Comparison of the fixation effect of formaldehyde and glutaraldehyde on the Ludox–QPS method

doi:10.3969/j.issn.1000-5641.2022.03.009

Abstract

Abstract:

Density gradient centrifugation in combination with quantitative protargol stain (Ludox–QPS) is an important research method for the separation, extraction, and staining of microbenthos. Glutaraldehyde solution is the most widely used fixative for this method; however, it is a hazardous chemical and transportation by civil aviation is forbidden. Hence, biogeography studies using glutaraldehyde solution on a large scale are greatly limited. In contrast, samples fixed with formaldehyde solution can be transported by air consignment under certain conditions. To test whether the results of a study on microbenthic communities fixed with formaldehyde and glutaraldehyde are comparable, we collected microbenthic samples from three natural habitats (bare mudflat, Scirpus mariqueter meadow, sandy beach) and fixed them with either formaldehyde (final concentration is 1%) or glutaraldehyde (final concentration is 2%). Then, we tested the significance of differences in species composition, diversity, and biomass of the communities using an ANOSIM two-way crossed analysis (fixative type and habitat type). The results of this study demonstrate that although significant differences exist in the community structures among the three habitats, the species composition, diversity indices (number of species, number of individuals, evenness, Shannon-Wiener diversity index), and biomass of the microbenthic communities between the two fixative types have no significant differences. NMDS analysis also shows similar results (stress = 0.11). In conclusion, the data indicates that the microbenthic communities fixed by the two fixatives are comparable. Therefore, formaldehyde is also suitable for ecological research of microbenthos as a fixative.

Key words: diversity, community structure, microbenthos, ciliates, fixative

CLC Number:

Miaoxun WANG, Yuan XU. Comparison of the fixation effect of formaldehyde and glutaraldehyde on the Ludox–QPS method[J]. Journal of East China Normal University(Natural Science), 2022, 2022(3): 82-89.

Figures/Tables 6

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Table 1

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References 38

1	CAREY P G. Marine interstitial ciliates: An illustrated key [M]. London: Chapman and Hall, 1992: 1-351.
2	杜永芬. 潮间带纤毛虫原生动物和小型底栖动物生态学研究 [D]. 北京: 中国科学院研究生院, 2008.
3	代仁海. 潮间带底栖纤毛虫分类学及生态学初步研究 [D]. 山东青岛: 中国海洋大学, 2009.
4	类彦立, 徐奎栋. 海洋微型底栖生物调查方法与操作规程. 海洋与湖沼, 2011, 42 (1): 157- 164. doi: 10.11693/hyhz201101024024
5	孟昭翠. 黄海夏季微型底栖生物群落结构与生态特点 [D]. 北京: 中国科学院研究生院, 2011.
6	类彦立, 徐奎栋. 海洋微型底栖生物调查规范: HY/T140—2011 [S]. 北京: 中国标准出版社, 2011.
7	方晓琪, 仇建标, 余海滨, 等. 海洋浮游植物调查中常见的固定和保存方法及其影响. 海洋湖沼通报, 2019, (6): 106- 111.
8	BURKILL P H. Ciliates and other microplankton components of a nearshore food–webs: Standing stocks and production processes. Ann Inst Oceanogr Paris N S, 1982, 58, 335- 350.
9	STOECKER D K, GIFFORD D J, PUTT M. Preservation of marine planktonic ciliates: Losses and cell shrink age during fixation. Marine Ecology Progress Series, 1994, 110 (2/3): 239- 299.
10	JEROME C A, MONTAGNES D J, TAYLOR F J. The effect of the quantitative protargol stain and Lugol’s and Bouin’s fixatives on cell size: A more accurate estimate of ciliate species biomass. Journal of Eukaryotic Microbiology, 2010, 40 (3): 254- 259.
11	XU K, DU Y, LEI Y, et al. A practical method of Ludox density gradient centrifugation combined with protargol staining for extracting and estimating ciliates in marine sediments. European Journal of Protistology, 2010, 46 (4): 263- 270. doi: 10.1016/j.ejop.2010.04.005
12	中国民用航空局. 旅客和机组携带危险品的航空运输规范: MH/T1030–2018 [S]. 北京: 中国民用航空局, 2018.
13	张武昌, 肖天, 王荣. 海洋微型浮游动物的丰度和生物量. 生态学报, 2001, 21 (11): 1893- 1908. doi: 10.3321/j.issn:1000-0933.2001.11.021
14	TURLEY C M, HUGHES D J. Effects of storage on direct estimates of bacterial numbers of preserved seawater samples. Deep Sea Research, 1992, 39 (3/4): 375- 394.
15	CHOI J W, STOECKER D K. Effects of fixation on cell volume of marine planktonic protozoa. Applied and Environmental Microbiology, 1989, 55 (7): 1761- 1765. doi: 10.1128/aem.55.7.1761-1765.1989
16	STOECKER D K. An experimentally determined carbon: Volume ratio for marine “Oligotrichous” ciliates from estuarine and coastal water. Limnology and Oceanography, 1989, 34 (6): 1097- 1103. doi: 10.4319/lo.1989.34.6.1097
17	OHMAN M D, SNYDER R A. Growth kinetics of the omnivorous oligotrich ciliate Strombidium sp. . Limnology and Oceanography, 1991, 36 (5): 922- 935. doi: 10.4319/lo.1991.36.5.0922
18	LEAKEY R J G, BURKLL P H, SLEIGH M A. A comparison of fixatives for the estimation of abundance and biovolume of marine planktonic ciliate populations. Journal of Plankton Research, 1994, (4): 375- 389.
19	曾钰婷. 少毛类纤毛虫定量方法比较——QPS与福尔马林固定液之数量体积及分类能力的评估 [D]. 台湾地区基隆市: 台湾海洋大学, 2012.
20	孟昭翠, 类彦立, 和莹莹, 等. 样品保藏方式与时间对海洋底栖细菌及原生生物荧光计数效能的影响. 海洋科学, 2010, 34 (5): 13- 20.
21	宋微波, 沃伦, 胡晓钟. 中国黄渤海的自由生纤毛虫 [M]. 北京: 科学出版社, 2009.
22	LYNN D H. The Ciliated Protozoa: Characterization, Classification, and Guide to the Literature [M]. 3th ed. Dordrecht, the Netherlands: Springer, 2008.
23	NOZAIS C, PERISSINOTTO R, TITA G. Seasonal dynamics of meiofauna in a South African temporarily open/closed estuary (Mdloti Estuary, Indian Ocean). Estuarine Coastal and Shelf Science, 2005, 62 (1/2): 325- 338.
24	HAMELS I, MUYLAERT K, SABBE K, et al. Contrasting dynamics of ciliate communities in sandy and silty sediments of an estuarine intertidal flat. European Journal of Protistology, 2005, 41 (4): 241- 250. doi: 10.1016/j.ejop.2005.02.002
25	周红, 张志南. 大型多元统计软件PRIMER的方法原理及其在底栖群落生态学中的应用. 青岛海洋大学学报(自然科学版), 2003, 3 (1): 58- 64. doi: 10.3969/j.issn.1672-5174.2003.01.023
26	XU Y, FAN X, WARREN A, et al. Functional diversity of benthic ciliate communities in response to environmental gradients in a wetland of Yangtze Estuary, China. Marine Pollution Bulletin, 2018, 127, 726- 732. doi: 10.1016/j.marpolbul.2017.12.068
27	VERISSIMO H, BREMNER J, GARCIA C, et al. Assessment of the subtidal macrobenthic community functioning of a temperate estuary following environmental restoration. Ecological Indicators, 2012, 23, 312- 322. doi: 10.1016/j.ecolind.2012.04.020
28	PAGANELLI D, MARCHINI A, OCCHIPINTI A. Functional structure of marine benthic assemblages using Biological Traits Analysis (BTA): A study along the Emilia–Romagna coastline (Italy, North–West Adriatic Sea). Estuarine Coastal and Shelf Science, 2012, 96, 245- 256. doi: 10.1016/j.ecss.2011.11.014
29	BREMNER J, ROGERS I S, FRID L C. Functional diversity of macrobenthic assemblages decreases in response to sewage discharges. Ecological Indicators, 2016, 66, 65- 75. doi: 10.1016/j.ecolind.2016.01.003
30	WONG M C, DOWD M. Patterns in taxonomic and functional diversity of macrobenthic invertebrates across seagrass habitats: A case study in Atlantic Canada. Estuaries and Coasts, 2015, 38 (6): 2323- 2336. doi: 10.1007/s12237-015-9967-x
31	张遐, 鞠躬, 董光皎. 甲醛和戊二醛应用于神经系统免疫组织化学组织固定的特性分析. 神经解剖学杂志, 1990, 6 (2): 249- 256.
32	马族航. 上海崇明岛东滩湿地底栖纤毛虫的物种多样性及其生态学研究 [D]. 上海: 华东师范大学, 2018.
33	OTA T, TANIGUCHI A. Standing crop of planktonic ciliates in the East China Sea and their potential grazing impact and contribution to nutrient regeneration. Deep Sea Research, 2003, 50 (2): 423- 442. doi: 10.1016/S0967-0645(02)00461-7
34	HWANG S J, HEATH R T. The distribution of Protozoa across a trophic gradient, factors controlling their abundance and importance in the plankton food web. Journal of Plankton Research, 1997, 19 (4): 491- 518. doi: 10.1093/plankt/19.4.491
35	XU Y, YAO S, SOETAERT K, et al. Effects of salt marsh restoration on eukaryotic microbenthic communities in Yangtze Estuary [J]. Marine Ecology Progress Series, 2020, 638: 39–50.
36	TURLEY C M, HUGHES D J. Effects of storage on direct estimates of bacterial numbers of preserved seawater samples. Deep Sea Research, 1992, 39 (3): 375- 394.
37	刘华雪, 谭烨辉, 黄良民, 等. 夏季南海北部纤毛虫群落组成及其水平分布. 生态学报, 2010, 30 (9): 2340- 2346.
38	周百灵, 孟昭翠, 赵峰, 等. 长江口外海域微型和小型底栖生物群落结构和时空变化. 生态学报, 2015, 35 (15): 5050- 5063.

	物种组成	物种数	个体数	均匀度	香农-维纳多样性指数	生物量
固定剂类型	0.104	–0.014	–0.052	0.045	–0.019	–0.051
生境类型	0.235*	0.063	0.127*	0.160*	0.041	0.234**

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