CHAPTER TWO
3.4 Results
3.4.2 Canonical correspondence analysis (CCA)
CCA is a multivariate statistical analysis used to elucidate the relationships between biological community and their environment (ter Braak and Verdonschot, 1995). In this study CCA was used to investigate the relationships between the water quality variables and the virus like particles, as well as the relationships between the bacteriophages and the physico-chemical parameters at all sites and seasons. The arrows representing the environmental variables indicate the direction of maximum change of that variable across the diagram. In essence, the length of the arrow is proportional to the rate of change, so a longer arrow indicated a larger change in environmental variable.
CCA ordination plot (Figure 3.3) revealed strong relationships between the overall VLP and phage populations at different sites and seasons measured as well as with the physico-chemical water quality variables.
CCA plot (blue circle), temperature, BOD5, turbidity, pH, conductivity, orthophosphate and sulphate were the most important variables (Figure 3.3- long arrows) that impacted the community
0 50 100 150 200 250 300 350 400 450 500 550 600 650 700
0 200 400 600 800 1000 1200 1400 1600 1800 2000 2200 2400
U1 U2 U3 U4 U5
Plaque Forming Units (PFU/ml)
Virus Like Particles (VLPs/ml)
SAMPLING POINTS ALONG UMGENI RIVER
AUTUMN (SOMATIC PFUs) WINTER (SOMATIC PFUs) SPRING (SOMATIC PFUs) SUMMER (SOMATIC PFUs) AUTUMN (F-RNA PFUs) WINTER (F-RNA PFUs) SPRING (F-RNA PFUs) SUMMER (F-RNA PFUs)
AUTUMN (VLPs) WINTER (VLPs) SPRING (VLPs) SUMMER (VLPs)
77 structures significantly at sites 1, 2 and 3 during the autumn, summer and spring seasons. Site 2, 4, 5 during autumn, winter and spring seasons correlated with the nitrate/nitrite profiles (green circle). Site 1 and 3 in autumn and winter showed no correlations with the rest of the sites and variables measured.
Figure 3. 3 CCA ordination plot for all the water quality variables and the total viral and bacteriophage growth at the five study sites and during autumn, winter, spring and summer seasons. Abbreviations: Sites 1= Umgeni River mouth, 2= Informal settlement at Reservoir Hills, 3= New Germany Waste Treatment Works, 4= Krantzkloof Nature Reserve, 5= Inanda Dam. Seasons: A=Autumn, W= Winter, Sp= Spring, S= Summer.
CCA axis 1 (Figure 3.3) accounted for 77.6 % of total variance of the species data set and in total the species – environment relation accounted for 94.1% of the cumulative variance suggesting that there may be a correlation between the VLP and phage populations at the various sites and water quality variables during all seasons. Positive significant (p<0.05) Pearson correlation of species and environmental scores for axis one and two were r = 0.955 and r = 0.962 respectively (Table 3.2).
78 Table 3. 2 Properties of the Canonical Correlation Analysis ordination bi-plot for all the water quality variables and the total viral and bacteriophage growth at the five study sites during autumn, winter, spring and summer seasons.
Canonical properties Axis
1 2
Canonical Eigen value 0.046 0.038
% Cumulative variance of species data 77.6 89.4
% Cumulative variance of species – environment relation 83.6 94.1
Monte Carlo test p – value 0.048 0.062
Pearson correlation of species and environmental Scores 0.955 0.962
The ordination plot revealed that the Somatic phage, FRNA phage and VLPs had no relationships witheach other and existed independently (Figure 3.4). The sample scores were scattered in the ordination with a number of denser clusters (physico-chemical) spread out around the origin. CCA axis 1 (Figure 3.4) accounted for 60.5% of total variance of the species data set and in total, the species – environment relation ordination accounted for 83.7% of the cumulative variance.
Figure 3. 4 CCA ordination plot for bacteriophage and virus-like particle populations and water quality variables at the five study sites during autumn, winter, spring and summer seasons.
Eigen values for axis point one and two were 0.043 and 0.021 respectively (Table 3.3). Positive significant (p<0.05) Pearson correlation of species and environmental scores for axis one and two were r = 0.759 and r =0.980 respectively.
79 Table 3. 3 Properties of the Canonical Correlation Analysis ordination bi-plot for bacteriophage and virus like particle populations and water quality variables at the five study sites during autumn, winter, spring and summer seasons.
Canonical properties Axis
1 2
Canonical Eigen value 0.043 0.021
% Cumulative variance of species data 60.5 98
% Cumulative variance of species – environment relation 83.7 94.6
Monte Carlo test p – value 0.065 0.048
Pearson correlation of species and environmental Scores 0.759 0.980
CCA shown in Figure 3.5 revealed strong relationship between the THB communities and VLP and phage communities (green circle). The VC and TC populations also had positive relationships with+ VLP and phage communities (yellow circle). The ordination plot revealed that SHIG, EC, SAL and FS did not impact on the presence of the viral communities. The sample scores were scattered in the ordination (Figure 3.4). FC had no correlation with VLP and phage communities.
Figure 3.5 CCA ordination plot for bacteriophage and virus populations and bacterial indicators at the five study sites during autumn, winter, spring and summer seasons.
CCA axis 1 (Figure 3.5) accounted for 79.7% of total variance of the species data set and in total the species – environment relation ordination accounted for 98.2% of the cumulative variance suggesting high correlation between bacterial community data and VLP and phage communities. Eigen values for axis point one and two were 0.039 and 0.024 respectively (Table 3.4). Positive significant (p<0.05) Pearson
80 correlation of species and environmental scores for axis one and two were r = 0.701 and r = 0.943 respectively.
Table 3.4 Properties of the Canonical Correlation Analysis ordination bi-plot for bacteriophage and virus populations and bacterial indicators at the five study sites during autumn, winter, spring and summer seasons.
Canonical properties Axis
1 2
Canonical Eigen value 0.039 0.024
% Cumulative variance of species data 79.7 91.1
% Cumulative variance of species – environment relation 94.2 98.2
Monte Carlo test p – value 0.051 0.042
Pearson correlation of species and environmental Scores 0.701 0.943
3.5 Visualisation of Virus-Like Particles (VLP) by Transmission Electron Microscopy (TEM)