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Collection and identification of freshwater zooplankton
Sampling and Identification of Zooplankton in Freshwater
CHEN Huihui1, 2, WANG Wenping1, 2, YANG Jun1 *
1 Research Group of Aquatic Ecology and Health, Key Laboratory of Urban Environment and Health, Chinese Academy of Sciences, Fujian Provincial Key Laboratory of Watershed Ecology, Institute of Urban Environment, Chinese Academy of Sciences, Xiamen, Fujian; 2 University of Chinese Academy of Sciences, Beijing
*Corresponding Author Email: [email protected]
Abstract: Zooplankton are an important part of aquatic ecosystems, and common zooplankton in freshwater include protozoa, rotifers, branches, copepods, etc. Taking zooplankton in lake reservoirs as an example, this paper introduces the workflow of collection, concentration, fixation, preservation and identification of zooplankton in inland water bodies, which can be used for quantitative analysis of zooplankton communities.
Keywords: zooplankton, plankton net, inland water bodies, collection, identification
Materials and reagents
1. Bonn reagent
2. 250 mL sample collection vials
3. 5 L sample collection vials
4. 60 mL sample preservation flask
5. Wash the bottles
6. 4# self-sealing bag bag
Instrumentation
1. Water Collector (5 L)
2.25# Plankton mesh (pore size 64 μm)
3. 25# Small Filtration Unit (Pore Diameter 64 μm Filtration Unit)
4. Invert the microscope
5. Dissecting mirror
6. Count box (0.1 mL, 1 mL)
Pipette gun (0.2 mL, 1 mL)
8. Counters
9. Dissecting needles
Experimental steps
1. Prepare before sampling
1.1 Sampling station settings
According to the research purpose, the appropriate sampling station is selected, and it is more difficult to form thermal stratification when the water depth is less than 3 meters, and the surface layer (0.5 m) water sample can be collected. At water depths of 3–10 m, surface layers (0.5 m) and bottom water bodies (sediment surface layers up 1–2 m) were collected, respectively. When the water depth is greater than 10 meters, according to the vertical profile distribution characteristics of the water temperature and dissolved oxygen detected in the field, samples of at least 3 water layers are generally collected, including the surface layer (0.5 m), the aquifer of the thermocline or oxygen jump, the thermocline or below the oxygen jump (the lower layer of the lake, but the sediment surface layer is 2 m upwards).
1.2 Preparation of sampling supplies
Prepare 2 5 L water collectors, 1 set of water pump sets (with 25 L vats for water sample quantification) for water sample collection; 25# plankton nets, 250 mL sample collection bottles for filtering enriched branch horns and copepod zooplankton; 5 L sample collection bottles for protozoa and rotifer samples.
Preparation of Bonn reagent (1500 mL saturated with picric acid, 660 mL formaldehyde, 139 mL glacial acetic acid).
Preparation of Rugo reagent (20 g potassium iodide, 10 g iodine, 20 mL glacial acetic acid, volume to 200 mL).
1.3 Check before sampling
Before sampling, check whether the water collector is intact, such as whether the bottom outlet is blocked, whether the hanging ear is loose, etc. Whether the pump set is complete, such as water pipe fittings, wire wear, graduated tow rope, battery power, and whether the 25 L vat has a marked 20 L tick line. 25# Whether the plankton net is intact, such as whether the interface is cracked, whether there are holes, and whether the switch is flexible. The sample bottle is written with the sample number in advance, and the sampling time and the sample person are marked.
2. Zooplankton collection and fixation
2.1 Quantitative sample collection of protozoa and rotifers
Individual protozoa and rotifers are smaller and more abundant, and water samples can be collected directly with a 5 L water collector or pump. A 5 L water sample was collected on site as protozoa and rotifer samples, and the samples were immediately fixed with Rugo reagent. If the eutrophication of the water body is more serious and the density of protozoa and rotifers is high, the amount of water harvesting can be appropriately reduced; if the density of protozoa and rotifers in the water body is low, the amount of water collection can be appropriately increased.
2.2 Quantitative sample collection of brachiocytopods and copepods
1) Quantitative: Collect water samples with a 5 L water collector or pump. Sampling with a water pump requires dosing in 25 L vats. Individuals with larger and lower abundances of clade and copepods usually take 60 L of water samples per sample in the reservoir for filtration as brachiopods and copepod samples. If the eutrophication of the water body is more serious and the density of branches and copepods is high, the amount of water harvesting can be appropriately reduced; if the density of branches and copepods in the water body is low, the amount of water collection can be appropriately increased.
2) Filtration: 60 L of water samples are filtered through the 25# plankton net, and the polygonals and copepods are concentrated and collected into the plankton net.
3) Collection: Transfer all filter-enriched branch and copepod samples into 250 mL vials.
4) Washing the net: The plankton net is rinsed 3 times by using the filtered water sample, and the remaining branches and copepods are collected together into the collection bottle for quantitative analysis of later branches and copepods.
2.3 Qualitative sample collection of zooplankton
Using the 25# plankton net to carry out multiple trawls at a water depth of less than 3 times the transparency, the zooplankton samples were collected into a 250 mL collection bottle, indicating the qualitative sample, the time and place of collection, the person who collected it, and the sample number.
2.4 Sample fixation
Immediately after sampling, Bonn reagent fixation brachiate and copepod samples were added to the 250 mL collection bottle on site, and the final concentration of the fixative was 5%. Add Lugo reagent to the 5 L collection bottle to fix protozoan and rotifer samples with a final fixative concentration of 1.5%.
3. Zooplankton concentration and preservation
3.1 Reconcentration of protozoa and rotifer samples
1) Let the 5 L collection bottle samples sit for more than 48 h and slowly aspirate the supernatant with a siphon.
2) The flow rate and flow rate during siphoning should be moderate, and the sedimentation and siphoning process should not be shaken, such as stirring the bottom should be re-settled.
3) When 1/3 of the clear liquid is aspirated, the flow rate should be gradually slowed down (or transferred to a 500 mL viale and then reconcented for more than 48 h), and the final concentrated water sample (including the precipitate) should be about 20–25 mL and placed in the sample preservation flask (60 mL).
4) Finally, rinse the precipitator 3 times with a small amount of aspirated supernatant, pour it into the sample preservation flask and set to 30 mL. If the amount of water in the sample exceeds 30 mL, it can be left to stand for another 24 h before the excess water (supernatant) is sucked up.
3.2 Reconcentration of dendritic and copepod samples
Further concentration of clades and copepods is carried out using a 64 μm filter made of 15 mL centrifuge tubes.
1) Pour the fixed branch and copepod samples in the 2 50 mL collection bottle into the filter and rinse the sample collection bottle 3 times.
2) Reach into the sample preservation bottle (60 mL) at the opening of the centrifuge tube and rinse the filter with the wash bottle.
3) Wash the bottle repeatedly to clean the filter 3 times, and continuously rotate the filter during the cleaning process to ensure that all branches and copepods are rinsed into the sample preservation bottle.
4) Check the number again, add Bonn reagent to the sample preservation bottle, fix the preservation, and the final concentration is 5%.
3.3 Preservation of zooplankton samples
1) After the cap of the sample preservation bottle (60 mL) is closed, put it into the 4# self-sealing bag, sealed and numbered.
2) Place in the crisper box in the order of sampling numbers.
3) Put the crisper boxes into the sorting box in turn.
4) Place the finishing boxes on the sample holder sequentially.
5) Make a sample table, including sample number, sampling time, sampling location, latitude and longitude, sampling water depth, water type, sample name, storage location, recorder, verifier and other information.
4. Zooplankton identification and counting
4.1 Zooplankton identification
1) Prepare the microscope, dissecting mirror, counter, dissecting needle, pipette gun (configured with 0.2 mL tip or 1 mL tip, pre-cut tip to enlarge the muzzle) and counting box (0.1 mL and 1 mL).
2) Refer to zooplankton taxonomy for species identification, requiring as much identification of species or genera as possible.
3) Dominant species and common species must be identified to the species, for species that are difficult to identify, it is necessary to further identify with the help of dissecting needles under the dissecting mirror or inverted microscope, and take pictures of key morphological characteristics to communicate with peer experts for advice.
4.2 Zooplankton Counting
The samples with a fixed capacity are counted for zooplankton, and the experimental record should record the sampling number, sampling volume, volume, and counting volume.
1) Shake the samples well and pipette 1 mL of bracepts and copepods with a 1 mL pipette into the 1 mL counting box. Pipette 0.1 mL protozoa and rotifer samples into a 0.1 mL counting box with a 0.2 mL pipette.
2) Observe the sample under the microscope or anatomical context, identify the species, count and record; usually at least 300 individuals are identified per sample to ensure the quality of community data analysis.
3) The number of zooplankton per unit water volume is calculated as follows
In the formula:
N represents the number of zooplankton in a 1 L water sample (pcs/L)
V represents the volume of the sample (L)
VS indicates the volume of the sample after concentration (mL)
VO indicates counting sample volume (mL)
n indicates the number of individuals obtained by microscopic counting
4.3 Calculation of zooplankton biomass
Using the volume method, each zooplankton can be approximated as a geometric stereoscopic figure, the biological volume is estimated according to the volume formula, and the biomass of the zooplankton is calculated by assuming that the density of the zooplankton is 1 g/cm3. With reference to Zhang Zongshi and Huang Xiangfei (1991), the volumetric formula measured the length, width and height of 30 individuals of the corresponding species, and substituted the formula to obtain wet weight biomass.
5. Data statistics and verification
Organize the zooplankton data of each sample, make a species abundance, biomass data table, and ask laboratory personnel to check the collated data, and if there is any doubt, the zooplankton sample should be immediately re-identified, counted and processed. The data after confirmation is corrected is formed into a final form and printed and saved.
Thanks
Thanks to the national natural science foundation of China (31370471, 31672312, 91851104) and the Fujian Provincial Natural Science Foundation of China (2019J02016) for their funding.
bibliography
1.Xi Lihong, Li Huiming, Lin Qiuqi, Han Boping. (2015) Phytoplankton community structure and seasonal changes in the open water area of tropical nutrient-rich reservoirs: A case study of Dashahe Reservoir in Guangdong Province. Journal of Lake Science, 27(6): 1049–1058.]
2.Shen Yunfen, Zhang Zongshi, Gong Xunju, Gu Manru, Shi Zhixin, Wei Yinxin. (1990) New technologies for microbiological monitoring. Beijing: China Architecture and Building Press.]
3. Zhang Zongshi, Huang Xiangfei. (1991) Research methods of freshwater plankton. Beijing: Science Press.]
4.Sommer, U., Adrian, R., Domis L. D. S., Elser, J., Gaedke, U., Ibelings, B., Jeppesen, E., Lurling, M., Molinero, J. C. and Mooij, W. M. (2012) Beyond the Plankton Ecology Group (PEG) model: mechanisms driving plankton succession. Annu. Rev. Ecol. Syst. 43: 429–448.
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