4. Bnviton, Science & Engg, Vol 52, No.4, p. 321-326, October 2010 Zooplankton’s Role in the Distributional Status of Autochthonous Vibrio cholerae : A Case Study Using Principal Component Analysis vs.s HANANDI P,P. 01 Miscellaneous statistial approaches were applied EPHAND K. NARENDRABABU to illustrate the influence exerted by different zooplankton groups on the survival and distributional status of autochthonous Vibrio cholerae in two diverse marine environments, Veli and Kochi, former with low nutrient loading and later with high nutrient loading. The data set has heen factorised using principal component analysis (PCA) for extracting linear relationships existing among a set of Variables. Box plots were derived from the PCA data and were graphically represented, Vibrio cholerae (VC) showed strong positive correlation to zooplanktons especially with erustacean copepods under low nutrient conditions Which confirmed a clear association between VC and zooplanktons for its surveillance and distribution under low nutrient concentration, while under high nutrient loading VC rely only on the available nutrients. Key words : Principal component analysis, Vibrio cholerae, distribution, nutrients, zooplankton Introduction The occurrence of outbreaks of cholera in Africa in 1970 and in Latin America in 1991, mainly in coastal ‘communities, and the appearance of the new serotype Vibrio cholerae 0139 in India and subsequently in Bangladesh have stimulated efforts to understand environmental factors Influencing the growth and geographic distribution of epidemic Vibrio cholerae. While most of these studies concentrated ‘on ecology of this organism and its survival under various physico-chemical conditions in the aquatic environment”, there is a paucity of information pertaining the influence of biotic components in the distributional status of VC. A variety of biological surfaces in water can bind bacteria, These associations can be accidental and transient, or they can be very specific and long lasting. It was reported that the role of Zooplankton, specifically copepods, in the survival and multiplication of Vibrio in an aqueous environment is of great important to its natura history, While ‘most of such studies use laboratory —based bioassays where ‘organisms are exposed to controlled conditions, studies using in vivo conditions are few. It was observed that the survival of Vibrio cholerae is enbanced when it is cultured with laboratory grown planktonic copepods originally isolated from fresh and estuarine waters*, Such studies performed under the controlled conditions ofthe laboratory may not accurately represent the prevailing environmental conditions, and their results can lead (o uncertainties that arise from the extrapolations that are required in ecological risk assessment”, ‘The study reported here was designed to find out any association between V. cholerae and zooplankton subsist ‘under different nutrient Ievels, one with high nutrient loading and another with low nutrient loading, only to ascertain ‘whether the variations of nutrients influence such association and to find out the role of Zooplankton groups in the survival and distributional status of V. cholerae in the aquatic environment Materials and methods The study area comprising two different stations, ‘Veli and Kochi (Fig. 1), each with 5 sampling locations (Table 1) nearshore, Ikm, 3km, Skm and 10km from shore respectively ‘was identified. Stations were selected based on the disparity in nutrient loading, Kochi with high nutrient concentration and Veli with low nutrient concentration, Samples were collected during the cruises of Sagar Purvi and Sagar Paschimi, the coastal research vessel of MoES (Ministry of Earth Science, Government of India) for a period of year (2008-2009), Water ‘Table 1: Geographic locations of the study area Centre for Earth Science Studies, Akkulam, Thiruvananthapuram, Sites Kochi Vai ‘Longitude | Latitude | Longitude Shore TswE | 829739N | T8STAs Tk, WI3'SS'E | $2939°N | 765212 3km WIDSVE | 82739°N | 7652 12"E | skm | 96 14°N | 761148"E | 829°39"N | 76'50'S6" 1okm | os618"N | 760913E | 8239'N | 764810 Kerala, India "Corresponding author : VS. Sudhanandh, Senior Research Fellow , Chemical Sciences Division, Centre for Earth Science Studies, Akkulam, Thiruvananthapuram, Kerala (India); e.mail: sudbanandhvs(@ gmail.com 3a‘Zooplankton’s role in the distributional status of autochthonous Vibrio cholerae Fig. 1: Thestudy area samples were collected using Niskin water sampler. Standard procedures were adopted forthe analysis of nutrient species (Si0,,NO,N,NO,N, PO,-P) and he results were expressed as amicromol per litre (umoVL.), Spread plate technique using 0.1 100.5 mL sample, was adopted forthe Vibvio cholerae and the results were reported in Colony Forming Units per milliitee (CFUImL). Alle samples were plated onto TCBS (thiosulphate citrate bile salts suerose agar), a medium that inhibits most other normal faecal flora but supports the growth ofthe vibrios and were incubated for18-24h, VC colonies appear as smooth yellow colonies with slightly raised centes". Zooplanktons ‘were collected using Bongonet (otal length 2m, mouth diameter 40cm, meshes size SSum). The net was equipped with flow meter estimate the volume of water filtered. The zooplankton collection was done by horizontal hauling at depth of 0.5 ~ 1.0 mo the surface ata speed of 2 knolshout, The net was hauled for 10 minutes at each time interval. The collected zooplankton was transferred from the plankton bucket to plastic container and was preserved in 4% formaldehyde and seawater solution. Zooplanktons were identified group wise” * using an inverted microscope (Ceti) at a magnification of 40x and results were expressed in No‘m’. ‘The water quality data was processed by principal component analysis (PCA) with statistical package, SPSS 11.0, Eigenvalues and Factor loadings for the correlation matrix ‘were also estimated. The principal component loading of two different stations were analyzed discretely and box plots were also derived from it Results and discussion To test the priori hypothesis, of a biological association of different zooplankton groups and bacterial characteristics, explanation of the variability observed among Vibrio cholerae population by dynamics of 9 zooplankton species were examined with different nutrient conditions using principal component analysis. The Eigen values > 1 were considered as prominent factors as per the Kaiser criterion’ Factor loadings were used to measure both the correlations and regression weights between factors and variables. A rule ff thumb frequently used is that the absolute value of the factor loading greater than 0.3 is considered significant, greater than 0.4 is more important and greater than 0.50 is very important ‘Table 2 represents the determined initisl PC and its eigenvalues and percent of variance contributed in each PC ‘with the cumulative variation of different PC , in lower nutrient condition of Veli. It is seen that the first four principal components (PC ,) capture a major portion 80.67% of the 322Sudhanandh et.al. J. Env. Sei. Eng., 52/4), 2010 ‘Table 2: Principal component matrix for different parameters val Parameter Prindpal componens ees Vir coteree | ota | 0382 | 0277 | on inte ‘tivo | ‘oo | cos | osan irae ‘tae | ou | ose | 023s anmonin | attus | 0357 | 021s | 030 Tora aivogen | anes. | 02s» | 032 [awe rong | saan | 0007 | comp | oz phosphate Ton asso | ows | oxs | ois phosphorous Sic cogss | oe | oe | ons Bractywace | oom | come | 016 |0330 Chctognats | oat | ost | co2et [031s Caiscenne’ | 0039 | 0am | ows | com coped” | ose | ots | cos | cow Fish ege oso | 0.671 | -0203 | os19 shen 8 Myatt sts | 0550 | ous? |oues oxplot of PCI showing the variation of major Nani ‘toa | oes | 035s [031s componensin Ve Polhace | oa@7 | o.gos | o207 | 00s rota | Ae | Oats | O87 Bk | tutte sean clave sundance of platon eps) Egan 9 38 [20 feranwion | 202 | ism | toe [oss | | Zmemnbtonarome [Stations Cumiave 20 | sao | mss Joon Cuma Badywnninae | 128) 38 Chastogatha, a5 | aa variance pron inthe dat Tking nt conterion he Claoceans te | an tno sigue variates in evlusog he component the | Copepods ao | axe few pip component (PCT accounted forneay hao | Feh eg eo | as the wl variance capsid an cxpesmenttwanance ot Mysas in| ast {2624 har high poravelonng valve for Capeposs, | Naw an | te None, Choctgnathe and Vriochoterac and nepasee | Pelyehacte in| ar loading for nutrients such as inorganic phosphate (-0.901), ‘Tintinnids 416 342 total phosphorous (-0.890) and silicate (-0,955), Boxplot of PCI scores of the zooplankton parameters (Fig. 2) shows that there is more variation in the score values of copepods compared to other zooplankton groups, vary from 8,00 No/ m'(nearshore) to 140 No/m’(10km from shore). Across all zooplankton groups in Veli, Copepod was the most commonly identified order (60% mean relative abundance), followed by fish eggs and cladocerans, with mean relative abundances of 16,20 and 8.98%, respectively (Table 3), According to the loadings of the variables in the second PC, the most contributing descriptors were polychacte, Nauplii, Fish egg and VC. VC showed a lowest value of 30 (CFU/mL (Skm) to a highest value of 250CFU/mL (3km) with a ‘mean of 14269.81CFU/mL (Fig. 3). Taking into consideration the first two PC which account for 58.40% of the variance, Vibrio cholerae showed strong positive correlation with most 323 of the zooplankton groups and significant negative or no correlation with nutrients, indicate that VC dependent on zooplankton groups whilst the nutrient supply is ‘comparatively low. For the third PC, the effective contributing descriptors were nitrite (0.628) and nitrate (0.549), which indicate the prevailing nittfication process involving active ‘microbial flora other than VC. Nitrite ranged from a lowest value of 0.02 pmol/L (3 & 5km) to highest value of 0.53 pmol/ L (10km) with a mesn of 0.150.14 jmoV/L and nitrate varied from 0.21 pmol/L (3km) to 3.87 mol/L (1m) with a mean of 1.812112 jmoVL (Fig. 4), During high nutrient condition, in Kochi, PCA. extracted five composite variables with Eigen values > 1 (Table) Of these composite variables the first three PC, captured a total of 76.83% of the variance in the original data. The first‘Zooplankton’s role inthe distributional status of autochthonous Vibrio cholerae ‘Table 4: Principal component matex for different parameters Fig. 4: Boxplot of PC 3 showing the variation of major components in Veli principal component explained 34.77% of the variance and ‘was strongly associated with positive loading for copepod, cladocerans, tintinnids, brachyurans, mysids, total nitrogen and ammonia (Fig. 5) with negative loading for inorganic phosphate and total phosphorous, indicates the variation in different zooplankton groups due (o the available total nitrogen concentration, The inverse relationship of total nitrogen with IP and TP suggests that they may arise from in Kochi a Parameter Principal components on T z 34 Vibrio cholerae | -0.142 a1 | 0230 Nitvite 0339 ‘0s4 | 0395 7 Nitrate 0510 024 | 0081 ‘Ammonia 0.728 0249 | 9.140 - Total nitrogen 0.843, 0.146 | 0.104 Inorganic phosphate | -0534 0297 | 0080 cod Total phosphorous | -0.754 0407 | 0.129 . Silicate -0.785 0272 | 9031 Brachyurans 0343 | 0404 a Chactognaths ost | 0246 Cladocerans 0248 | 0.037 . Copepod 0070 | 0.006 : = —— Fish ceg 460 | 0499 ceait be “i Mysids 0623 | 0.148 > 5 5 Nauplit 0432. | 0361 Fig.3 “BoxplotofPC25 ‘owing he variation of major Polnchacte om | 0780 ‘Tintinnids 0s80_| 0384 igen values 198 | 160 = _ % variation 1166 | 940 (Cumulative variation 5576 | 6742 | 7683 different sources. Both copepods and cladocerans showed ‘their maximum values at Skm from shore respectively of 172 ‘Nol’ and 192 No/m’. In the second principal component (PC- I) it explained 20.99% of the variance, in which VC, nitrite, Fig.5 joxplot of PCI showing the variation of major ‘components in KochiSudhanandh er. al. / J. Env, Sci. Eng., 52/4), 2010 nitrate and inorganic phosphate showed strong positive loadings, indicating the increase in VC population due to nutrient enrichment. Vibrio cholerae showed a maximum ‘enumeration of 820 CFU/ml (near shore) where the nutrient species like nitrite and nitrate also reported their maximum concentration, respectively of 1.08 and 9.37 mol/L. (Fig. 6) Thus, high nutrient condition seems tobe ideal for proliferation of VC as evidenced by their higher population, respective of zooplankton abundance. Principal component third (PC- 111) and fourth (PC-IV) are not self explanatory and was not taken into consideration. In Kochi among the zooplankton studied, copepods were the most frequently identified order (62.36% relative abundance) followed by brachyuran (decapoda) larvae at 10.49% mean relative abundance (Table 3) Fig.6: Boxplot of PC 2 showing the variation of major ‘components in Kochi PCA revealed that VC population were directly related to zooplankton especially with copepod under low nutrient condition, whereas under higher nutrient loading VC rely only fon available nutrients. It was suggested that attachment (0 zooplankton enbances environmental survival of Vibrio spp. probably by providing both an abundant source of carbon and nitrogen and protection from numerous environmental challenges". I is reported that during the periods of reduced nutrient levels Vibrio spp, undergo physiological and ‘morphological changes, These include the production of novel bacterial proteins and changes in faty acids” and with that adherence properties can also be enhanced”. V. cholerae also possess secreted chitinase enzyme(s) which ate probably needed to utilize chitin (homopolymer of Nacetylglucosamine) as « carbon source”; the hypothetical chi gene products were identified in the genome sequence”. These findings are suggestive of a close association of V. cholerae with chitin structures in the environment (e.g. zooplankton such as copepods)”. Isolation of this bacterium below at a very low level in water samples in Vel suggested that zooplankton might be associated with this pathogen during unfavorable condition. In particular, parameter estimates indicated a strong direct relationship between VC and copepods with respect to the relative decrease in nutrient loading. In addition we have shown that plankton composition plays an important and independent role as a driver of the (otal culturable Vibrio cholerae community in marine aquatic systems under low nnutsient condition, Conclusion tis concluded from the results of this study that distribution and survival of Vibrio cholerae is influenced by the degree of nutrients rather than by the association with zooplankton, However, under low nutrient condition VC seems to be associated with crustacean copepods compared to other zooplankton groups which indicate that crustacean copepods can also be used as an indicator of VC pollution under low nutrient condition. 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