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Abstract In order to investigate the influence of season changes of environmental factors in closed shrimp ponds on the community structure of planktonic microalgae， this paper conducted isolated tests to the temperature， pH， total nitrogen （TN）， total phosphorus （TP） and chlorophyll a in the shrimp culture ponds， analyzed the planktonic microalgae community structure and changes of dominant species in water over the same period， with the aim to explain the responses of microalgae community to changes of environmental factors in the pond without foreign water sources. The results showed that pond water temperature and TP content gradually increased from April to the end of September and then declined in October; water pH first decreased and then increased; TN and chlorophyll a first increased and then decreased. The dominant species in the pond were Cyclotella sp.， Navicula sp.， Oocystis borgei and S. quadricauda in the early stage （Apirl-May）， Chlamydomonas sp.， Chlorella sp.， S. quadricauda， Golenkinia radiata and Pseudanabeanain in medium stage （June-July）， Microcystis， Pseudanabeana， Chlorella sp. And S. quadricaudain in the midlate stage （August-September）， Microcystis， Pseudanabeana， Oscillatoria sp. and Chlorella sp. in the late stage （October-December）， and Chlorella sp.， Golenkinia radiate， S. quadricauda in the last stage. Diatoms and green algae were the dominant species in the early stage when the water had low temperature， low N and P nutrition but high N/P and a certain salinity. With the increase of temperature， water desalination and accumulation of N and P nutrition， algae became the dominant species. Cyanobacteria became the strong dominant stages in the midlate stage when the water had high temperature in the state of eutrophication， and there were also some green algae which were fond of high temperature and had resistant to pollution. In the late stage， cyanobacteria were the absolute dominant stage， but with the decrease of water temperature in the last stage， green algae became the dominant species， and planktonic microalgae biomass in the pool decreased.
Key words Closed ponds; Environmental factors; Microalgae; Population; Succession
In shrimp culture， the optimization of water ecological environment is the fundamental guarantee for the healthy growth of shrimp. Planktonic microalgae are an important part of aquaculture water. The community structure of microalgae is closely related to water environment factors[1]. Investigations have been made to the community distribution of planktonic microalgae and changes of environmental factors in the shrimp ponds with different aquaculture modes， seasons and regions， and studies have been conducted to the ecological niches of microalgae that can easily form dominant populations in shrimp ponds[2-5]. The excellent microalgae community plays an important role in the construction of a good aquaculture ecosystem. As a primary productivity， it synthesizes organic matter through photosynthesis， continuously supplies food to the system， increases the dissolved oxygen content of the water body， accelerates the oxidative decomposition of the reducing harmful substances in the water， absorbs ammonia nitrogen， nitrite and other substances which are toxic to shrimps， and also absorbs dissolved nutrients such as active phosphate， nitrate nitrogen and amino acids， which can maintain a virtuous cycle of ecosystems and regulate water purification. The poor microalgae community can cause stress to the healthy growth of shrimps， such as some species of blue algae like Microcystis， Anabaena and Oscillaria. The secreted toxins may cause harm to the health of the shrimp， and it is easy to form water blooms in high temperature water bodies with high nitrogen content and rich organic matters， which can result in the release of a large number of toxic amines such as hydroxylamines and sulfides， resulting in stressinduced death of shrimps[6-7]. Therefore， it is of important significance for the construction of a healthy aquaculture ecosystem to study the changes of environmental factors and the succession rules of microalgae communities in shrimp ponds. In this study， to completely eliminate the interference of external water sources， closed shrimp ponds were used to analyze the changes of environmental factors and the succession of the dominant communities of microalgae in shrimp ponds， which more accurately reflected the correlations， so as to provide the basis for the controlling technology of shrimp cultured algae. Materials and Methods
Basic situation of shrimp ponds
Located in Laiwuwei of Chenghai District， Shantou City， Guangdong Province， the test shrimp ponds had a total area of 1.452 hm 2， consisting of 2 sand sediment ponds of different sizes with an area of 0.924 and 0.528 hm 2， respectively. The aquaculture water was the seeping surface water. Since the shrimp ponds were built from the sand by the seaside， the seeping surface water contained a small amount of salt during winter when the rainfall was low. The salinity of the ponds was 1-2， and the salinity was 0 after the rainy season. There was no external water source in the whole aquaculture process， and the water lost by pumping and drainage in the middle and late period was supplemented by surface water seepage. The water depth of the shrimp pond was 1.3-1.8 m， and there was no water inlet in the pond but 1 drainage ditch for discharge waste water. The 2 ponds were equipped with 5 and 3 waterwheel aerators with the power of 1.5 kW according to the sizes， which were used during night and rainy days in the aquaculture process. Before the aquaculture， the shrimp ponds were drained and exposed for 1 month， and young Litopenaeus vannamei was thrown in the ponds in early April. For the first time， 2 million young shrimps were put into the pond， and then 0.3 million more were added to the ponds in the middle of each month until October， when no more shrimps were added. There were a total of about 3.8 million young shrimps cultured in the ponds every year. Adult shrimps were harvested from late June and ended in January of the next year. Artificial compound feed was delivered to the ponds during the whole process of aquaculture. There were 160 silver carps and 700 grass carps mixed cultured in the shrimp ponds.
Collection and analysis of planktonic microalgae
From April 2015 to January 2016， sampling and analysis of planktonic microalgae in shrimp ponds were carried out every 15 d throughout the whole aquaculture year. Water samples were collected from the 4 sides and the centers of the 2 ponds using a 2.5 L sampler， 1 for each site. The collected 5 water samples from each pond were merged and mixed， and then filtered through the No. 25 planktonic microalgae screen. The filtrate was transferred to a 250 ml sample bottle， fixed with 5% formalin， followed by microscopic examination and counting after fully shaking[8]. The qualitative analysis of the microalgae samples was carried out under the light microscope to identify their species with reference to the method of Hu et al[9]. The sample solution was precipitated and concentrated for quantitative analysis， and the 0.1 ml planktonic micrometer was used to count the planktonic microalgae under a light microscope[10]， and the average of 3 repetitions was taken. The degree of dominance was calculated using the following formula：
pi=ni/N·fi
Where， ni is the number of individuals of the i species; N is the total number of individuals in the community sample; fi is the frequency at which the species appears in the region. The species with the total number accounting for over 10% of the total individual numbers in each sampling was the dominant species， 1%-10% was the common species， and less than 1% was the rare species[11].
Physical and chemical factor determination and water sample collection
The water temperature was measured according to the Water Quality-Determination of Water Temperature-Thermometer or Reversing Thermometer Method （GB1319591）; pH was determined by the glass electrode method （GB6920 86） of the Environmental Quality Standards for Surface Water （GB38382002）; total nitrogen （TN） was determined using the alkaline potassium persulfate digestion UV spectrophotometric method of the Environmental Quality Standards for Surface Water （GB38382002）; total phosphorus （TP） using the ammonium molybdate spectrophotometric method （GB1189389） in the the Environmental Quality Standards for Surface Water （GB38382002; chlorophyll a content was determined by dimethyl sulfoxideacetone extraction spectrophotometry.
The collecting time and frequency of water samples for detecting environmental factors were synchronized with the collection of water samples from planktonic microalgae. Water samples were collected from the upper layer （20 cm from the water surface） and the lower layer （30 cm from the bottom of the pool） using a 2 L column glass sampler， and after mixed， the collected water was put into a 1 L polyethylene bottle for the determination of various physical and chemical factors. Then， the 500 ml polyethylene bottle was used to collect the water from the upper and lower layers， and after mixed， 500 ml was taken for the determination of chlorophyll a content. The bottles with water samples were placed in a foam box with ice cubes in advance and brought back to the laboratory for testing. The temperature was tested on site.
Results and Analysis
Determination of environmental factors
From April， samples were taken once in the middle of each month and at the end of each month. Each time， three parallel tests were performed， and the averages of the test results were taken （Table 1）. The water temperature change in the shrimp pond was synchronized with the air temperature. TP content remained at a low level when threw was no shrimp released in April， and gradually increased from May to the end of September until October， when it began to decline. In addition to the release of residual bait and animal waste and the transport of phosphorus to water bodies due to temperature factors， it may also be related to the accelerated rate of phosphorus uptake after cyanobacteria became the absolute dominant populations. pH declined slowly before September， and then rose after October， because microalgae consumed CO2-3 and PO3-4 during the rapid growth period， leading to the reverse increase of pH. TN and chlorophyll a content increased from the beginning to November. The accumulation of residual bait and animal waste led to the increase of TN， while the increase of chlorophyll a content was related with the biomass of planktonic microalgae in the water. Analysis of dominant populations of planktonic microalgae
From April， samples were taken once in the middle of each month and at the end of each month. Each time， three parallel tests were performed， and the averages of the test results were taken and calculated according to the dominance formula （Table 2）. Combined the environmental factor testing results with the data in Table 2， the population succession of dominant microalgae was closely related to the change of environmental factors. In the early waters with low temperature， low nitrogen and phosphorus nutrition， high N/P and certain salinity， the dominant species were green algae and diatoms like Chlorella sp.， Navicula sp.， Oocystis borgei and S. quadricauda. With the increase of temperature， complete water desalination and accumulation of N and P nutrition， green algae like S. quadricauda， Chlamydomonas sp.， Chlorella sp.， and Golenkinia radiate became the dominant species. In the midlate stage when the water had high temperature in the state of eutrophication， cyanobacteria became the dominant species， and there were also some green algae which were fond of high temperature and had resistant to pollution. In the late stage， cyanobacteria such as Microcystis， Pseudanabeana and Oscillatoria sp. were in the absolute dominant status with the dominance degree beyond or close to 20%. With the decrease of water temperature in the last stage， green algae such as Chlorella sp.， G. radiate became the dominant species， and planktonic microalgae biomass in the pool decreased.
Conclusion and Discussion
The enclosed shrimp pond is isolated from the outside， which relies only on surface water seepage as aquaculture water， eliminating the interference of external water sources， and thereby forming a completely independent pond culture ecosystem. In this way， the analysis on the interaction between environmental factors and microalgae populations can reflect the correlations between the changes of environmental factors and the succession of microalgae community more truly， making it better to have a true understanding of the niche of various microalgae. There are interactions and mutual constraints between biological factors and physical and chemical factors in the shrimp pond ecosystem. Changes in environmental factors can change the structure of planktonic microalgae community， and the succession of microalgae communities can also result in the changes of environmental factors[12]. In the shrimp pond culture ecosystem， the succession of microalgae community is a very complicated process， which is affected by various environmental factors. The results of this study indicate that temperature and N and P nutrition changes are the main factors affecting the succession of microalgae communities.
Key words Closed ponds; Environmental factors; Microalgae; Population; Succession
In shrimp culture， the optimization of water ecological environment is the fundamental guarantee for the healthy growth of shrimp. Planktonic microalgae are an important part of aquaculture water. The community structure of microalgae is closely related to water environment factors[1]. Investigations have been made to the community distribution of planktonic microalgae and changes of environmental factors in the shrimp ponds with different aquaculture modes， seasons and regions， and studies have been conducted to the ecological niches of microalgae that can easily form dominant populations in shrimp ponds[2-5]. The excellent microalgae community plays an important role in the construction of a good aquaculture ecosystem. As a primary productivity， it synthesizes organic matter through photosynthesis， continuously supplies food to the system， increases the dissolved oxygen content of the water body， accelerates the oxidative decomposition of the reducing harmful substances in the water， absorbs ammonia nitrogen， nitrite and other substances which are toxic to shrimps， and also absorbs dissolved nutrients such as active phosphate， nitrate nitrogen and amino acids， which can maintain a virtuous cycle of ecosystems and regulate water purification. The poor microalgae community can cause stress to the healthy growth of shrimps， such as some species of blue algae like Microcystis， Anabaena and Oscillaria. The secreted toxins may cause harm to the health of the shrimp， and it is easy to form water blooms in high temperature water bodies with high nitrogen content and rich organic matters， which can result in the release of a large number of toxic amines such as hydroxylamines and sulfides， resulting in stressinduced death of shrimps[6-7]. Therefore， it is of important significance for the construction of a healthy aquaculture ecosystem to study the changes of environmental factors and the succession rules of microalgae communities in shrimp ponds. In this study， to completely eliminate the interference of external water sources， closed shrimp ponds were used to analyze the changes of environmental factors and the succession of the dominant communities of microalgae in shrimp ponds， which more accurately reflected the correlations， so as to provide the basis for the controlling technology of shrimp cultured algae. Materials and Methods
Basic situation of shrimp ponds
Located in Laiwuwei of Chenghai District， Shantou City， Guangdong Province， the test shrimp ponds had a total area of 1.452 hm 2， consisting of 2 sand sediment ponds of different sizes with an area of 0.924 and 0.528 hm 2， respectively. The aquaculture water was the seeping surface water. Since the shrimp ponds were built from the sand by the seaside， the seeping surface water contained a small amount of salt during winter when the rainfall was low. The salinity of the ponds was 1-2， and the salinity was 0 after the rainy season. There was no external water source in the whole aquaculture process， and the water lost by pumping and drainage in the middle and late period was supplemented by surface water seepage. The water depth of the shrimp pond was 1.3-1.8 m， and there was no water inlet in the pond but 1 drainage ditch for discharge waste water. The 2 ponds were equipped with 5 and 3 waterwheel aerators with the power of 1.5 kW according to the sizes， which were used during night and rainy days in the aquaculture process. Before the aquaculture， the shrimp ponds were drained and exposed for 1 month， and young Litopenaeus vannamei was thrown in the ponds in early April. For the first time， 2 million young shrimps were put into the pond， and then 0.3 million more were added to the ponds in the middle of each month until October， when no more shrimps were added. There were a total of about 3.8 million young shrimps cultured in the ponds every year. Adult shrimps were harvested from late June and ended in January of the next year. Artificial compound feed was delivered to the ponds during the whole process of aquaculture. There were 160 silver carps and 700 grass carps mixed cultured in the shrimp ponds.
Collection and analysis of planktonic microalgae
From April 2015 to January 2016， sampling and analysis of planktonic microalgae in shrimp ponds were carried out every 15 d throughout the whole aquaculture year. Water samples were collected from the 4 sides and the centers of the 2 ponds using a 2.5 L sampler， 1 for each site. The collected 5 water samples from each pond were merged and mixed， and then filtered through the No. 25 planktonic microalgae screen. The filtrate was transferred to a 250 ml sample bottle， fixed with 5% formalin， followed by microscopic examination and counting after fully shaking[8]. The qualitative analysis of the microalgae samples was carried out under the light microscope to identify their species with reference to the method of Hu et al[9]. The sample solution was precipitated and concentrated for quantitative analysis， and the 0.1 ml planktonic micrometer was used to count the planktonic microalgae under a light microscope[10]， and the average of 3 repetitions was taken. The degree of dominance was calculated using the following formula：
pi=ni/N·fi
Where， ni is the number of individuals of the i species; N is the total number of individuals in the community sample; fi is the frequency at which the species appears in the region. The species with the total number accounting for over 10% of the total individual numbers in each sampling was the dominant species， 1%-10% was the common species， and less than 1% was the rare species[11].
Physical and chemical factor determination and water sample collection
The water temperature was measured according to the Water Quality-Determination of Water Temperature-Thermometer or Reversing Thermometer Method （GB1319591）; pH was determined by the glass electrode method （GB6920 86） of the Environmental Quality Standards for Surface Water （GB38382002）; total nitrogen （TN） was determined using the alkaline potassium persulfate digestion UV spectrophotometric method of the Environmental Quality Standards for Surface Water （GB38382002）; total phosphorus （TP） using the ammonium molybdate spectrophotometric method （GB1189389） in the the Environmental Quality Standards for Surface Water （GB38382002; chlorophyll a content was determined by dimethyl sulfoxideacetone extraction spectrophotometry.
The collecting time and frequency of water samples for detecting environmental factors were synchronized with the collection of water samples from planktonic microalgae. Water samples were collected from the upper layer （20 cm from the water surface） and the lower layer （30 cm from the bottom of the pool） using a 2 L column glass sampler， and after mixed， the collected water was put into a 1 L polyethylene bottle for the determination of various physical and chemical factors. Then， the 500 ml polyethylene bottle was used to collect the water from the upper and lower layers， and after mixed， 500 ml was taken for the determination of chlorophyll a content. The bottles with water samples were placed in a foam box with ice cubes in advance and brought back to the laboratory for testing. The temperature was tested on site.
Results and Analysis
Determination of environmental factors
From April， samples were taken once in the middle of each month and at the end of each month. Each time， three parallel tests were performed， and the averages of the test results were taken （Table 1）. The water temperature change in the shrimp pond was synchronized with the air temperature. TP content remained at a low level when threw was no shrimp released in April， and gradually increased from May to the end of September until October， when it began to decline. In addition to the release of residual bait and animal waste and the transport of phosphorus to water bodies due to temperature factors， it may also be related to the accelerated rate of phosphorus uptake after cyanobacteria became the absolute dominant populations. pH declined slowly before September， and then rose after October， because microalgae consumed CO2-3 and PO3-4 during the rapid growth period， leading to the reverse increase of pH. TN and chlorophyll a content increased from the beginning to November. The accumulation of residual bait and animal waste led to the increase of TN， while the increase of chlorophyll a content was related with the biomass of planktonic microalgae in the water. Analysis of dominant populations of planktonic microalgae
From April， samples were taken once in the middle of each month and at the end of each month. Each time， three parallel tests were performed， and the averages of the test results were taken and calculated according to the dominance formula （Table 2）. Combined the environmental factor testing results with the data in Table 2， the population succession of dominant microalgae was closely related to the change of environmental factors. In the early waters with low temperature， low nitrogen and phosphorus nutrition， high N/P and certain salinity， the dominant species were green algae and diatoms like Chlorella sp.， Navicula sp.， Oocystis borgei and S. quadricauda. With the increase of temperature， complete water desalination and accumulation of N and P nutrition， green algae like S. quadricauda， Chlamydomonas sp.， Chlorella sp.， and Golenkinia radiate became the dominant species. In the midlate stage when the water had high temperature in the state of eutrophication， cyanobacteria became the dominant species， and there were also some green algae which were fond of high temperature and had resistant to pollution. In the late stage， cyanobacteria such as Microcystis， Pseudanabeana and Oscillatoria sp. were in the absolute dominant status with the dominance degree beyond or close to 20%. With the decrease of water temperature in the last stage， green algae such as Chlorella sp.， G. radiate became the dominant species， and planktonic microalgae biomass in the pool decreased.
Conclusion and Discussion
The enclosed shrimp pond is isolated from the outside， which relies only on surface water seepage as aquaculture water， eliminating the interference of external water sources， and thereby forming a completely independent pond culture ecosystem. In this way， the analysis on the interaction between environmental factors and microalgae populations can reflect the correlations between the changes of environmental factors and the succession of microalgae community more truly， making it better to have a true understanding of the niche of various microalgae. There are interactions and mutual constraints between biological factors and physical and chemical factors in the shrimp pond ecosystem. Changes in environmental factors can change the structure of planktonic microalgae community， and the succession of microalgae communities can also result in the changes of environmental factors[12]. In the shrimp pond culture ecosystem， the succession of microalgae community is a very complicated process， which is affected by various environmental factors. The results of this study indicate that temperature and N and P nutrition changes are the main factors affecting the succession of microalgae communities.