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Abstract In order to find out the change pattern of phytoplankton community structure in pond industrial ecoaquaculture system and explain its ecological mechanism， tests were carried out using Megalobrama amblycephala as aquaculture species with two stocking densities. ShannonWiener diversity index and Pielou uniformity index were used to study the phytoplankton community structure in aquaculture area （including low stocking density aquaculture area and high stocking density aquaculture area）， inlet area， outlet area， purification area， recirculation area， back area of the pond industrial ecoaquaculture system. The results showed that a total of 92 species of 5 phyla were identified， including 46 species of Chlorophyta， 15 species of Cyanophyta， 15 species of Euglenophyta， 10 species of Bacillariophyta， 5 species of Cryptophyta， and 1 species of Pyrrophyta. The numbers of phytoplankton species， biodiversity indexes， uniformity indexes， Chlorophyta biomass and its proportion in total phytoplankton followed the order of inlet area， purification area， recirculation area， back area > low stocking area > high stocking area > outlet area. The total biomass of phytoplankton， Cyanophyta biomass and its proportion in total phytoplankton followed the order of inlet area， purification area， recirculation area， back area < low stocking area < high stocking area < outlet area. However， all these parameters showed no significant differences or change rules among inlet area， purification area， recirculation area， back area. It could be concluded according to the biodiversity that the water quality in outlet area was at middle pollution level， the water quality in high stocking area was in light pollution level， and the water quality in other 5 areas were at clean level， which suggested that the water quality was in good states except that in outlet area， and the pond industrial ecoaquaculture system functioned well.
Key words Pond industrial ecoaquaculture system; Water quality assessment; Megalobrama amblycephala; Phytoplankton; Structure characteristics
China is a big pond aquaculture country in the world， and pond aquaculture plays an important role in the national economy of China[1]. However， the current pond aquaculture in China faces 2 major bottlenecks， namely， serious water pollution[2-3] and low mechanization[4]， thus limiting the healthy and sustainable development of pond aquaculture. Therefore， it has become the key issue to be solved urgently in modern pond aquaculture to create new aquaculture modes， improve the modernization degree of pond culture and reduce the pollution of pond aquaculture to the internal and external environment. To this end， scholars in China have proposed the pond industrial ecoaquaculture system based on the partition aquaculture system of the foreign countries. In the system， a series of raceways with airpushing water aeration and sewage disposal equipment are constructed by using 2%-5% of the pond surface， and the raceways are used as the aquaculture area to proceed high stocking density aquaculture similar to “factories” to carry out industrial management， and the remaining 95%-98% of the pond water surface is used as the purification area to carry out biological purification to the culture tail water remaining in the pond after proper modification， which realizes the zero discharge or standardized discharge[5]. Thus， the partition of the aquaculture units and the water quality purification unit makes the yield of a small area equal to that of the original total water area， which completely reforms the traditional pond fish aquaculture mode. As a new pond ecoaquaculture mode， pond industrial ecoaquaculture system has been promoted and applied in some areas， but its fundamental research is very poor. There is still no report on the phytoplankton community structure in pond industrial ecoaquaculture system. Phytoplankton are the primary producers in aquatic ecosystems， and also important ecological indicators for monitoring and evaluating water quality[6]. Phytoplankton can respond quickly to changes in water nutrient status[7]， and their community structure is closely related to water quality， which can comprehensively and truly reflect the ecological and nutritional status of water bodies[7-9]. Therefore， studying phytoplankton community structure in aquaculture water can not only understand the primary productivity of pond water， but also determine the environmental conditions of water. Therefore， this study investigated the changes of phytoplankton community structure in the pond industrial ecoaquaculture system for Megalobrama amblycephala， with the aim to provide scientific bases for clarifying the ecological mechanism of the pond industrial ecoaquaculture system.
Materials and Methods
Test design
The test was carried out at Yancheng Zhengrong Ecological Fishery Co.， Ltd.， located in Jianhu County， Jiangsu Province. The pond industrial ecoaquaculture system included 6 test partitions （Fig.1）， namely， Aquaculture area （AA）， Inlet area （IA）， Outlet area （OA）， Purification area （PA）， Recirculation area （RA）， and Back area （BA）， in which the aquaculture area was divided into high stocking density aquaculture area and low stocking density culture area according to the test needs. The areas of the inlet area， outlet area， purification area， recirculation area and back area were respectively 3 000， 900， 15 000， 170 000 and 55 000 m 2， and the water depths were 4.0， 2.0， 1.2， 1.5 and 2.0 m， respectively. The aquaculture area had an area of 5 720 m 2 and consists of 52 cement ponds with an area of 110 m 2， pond depth of 2.5 m and water depth 2.0 m.
The test began on July 13， 2016， and there were 2 different aquaculture densities， namely， the Low stocking density aquaculture area （LA） and the High stocking density aquaculture area （HA）. The cultured species was Megalobrama amblycephala with an initial average body weight of （3.33 ± 0.52） g and an average body length of （56.13 ± 3.45） mm. The stocking densities for the low stocking and high stocking groups were 200 fish/m 2 and 300 fish/ m 2， respectively. Aquaculture test
The daily feeding amount during the aquaculture period is about 3% to 5% of the fish weight， which was fed for 3 times at 9：00， 12：30 and 16：00. During the aquaculture， the ponds were maintained with microflowing water， and the water velocity at the front end of the push water was 12 cm/s. The microtube aeration system was equipped for standby. The microtube aeration system was normally not open and only activated when the dissolved oxygen in the water was below 3 mg/L.
Phytoplankton collection， identification and counting
Phytoplankton samples were collected since July 2016. The samples were collected once a month in summer and autumn， and once every two months in winter and spring. A total of 23 sampling points were set up in the test area， including 3 high stocking density aquaculture areas， 3 low stocking density aquaculture areas， 3 inlet areas， 3 outlet areas， 5 purification areas， 3 recirculation areas and 3 back areas. The sampling points in each sampling site were shown in Fig.1. The phytoplankton samples were collected according to the method of Meng et al.[10]. The identification of phytoplankton was referred to Zheng et al.[11]， Han et al.[12]， Hu et al.[13].
Evaluation methods
Pielou uniformity index （J， Pielou） and ShannonWiener diversity index （D， ShannonWiener index） were used to analyze and evaluate the ecological characteristics of phytoplankton in water. The calculation methods of the above indexes were referred to Meng et al.[10， 14]， Liu et al.[15].
Results and Analysis
Species composition and biomass of phytoplankton
A total of 92 species （including mutants and variants） of 5 phyla were identified in the back areas of all the partitions， namely， Chlorophyta， Bacillariophyta， Cyanophyta， Euglenophyta， Cryptophyta， and Pyrroptata. The number of species of Chlorophyta was the largest with a total of 46， which accounted for 50.0% of the total species of the identified phytoplankton， followed by Cyanophyta and Euglenophyta， 15 species for each， which accounted for 16.3% of the total. There were 10 species of Bacillariophyta， accounting for 10.9% of the total， 5 species of Cryptophyta， accounting for 5.4% of the total， 1 species of Pyrroptata， accounting for 1.1% of the total. The species composition of phytoplankton was shown in Table 1. As shown in Table 1， 57 species of 6 phyla were identified in the low stocking area， 55 species of 5 phyla in the high stocking area， 64 species of 5 phyla in the inlet area， 51 species of 5 phyla in the outlet area， 64 species of 5 phyla in the purification area， 64 species of 6 phyla in the recirculation area and 63 species of 6 phyla in the back area. The number of species of phytoplankton in the low stocking area， high stocking area， inlet area， outlet area， purification area， recirculation area and back area was 40-52， 40-53， 45-62， 33-51， 45-55， 46-58， and 45-59， respectively （Fig. 2）. The number of phytoplankton species in each test partition was in the order of inlet area， purification area， recirculation area， back area > low stocking area > high stocking area > outlet area， and there were no significant difference and change rules among the inlet area， purification area， recirculation area and back area. In terms of seasonal changes， the number of species in each test partition was generally in the order of summer > autumn > winter. From July 2016 to April 2017， the total biomass of phytoplankton in low stocking area， high stocking area， inlet area， outlet area， purification area， recirculation area and back area was 5.25×10 7-13.42×10 7， 5.74×10 7-16.54×10 7， 5.08×10 7-10.36×10 7， 6.01×10 7-17.82×10 7， 5.46×10 7 - 11.31×10 7， 4.84×10 7-11.96×10 7， 4.84×10 7-10.75×10 7 cells/L （Fig. 2）. The total biomass in each partition was in the order of outlet area > high stocking area > low stocking area > inlet area， purification area， recirculation area and back area， and there were no significant difference and change rules among the inlet area， purification area， recirculation area and back area. As for seasonal changes， the total biomass of each partition was generally in the order of summer > autumn > winter.
Biomass of Cyanophyta and Chlorophyta and their proportions
The biomass of Cyanophyta was respectively 3.28×10 7-10.08×10 7， 4.07×10 7-13.04×10 7， 2.54×10 7-7.25×10 7， 4.78×10 7-15.63×10 7， 3.08×10 7-7.94×10 7， 2.52×10 7-8.66×10 7， 2.62×10 7 - 6.91×10 7 cells/L in the low stocking area， high stocking area， inlet area， outlet area， purification area， recirculation area and back area （Fig. 3）. Both the biomass of Cyanophyta and the proportion of Cyanophyta in total phytoplankton were in the order of outlet area > high stocking area > low stocking area > inlet area， purification area， recirculation area， back area， and there were no significant difference and change rules among the inlet area， purification area， recirculation area and back area. As for seasonal changes， the total biomass of Cyanophyta in each test partition was generally in the order of summer > autumn > winter， while there was no significant difference in the proportion of Cyanophyta in total phytoplankton.
The biomass of Chlorophyta was respectively 1.23×10 7-3.04×10 7， 1.03×10 7-2.96×10 7， 1.53×10 7-3.69×10 7， 0.99×10 7-2.87×10 7， 1.52×10 7-3.01×10 7， 1.43×10 7-3.03×10 7， 1.36×10 7-3.33×10 7 in the low stocking area， high stocking area， inlet area， outlet area， purification area， recirculation area and back area （Fig. 3）. Both the biomass of Chlorophyta and the proportion of Chlorophyta in total phytoplankton were in the order of inlet area， purification area， recirculation area， back area > low stocking area > high stocking area > outlet area， and there were no significant difference and change rules among the inlet area， purification area， recirculation area and back area. As for seasonal changes， the total biomass of Chlorophyta in each test partition was generally in the order of summer > autumn > winter， while there was no significant difference in the proportion of Chlorophyta in total phytoplankton. ShannonWiener diversity index is also an important indicator for assessing water quality through biomonitoring. The water with ShannonWiener diversity index of above 3 is clean water， of 2-3 is in light pollution level， of 1-2 is at middle pollution level， and of less than 1 is of heavy pollution level. The water quality is better with larger diversity index[34]. The results of this study show that the diversity indexes of phytoplankton in the inlet area， purification area， recirculation area and back area are higher than those in the aquaculture area and outlet area， indicating that the water quality is better in the inlet area， purification area， recirculation area and back area than in the aquaculture area and outlet area. Moreover， according to the biodiversity， the water quality in outlet area is at middle pollution level， the water quality in high stocking area is in light pollution level， and the water quality in other 5 areas is at clean level， which suggests that the water quality is in good states except that in outlet area， and the pond industrial ecoaquaculture system functioned well.
Conclusion
The total biomass of phytoplankton， the biomass of Cyanophyta and the proportion of Cyanophyta are all in the order of outlet area > high stocking area > low stocking area > inlet area， purification area， recirculation area and back area. The biomass of Chlorophyta and its proportion are in the order of inlet area， purification area， recirculation area and back area > low stocking area > high stocking area > outlet area. These parameters show no significant difference and change rules among the inlet area， purification area， recirculation area and back area.
The number of phytoplankton species， diversity index and uniformity index are all in the order of inlet area， purification area， recirculation area and back area > low stocking area > high stocking area > outlet area. These parameters show no significant difference and change rules among the inlet area， purification area， recirculation area and back area.
The water in the outlet area is close to mille pollution， water in the high stocking area is in light pollution level， and the water in the other 5 areas are ate clear level， indicating that the water quality is in good states except that in outlet area， and the pond industrial ecoaquaculture system functioned well.
References
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Key words Pond industrial ecoaquaculture system; Water quality assessment; Megalobrama amblycephala; Phytoplankton; Structure characteristics
China is a big pond aquaculture country in the world， and pond aquaculture plays an important role in the national economy of China[1]. However， the current pond aquaculture in China faces 2 major bottlenecks， namely， serious water pollution[2-3] and low mechanization[4]， thus limiting the healthy and sustainable development of pond aquaculture. Therefore， it has become the key issue to be solved urgently in modern pond aquaculture to create new aquaculture modes， improve the modernization degree of pond culture and reduce the pollution of pond aquaculture to the internal and external environment. To this end， scholars in China have proposed the pond industrial ecoaquaculture system based on the partition aquaculture system of the foreign countries. In the system， a series of raceways with airpushing water aeration and sewage disposal equipment are constructed by using 2%-5% of the pond surface， and the raceways are used as the aquaculture area to proceed high stocking density aquaculture similar to “factories” to carry out industrial management， and the remaining 95%-98% of the pond water surface is used as the purification area to carry out biological purification to the culture tail water remaining in the pond after proper modification， which realizes the zero discharge or standardized discharge[5]. Thus， the partition of the aquaculture units and the water quality purification unit makes the yield of a small area equal to that of the original total water area， which completely reforms the traditional pond fish aquaculture mode. As a new pond ecoaquaculture mode， pond industrial ecoaquaculture system has been promoted and applied in some areas， but its fundamental research is very poor. There is still no report on the phytoplankton community structure in pond industrial ecoaquaculture system. Phytoplankton are the primary producers in aquatic ecosystems， and also important ecological indicators for monitoring and evaluating water quality[6]. Phytoplankton can respond quickly to changes in water nutrient status[7]， and their community structure is closely related to water quality， which can comprehensively and truly reflect the ecological and nutritional status of water bodies[7-9]. Therefore， studying phytoplankton community structure in aquaculture water can not only understand the primary productivity of pond water， but also determine the environmental conditions of water. Therefore， this study investigated the changes of phytoplankton community structure in the pond industrial ecoaquaculture system for Megalobrama amblycephala， with the aim to provide scientific bases for clarifying the ecological mechanism of the pond industrial ecoaquaculture system.
Materials and Methods
Test design
The test was carried out at Yancheng Zhengrong Ecological Fishery Co.， Ltd.， located in Jianhu County， Jiangsu Province. The pond industrial ecoaquaculture system included 6 test partitions （Fig.1）， namely， Aquaculture area （AA）， Inlet area （IA）， Outlet area （OA）， Purification area （PA）， Recirculation area （RA）， and Back area （BA）， in which the aquaculture area was divided into high stocking density aquaculture area and low stocking density culture area according to the test needs. The areas of the inlet area， outlet area， purification area， recirculation area and back area were respectively 3 000， 900， 15 000， 170 000 and 55 000 m 2， and the water depths were 4.0， 2.0， 1.2， 1.5 and 2.0 m， respectively. The aquaculture area had an area of 5 720 m 2 and consists of 52 cement ponds with an area of 110 m 2， pond depth of 2.5 m and water depth 2.0 m.
The test began on July 13， 2016， and there were 2 different aquaculture densities， namely， the Low stocking density aquaculture area （LA） and the High stocking density aquaculture area （HA）. The cultured species was Megalobrama amblycephala with an initial average body weight of （3.33 ± 0.52） g and an average body length of （56.13 ± 3.45） mm. The stocking densities for the low stocking and high stocking groups were 200 fish/m 2 and 300 fish/ m 2， respectively. Aquaculture test
The daily feeding amount during the aquaculture period is about 3% to 5% of the fish weight， which was fed for 3 times at 9：00， 12：30 and 16：00. During the aquaculture， the ponds were maintained with microflowing water， and the water velocity at the front end of the push water was 12 cm/s. The microtube aeration system was equipped for standby. The microtube aeration system was normally not open and only activated when the dissolved oxygen in the water was below 3 mg/L.
Phytoplankton collection， identification and counting
Phytoplankton samples were collected since July 2016. The samples were collected once a month in summer and autumn， and once every two months in winter and spring. A total of 23 sampling points were set up in the test area， including 3 high stocking density aquaculture areas， 3 low stocking density aquaculture areas， 3 inlet areas， 3 outlet areas， 5 purification areas， 3 recirculation areas and 3 back areas. The sampling points in each sampling site were shown in Fig.1. The phytoplankton samples were collected according to the method of Meng et al.[10]. The identification of phytoplankton was referred to Zheng et al.[11]， Han et al.[12]， Hu et al.[13].
Evaluation methods
Pielou uniformity index （J， Pielou） and ShannonWiener diversity index （D， ShannonWiener index） were used to analyze and evaluate the ecological characteristics of phytoplankton in water. The calculation methods of the above indexes were referred to Meng et al.[10， 14]， Liu et al.[15].
Results and Analysis
Species composition and biomass of phytoplankton
A total of 92 species （including mutants and variants） of 5 phyla were identified in the back areas of all the partitions， namely， Chlorophyta， Bacillariophyta， Cyanophyta， Euglenophyta， Cryptophyta， and Pyrroptata. The number of species of Chlorophyta was the largest with a total of 46， which accounted for 50.0% of the total species of the identified phytoplankton， followed by Cyanophyta and Euglenophyta， 15 species for each， which accounted for 16.3% of the total. There were 10 species of Bacillariophyta， accounting for 10.9% of the total， 5 species of Cryptophyta， accounting for 5.4% of the total， 1 species of Pyrroptata， accounting for 1.1% of the total. The species composition of phytoplankton was shown in Table 1. As shown in Table 1， 57 species of 6 phyla were identified in the low stocking area， 55 species of 5 phyla in the high stocking area， 64 species of 5 phyla in the inlet area， 51 species of 5 phyla in the outlet area， 64 species of 5 phyla in the purification area， 64 species of 6 phyla in the recirculation area and 63 species of 6 phyla in the back area. The number of species of phytoplankton in the low stocking area， high stocking area， inlet area， outlet area， purification area， recirculation area and back area was 40-52， 40-53， 45-62， 33-51， 45-55， 46-58， and 45-59， respectively （Fig. 2）. The number of phytoplankton species in each test partition was in the order of inlet area， purification area， recirculation area， back area > low stocking area > high stocking area > outlet area， and there were no significant difference and change rules among the inlet area， purification area， recirculation area and back area. In terms of seasonal changes， the number of species in each test partition was generally in the order of summer > autumn > winter. From July 2016 to April 2017， the total biomass of phytoplankton in low stocking area， high stocking area， inlet area， outlet area， purification area， recirculation area and back area was 5.25×10 7-13.42×10 7， 5.74×10 7-16.54×10 7， 5.08×10 7-10.36×10 7， 6.01×10 7-17.82×10 7， 5.46×10 7 - 11.31×10 7， 4.84×10 7-11.96×10 7， 4.84×10 7-10.75×10 7 cells/L （Fig. 2）. The total biomass in each partition was in the order of outlet area > high stocking area > low stocking area > inlet area， purification area， recirculation area and back area， and there were no significant difference and change rules among the inlet area， purification area， recirculation area and back area. As for seasonal changes， the total biomass of each partition was generally in the order of summer > autumn > winter.
Biomass of Cyanophyta and Chlorophyta and their proportions
The biomass of Cyanophyta was respectively 3.28×10 7-10.08×10 7， 4.07×10 7-13.04×10 7， 2.54×10 7-7.25×10 7， 4.78×10 7-15.63×10 7， 3.08×10 7-7.94×10 7， 2.52×10 7-8.66×10 7， 2.62×10 7 - 6.91×10 7 cells/L in the low stocking area， high stocking area， inlet area， outlet area， purification area， recirculation area and back area （Fig. 3）. Both the biomass of Cyanophyta and the proportion of Cyanophyta in total phytoplankton were in the order of outlet area > high stocking area > low stocking area > inlet area， purification area， recirculation area， back area， and there were no significant difference and change rules among the inlet area， purification area， recirculation area and back area. As for seasonal changes， the total biomass of Cyanophyta in each test partition was generally in the order of summer > autumn > winter， while there was no significant difference in the proportion of Cyanophyta in total phytoplankton.
The biomass of Chlorophyta was respectively 1.23×10 7-3.04×10 7， 1.03×10 7-2.96×10 7， 1.53×10 7-3.69×10 7， 0.99×10 7-2.87×10 7， 1.52×10 7-3.01×10 7， 1.43×10 7-3.03×10 7， 1.36×10 7-3.33×10 7 in the low stocking area， high stocking area， inlet area， outlet area， purification area， recirculation area and back area （Fig. 3）. Both the biomass of Chlorophyta and the proportion of Chlorophyta in total phytoplankton were in the order of inlet area， purification area， recirculation area， back area > low stocking area > high stocking area > outlet area， and there were no significant difference and change rules among the inlet area， purification area， recirculation area and back area. As for seasonal changes， the total biomass of Chlorophyta in each test partition was generally in the order of summer > autumn > winter， while there was no significant difference in the proportion of Chlorophyta in total phytoplankton. ShannonWiener diversity index is also an important indicator for assessing water quality through biomonitoring. The water with ShannonWiener diversity index of above 3 is clean water， of 2-3 is in light pollution level， of 1-2 is at middle pollution level， and of less than 1 is of heavy pollution level. The water quality is better with larger diversity index[34]. The results of this study show that the diversity indexes of phytoplankton in the inlet area， purification area， recirculation area and back area are higher than those in the aquaculture area and outlet area， indicating that the water quality is better in the inlet area， purification area， recirculation area and back area than in the aquaculture area and outlet area. Moreover， according to the biodiversity， the water quality in outlet area is at middle pollution level， the water quality in high stocking area is in light pollution level， and the water quality in other 5 areas is at clean level， which suggests that the water quality is in good states except that in outlet area， and the pond industrial ecoaquaculture system functioned well.
Conclusion
The total biomass of phytoplankton， the biomass of Cyanophyta and the proportion of Cyanophyta are all in the order of outlet area > high stocking area > low stocking area > inlet area， purification area， recirculation area and back area. The biomass of Chlorophyta and its proportion are in the order of inlet area， purification area， recirculation area and back area > low stocking area > high stocking area > outlet area. These parameters show no significant difference and change rules among the inlet area， purification area， recirculation area and back area.
The number of phytoplankton species， diversity index and uniformity index are all in the order of inlet area， purification area， recirculation area and back area > low stocking area > high stocking area > outlet area. These parameters show no significant difference and change rules among the inlet area， purification area， recirculation area and back area.
The water in the outlet area is close to mille pollution， water in the high stocking area is in light pollution level， and the water in the other 5 areas are ate clear level， indicating that the water quality is in good states except that in outlet area， and the pond industrial ecoaquaculture system functioned well.
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