23.7±1.1	1.41	184

图1 3种除藻剂对藻细胞的去除效果图中数据经Tukey检验，柱子上不同字母表示在0.05水平差异显著

Fig.1 Removal efficiencies of algal cells by 3 algicides Data of the bar marked by different letters indicate significant difference at 0.05 levels by Tukey’s test

图2 3种除藻剂对藻类群落组成（门水平）的影响 Initial代表初始时刻藻类群落；CK代表对照组；Cl、Cu和Bio分别代表Ca(ClO)2、CuSO4和生物除藻剂；数字代表除藻剂的投加量（单位：mg/L）；NPMA代表未经PMA处理的样本；PMA代表经PMA处理的样本，下同

Fig. 2 Influences of 3 algicides on algal community compositions（phylum level） “Initial” refers to the initial algal community；“CK” refers to control check；“Cl”，“Cu” and “Bio” refers to Ca(ClO)2，CuSO4 and bio-algicide，respectively；number indicates the dosage of algicide（mg/L）；NPMA refers to the samples without PMA pre-treatment；PMA refers to the samples with PMA pre-treatment，the same below

图3 3种除藻剂对藻类群落组成（属水平）的影响

Fig. 3 Influences of 3 algicides on algal community compositions（genus level）

表2 3种除藻剂处理下藻类在种水平的群落组成（相对丰度前10的物种）

Table 2 Algal community compositions in species level under 3 algicides treatment（species with top 10 relative abundance）

物种名称 Species name	Initial /%	CK_NPMA /%	Cl-0.5_NPMA /%	Cl-5.0_NPMA /%	Cu-0.1_NPMA /%	Cu-1.0_NPMA /%	Bio-10_NPMA /%	Bio-50_NPMA /%	CK_PMA /%	Cl-0.5_PMA /%	Cl-5.0_PMA /%	Cu-0.1_PMA /%	Cu-1.0_PMA /%	Bio-10_PMA /%	Bio-50_PMA /%
Leptolyngbya boryana^*	0.27	0.44	0.33	0.49	0.22	0.23	0.50	0.87	20.60	28.01	28.67	41.52	18.18	27.85	28.01
Chamaesiphon minutus^*	<0.01	0.02	<0.01	0.03	<0.01	0.01	<0.01	0.02	0.29	0.62	0.56	0.66	0.79	0.65	1.51
Nannochloris normandinae^**	<0.01	<0.01	<0.01	<0.01	<0.01	<0.01	<0.01	<0.01	0.047	0.33	0.73	0.43	0.27	0.12	0.41
Xylochloris irregularis^**	0.02	0.02	0.02	0.04	<0.01	0.01	<0.01	<0.01	0.07	0.07	0.61	0.31	0.20	0.24	0.27
Pectinodesmus pectinatus^**	<0.01	0.06	<0.01	<0.01	<0.01	<0.01	<0.01	<0.01	0.14	0.25	0.11	<0.01	0.46	0.18	0.44
Lobosphaera incisa^**	0.57	0.02	0.02	0.18	<0.01	0.08	<0.01	0.18	0.03	0.03	0.16	0.18	<0.01	0.03	0.18
Dinophysis acuta^***	<0.01	<0.01	<0.01	<0.01	<0.01	<0.01	<0.01	<0.01	<0.01	<0.01	<0.01	<0.01	0.56	<0.01	<0.01
Pleurocapsa sp. 04302-00001^*	<0.01	<0.01	<0.01	<0.01	<0.01	<0.01	<0.01	<0.01	<0.01	<0.01	<0.01	0.57	<0.01	<0.01	<0.01
Tetradesmus obliquus^**	0.04	0.05	0.02	0.04	0.02	0.01	0.04	0.06	0.01	<0.01	<0.01	0.01	<0.01	<0.01	0.10
Neochloris aquatica^**	<0.01	0.02	0.03	0.05	<0.01	0.02	0.02	0.03	0.05	<0.01	0.03	<0.01	<0.01	0.02	<0.01

图4 3种除藻剂对藻类群落4种α多样性指数的影响

Fig. 4 Influences of 3 algicides on 4 alpha diversity indexes of algal community

图5 基于OTU水平下藻类群落的PCoA分析图实心三角形代表NPMA样本，空心三角形代表PMA样本

Fig. 5 PCoA analysis of algal communities based on OTU level Filled triangles refer to NPMA samples，and hollow triangles refer to PMA samples

图6 NMPA样本和PMA样本中的OTU数

Fig. 6 OTU numbers in NMPA samples and PMA samples

图7 不同除藻剂处理下藻类群落中物种差异（OTU水平）的随机森林分析

Fig. 7 Random forest analysis of species differences in algal communities under different algicides treatment

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