Planktonic ciliates are a significant component of microplankton communities in the ocean and they play a crucial role in microbial food webs (Azam et al. 1983). They have long been considered mediators of production and energy transfer from primary producers (e.g. pico- and nano-phytoplankton) to higher trophic levels (Ellumi et al. 2006; Xu et al. 2008). Moreover, ciliates respond more quickly to environmental factors compared to other organisms, because of their sensitivity, higher reproduction rates, and delicate cell membranes (Madoni et al. 2005; Kchaou et al. 2009). Therefore, changes in ciliate communities are integral to aquatic habitats, especially to chemical and physical conditions in aquatic ecosystems (Xu et al. 2009; Jiang et al. 2011a; Kim et al. 2012).
Daya Bay is a shallow semi-enclosed bay with an area of approximately 600 km2, located in the northwestern part of the South China Sea. Daya Bay and its surrounding area have been listed as an important economic development district and mariculture areas in Guangdong Province, China. Mariculture has developed rapidly since the 1990s and is responsible for the increasing nutrient loadings (Wang et al. 2008). Petrochemical, plastic, printing and other industries, as well as harbors are present in the province (Song et al. 2004). Additionally, the Daya Bay Nuclear Power Station (DNPS) is located in the bay, which has operated since 1994 and discharges heated water at the rate of 2.9 × 107 m3 yr-1 (Liu et al. 2006). Another nuclear power station, the Lingao Nuclear Power Station, has operated since 2002. All these activities generate an increasing anthropogenic impact on the ecological environment of Daya Bay, and environmental changes (e.g. nutrients, water temperature, salinity and pH) cause significant changes in the abundance, biomass, diversity and the community structure of microplanktonic organisms in Daya Bay (Wang et al. 2009; Ma et al. 2014).
There have been a number of studies dealing with plankton community dynamics in Daya Bay (Song et al. 2004; Sun et al. 2011, Wang et al. 2014a; Li et al. 2014). However, there are no data available on planktonic ciliate community structures. In this study, we investigated the spatial and seasonal variation in the species composition and abundance. The main objectives were to clarify the spatial and seasonal pattern of planktonic ciliate communities on a local scale and to identify the most important environmental factors affecting the spatial and seasonal pattern.
Twelve sampling sites were located in four areas of Daya Bay where different types of human activities occur (Fig. 1). Sampling sites 1-3 were located in the Dapeng Cove aquaculture area (DC), sites 4-6 near the Daya Bay Nuclear Power Station (DNPS), sites 7-9 at artificial reefs (ARs), and sites 10-12 near the water outlets of the Pinghai fossil fuel-fired Power Station (PFPS).
Location of the study area and the sampling sites of planktonic ciliates in Daya BayFigure 1
Planktonic ciliate samples were collected with a Niskin water sampler at a depth of 0.5 m from Daya Bay in January, April, August and November 2014, representing the temporal ciliate community in winter, spring, summer and autumn, respectively. Seawater samples of 2500 ml (fixed with 1% formaldehyde in plastic bottles) were used for quantitative analysis and identification of ciliates. A total of 48 ciliate samples were fixed and analyzed. Environmental factors, such as pH, salinity (Sal.), and temperature (Temp. oC) were measured in situ at sampling sites, using a multi-parameter sensor (YSI Professional Plus, USA). Dissolved oxygen concentration (DO, mg l-1), nitrate nitrogen (NO3-, μmol l-1), nitrite nitrogen (NO2-, μmol l-1), ammonium nitrogen (NH4+, μmol l-1) and soluble reactive phosphate (PO43-, μmol l-1) were determined using a UV-visible spectrophotometer (UV2450, Shimadzu) according to marine monitoring specifications (GB 17378.4-2007, 2007). DIN is the sum of NO3-, NO2-, and NH4+. Chlorophyll-
Formaldehyde fixed samples were settled for at least 48 h and then concentrated to 50 ml (Utermöhl 1958). Each time, 0.1 ml of a well-mixed concentrated sample was placed on a microscope slide and ciliates were counted under a light microscope at 200× or 400×. Ten slide replicates were examined for each concentrated sample. Tintinnids were identified on the basis of lorica morphology and species description provided by Kofoid and Campbell (1929, 1939) and Lynn (2008). Other ciliates were identified following Song et al. (2008). The taxonomic scheme mainly refers to Lynn (2008). All ciliates were finally identified to the lowest possible taxa.
The number of species and ciliate abundance were calculated for each sample. The dominance (
where n
Cluster analysis from the Primer v5.0 software package was used to investigate the ciliate community structure (Clarke and Gorley 2001). Bray-Curtis similarity matrices were computed on the fourth-root transformed ciliate abundance data and the Euclidean distance matrix was computed on log(x+1)-transformed environmental data (including Chl
Detailed relationships between ciliate communities and environmental factors were analyzed by CANOCO 4.5 (ter Braak and Smilauer 2002). To prevent any disproportionate influence of rare species in the subsequent analysis, only species with incidence >10% in all samples (or total sites) and abundance >1.0% of the total ciliate abundance were considered. First, detrended correspondence analysis (DCA) was employed on the ciliate abundance data to decide whether linear or unimodal ordination methods should be applied. If the length of the gradient was greater than 3, Canonical Correspondence Analysis (CCA) was used, otherwise Redundancy Analysis (RDA) was applied. CCA or RDA analysis was conducted based on log(x+1)-transformed species abundance and environmental data (except for pH). In addition, Spearman correlation analysis between ciliate communities and environmental factors was computed by the SPSS v20.0 statistical program.
Mean values of ten environmental factors were summarized in Table 1. Water temperature followed a clear seasonal pattern, ranging from 16.93oC to 27.75oC (mean 23.58oC), and the maximum was recorded in DNPS areas in four seasons. Salinity peaked in summer (33.94 PSU). Values of pH ranged from 8.07 to 8.27, on average 8.21. Values of DO varied inversely with temperature, and the maximum was recorded in winter. Concentration of NO2-, NH4+, NO3- and DIN peaked in summer, and was relatively low in winter. Soluble reactive phosphate (PO43-) ranged from 0.20 μmol l-1 to 0.41 μmol l-1, which was relatively high in summer. Chl
Environmental variables for Daya Bay water samplings in winter 2013, spring, summer and autumn 2014
Parameters
Unit
Winter
Spring
Summer
Autumn
Temp.
°C
16.93
24.73
27.75
24.89
Sal.
PSU
32.83
31.86
33.94
33.80
pH
8.27
8.31
8.07
8.20
DO
mg l-1
7.21
5.98
5.45
5.46
NO2-
μmol l-1
0.39
0.27
0.63
0.20
NO3-
1.57
1.73
7.05
2.09
NH4+
1.53
1.74
3.12
1.78
DIN
3.49
3.74
10.8
4.08
PO43-
0.29
0.20
0.41
0.22
Chl
μg l-1
1.78
2.07
3.92
2.48
The taxonomic composition of ciliate communities observed during the study period is summarized in Table 2. A total of 41 planktonic ciliate species representing 22 genera and eight orders (Tintinnida, Oligotrichida, Euplotida, Cyclotrichida, Haptorida, Pleurostomatida, Prostomatida, Pleuronematida) were recorded during the one-year survey. Tintinnida and Oligotrichida were represented by the highest number of species, accounting for 70% and 15% of the total number of species, respectively (Fig. 2a). However, Oligotrichida was the most abundant order and accounted for up to 54.35% of the total ciliate abundance, followed by Tintinnida and Cyclotrichida (Fig. 2b).
List of ciliate species encountered in Daya Bay
Ciliate species
1
22
2
23
3
24
4
25
5
26
6
27
7
28
8
29
9
30
10
31
11
32
12
33
13
34
14
35
15
36
16
37
17
38
18
39
19
40
20
41
21
Proportions of the number of species (a) and abundance (b) of planktonic ciliates in Daya BayFigure 2
The dominant species (dominance
Dominant ciliate species in each season in Daya Bay (species dominance,
Dominant species
Winter
Spring
Summer
Autumn
0.5716
-
-
0.0215
0.0863
-
-
-
0.0387
0.4583
0.0336
0.1792
0.1165
0.0538
0.0413
0.1481
-
0.3678
0.0656
0.0443
-
0.0687
-
-
-
-
0.1321
-
-
-
0.0407
-
-
-
0.0200
0.0447
-
-
0.0235
-
-
-
0.3221
-
-
-
-
0.0937
-
-
-
0.0507
-
-
-
0.0948
The total number of ciliate species ranged from 6 to 32 (Fig. 3a). The smallest number of species was observed in spring, and the highest one in summer. In terms of spatial distribution, the maximum number of species was recorded at ARs and the minimum at DC (Fig. 3a).
Variation in the number of species (a) and abundance (b) of planktonic ciliates in Daya BayFigure 3
The abundance of ciliates showed a marked seasonal change, with the highest values observed in spring and the lowest in summer (Fig. 3b). In spring, their abundance ranged from 350 to 3120 ind. l-1, with an average of 1409.1 ± 92.6 ind. l-1. Interestingly,
Cluster analysis divided the ciliate communities into four groups at a similarity level of 40% in terms of their temporal distribution in Daya Bay during the study period (Fig. 4). Group 1 comprised some of the samples collected in autumn. Group 2 included all winter samples, while Group 3 consisted of all spring samples except for the sample collected from site 3 in autumn. Group 4 contained all the remaining autumn and all summer samples. The multidimensional scaling test (MDS) produced a similar result as mentioned above (Fig. 5). ANOSIM demonstrated that there were significant differences between each group (global R = 0.836,
Cluster analysis of ciliate communities on the Bray-Curtis similarity matrix from the fourth-root transformed biotic data of 48 surface samples from Daya Bay. Wi: winter; Sp: spring; Su: summer; Au: autumnFigure 4
Multidimensional scaling (MDS) analysis of ciliate communities on the Bray-Curtis similarity matrix from the fourth-root transformed biotic data of 48 surface samples in Daya Bay. Wi: winter; Sp: spring; Su: summer; Au: autumnFigure 5
BIOENV analysis revealed that ciliate communities were significantly correlated with abiotic environmental factors, i.e. temperature, salinity, NO2-, NO3- and PO43- (Table 4). Spearman correlation analysis demonstrated that ciliate abundance was significantly positively correlated with pH and DO, and negatively correlated with water salinity (Table 5). Similarly, there was a significant negative correlation between ciliate abundance and salinity in spring. In summer, temperature, DO, NO2- and PO43- were the main factors affecting the ciliate abundance, whereas NH4+ was significantly positively correlated with ciliate abundance in autumn. As can be observed in the CCA plot of four seasons (Fig 6a), samples collected in winter were generally gathered on positive Axis 2, indicating higher optima for DO, NO3-, PO43-, NO2- and DIN, and lower values for temperature and pH. Samples collected in summer were entirely on positive Axis 1, indicating higher optima for temperature and NH4+, whereas lower optima for pH, NO3-, DIN and DO. Spring and autumn samples were located on negative Axis 2, indicating higher optimum values for temperature and pH, but lower values for PO43-, NO3-, NO2-, DO and DIN. Results of RDA implied that environmental variables significantly affecting ciliate communities during four seasons were different. Environmental variables that significantly affected the spatial pattern of ciliate communities in winter were DO, Chl
Summary of results from biota-environment (BIOENV) analysis, with the top ten correlations corresponding to different variables (R is Spearman correlation coefficient)
Rank
R
Environmental variables
1
0.391
Sal., pH
2
0.388
Sal., NO2-, NO3-, NH4+, PO43-
3
0.387
DO, NO2-, NO3-, NH4+, PO43-
4
0.386
Temp., Sal., pH
5
0.386
Temp., NO2-, NO3-, NH4+, PO43-
6
0.386
Sal., DO, NO2-, NO3-, PO43-
7
0.384
pH, NO2-, NO3-, NH4+, PO43-
8
0.384
Sal., pH, NO2-, NO3-, PO43-
9
0.383
Temp., Sal., NO2-, NO3-, PO43-
10
0.383
pH, DO, NO2-, NO3-, PO43-
Correlation between environmental variables and ciliate abundance (
parameters
Winter
Spring
Summer
Autumn
Temp.
-0.448
0.088
0.374
-0.181
0.282
-0.751**
-0.470
-0.083
Sal.
-0.606*
0.441
-0.840**
-0.103
0.881**
-0.720**
0.231
0.431
pH
0.748**
-0.537
-0.448
-0.061
0.342
-0.241
0.309
-0.169
DO
0.672*
-0.352
-0.379
-0.330
0.757**
-0.560
-0.214
-0.064
NO2-
-0.469
0.454
0.331
-0.088
0.772**
-0.549
0.499
0.395
NO3-
-0.489
0.185
0.341
-0.02
-0.346
0.049
-0.161
-0.511
NH4+
-0.113
0.543
0.434
-0.105
-0.088
0.225
0.590*
0.688*
DIN
-0.484
0.247
0.468
-0.091
0.179
-0.155
0.474
0.338
PO43-
-0.329
0.693*
0.547
-0.165
0.677**
-0.278
-0.423
-0.794**
Chl
0.395
0.179
0.244
0.432
0.026
0.047
-0.065
-0.298
Canonical Correspondence Analysis (CCA) and Redundancy Analysis (RDA) ordination plots show the relationship between ciliate communities and environmental factors during four seasons (a), in winter (b), in spring (c), in summer (d) and in autumn (e). The length of vectors indicates the marginal effects of environmental variables they represent. The sample points can be projected perpendicularly onto the line overlaying the arrow of a given environmental variable. The sample points are arranged according to the predicated increase in values of a given environmental variable and the predicted increase occurs in the direction indicated by the arrow. (a) expresses mainly seasonal variation in ciliate communities in relation to environmental factors. (b)-(e) express spatial variation in ciliate communities in each season in relation to environmental factors. Wi: winter; Sp: spring; Su: summer; Au: autumnFigure 6
Our study revealed that planktonic ciliate communities exhibited an obvious seasonal and spatial variation in terms of species composition and abundance in Daya Bay. The number of ciliate species was high in summer and autumn, while low in winter and spring. In contrast, the ciliate abundance in spring was significantly higher compared to other three seasons. Similar results were obtained in other marine systems, such as the Bay of Biscay (Urrutxurtu et al. 2003) and the Bay of Baisha (our unpublished data), where one peak of ciliate abundance was observed in spring. However, in the Helgoland Roads (the North Sea), Yang et al. (2015) reported two peaks of ciliate abundance in spring and summer, respectively. Yu et al. (2013) reported two peaks of ciliates in spring and autumn on Zhangzi Island (the northern Yellow Sea). Jiang et al. (2011b) found that the abundance was high in summer and winter, while low in spring and autumn in Jiaozhou Bay. These differences in temporal variation patterns of ciliate communities may result from different locations of the study sites, different sampling seasons or different study methods applied. For example, samples in our study were collected from the surface water (at a depth of 0.5 m), while the water depth of ciliate samples in another study (Yu et al. 2013) was between 32 m and 58 m. In addition, environmental factors in marine regions were different, for example Jiang et al. (2011b) reported three peaks in Chl
Higher abundance of ciliates occurred in the DC area. Intensive aquaculture activities led to elevated nutrient concentrations (Wang et al. 2009) and phytoplankton biomass (Chl
Multivariate correlation analysis demonstrated that seasonal and spatial variation in planktonic ciliate communities were significantly related to environmental variations, especially water temperature, pH, dissolved oxygen, nutrients (e.g. NO2-, NO3-, NH4+ and PO43-) and Chl
In addition, the BIOENV analyses, spearman correlation analysis and CCA have suggested that ciliate communities in our study were significantly correlated with nutrient levels (e.g. PO43-, NO2-, NO3-, NH4+). Consistent with our results, previous studies indicated that the total abundance of ciliates was significantly affected by the level of phosphorus and nitrogen (Wang et al. 2013, 2014c). Simultaneously, Jiang et al. (2011b) suggested that the ciliate community variation in Jiaozhou Bay was correlated with NO3- and soluble reactive phosphates. Therefore, these findings demonstrated that the nutrient level is also an important factor influencing the seasonal and spatial variations in ciliate abundance. Moreover, other factors such as salinity, DO and water temperature also played an important role in determining both the spatial and seasonal pattern of ciliate communities in Daya Bay.
Furthermore, it is worth noting that the nutrient level and Chl
In conclusion, the ciliate communities show a significant seasonal and spatial variation in species composition and abundance in surface waters of Daya Bay. In addition, environmental factors, such as the nutrient level, Chl