Phytoplankton and Bacterial Communities in South Harbour , Manila Bay , Philippines

In line with the ASEAN-India project “Extent of Transfer of Alien Invasive Organisms in South/Southeast Asia via Shipping”, phytoplankton and bacterial communities in the waters off South Harbour, Manila Bay were investigated. Sampling was done in July and August 2012 and in April and May 2013. A total of 67 phytoplankton species including 29 diatoms and 38 dinoflagellates were identified. Potentially toxic Pseudo-nitzschia spp. were among the diatoms found as well as dinoflagellates Alexandrium spp., and Gymnodinium spp. The diatom Skeletonema costatum appeared to be the dominant species in July and August 2012, whereas Chaetoceros spp. constituted over 85% of the total phytoplankton assemblage in April and May 2013. Mean bacterial abundance ranged from 9.53 x 102–3.18 x 105 cells/mL in July 2012. In addition, 93 bacterial isolates were identified using 16S rDNA, several of which belonged to the following phyla: Actinobacteria, Bacteriodetes, Firmicutes, and Proteobacteria; whereas, others were determined as uncultured bacterial clones. These results will serve as a valuable baseline for future studies on phytoplankton and bacterial community structure in Manila Bay.


INTRODUCTION
Among the major ports in the Philippines, South Harbour in Manila Bay has catered to over 1500 foreign and over 5000 domestic vessels from year 2000 to 2012 (Philippine Ports Authority 2015) indicating high vessel traffic.Since cargo and other shipping vessels carry thousands of potentially harmful organisms and bacteria, the process of ballast water exchange present high risks of introduction and transport of these species from port to port and from region to region (Drake et al. 2007;Altug et al. 2012).In addition, the issues on alien invasive species that have been long implicated with ballast water movement need further assessment on the native species and potentially introduced species present in the receiving regions (Smayda 2007).
Recent studies regarding port water surveys have focused on the risks and hazards of transfer of Harmful Algal Blooms (HABs), pathogenic bacteria/viruses, and alien biota from one region to another (e.g., Rao and Mohanchand 1988;Varela and Prego 2003;Webber et al. 2003;Zamora-Ley et al. 2006;Ho et al. 2008;Chandrasekera and Fernando 2009).In the A Coruña Harbour (northwestern Spain) for example, high biomass of large diatom dominated blooms was observed, indicative of eutrophication (Varela and Prego, 2003).The high risks of transfer of HAB species into other regions pose threats to all receiving areas wherein highly adaptive, introduced species successfully establish populations and outcompete native species (Butron et al. 2011).
Despite studies on surveys of Manila Bay regarding recurrent HABs and eutrophication (Azanza et al. 2004;Hansen et al. 2004;Chang et al. 2009), there has been no assessment conducted on phytoplankton and bacterial assemblages in major ports of the country.Therefore, as part of the ASEAN-India project "Extent of Transfer of Alien Invasive Organisms in South/Southeast Asia via Shipping", this paper presents the baseline information on microalgae and bacteria necessary for identifying potentially toxic and harmful/pathogenic species already been present in the coastal waters of South Harbour, Manila Bay.

Phytoplankton analysis
To determine the abundance and composition of phytoplankton in South Harbour, 1 L water samples were collected at near surface (1 m below the surface) using a 5 L Niskin water sampler.Subsequently, samples were preserved with Lugol's solution and used for qualitative analysis.Enumeration and counting of phytoplankton was done using Sedgewick-Rafter counting chamber based on microscopic and molecular methods for quantitative phytoplankton analysis following the technique of Azanza (1997) and Corrales et al. (1995).Phytoplankton species were identified to the lowest taxonomic level possible using Tomas (1997) and Yamaji (1984).

Total bacterial abundance
Water samples collected in July 2012 were analyzed for total bacterial count.Five-milliliter (5 mL) seawater samples from each station were fixed with 250 µL formalin and were filtered through 0.22 µm syringe filter (Whatman TM , PURADISC TM 25 NYL Disposable Filter Device with 0.22 µm Nylon Membrane with 25 mm diameter polypropylene housing).Fixed samples were analyzed at the Council of Scientific and Industrial Research (CSIR), National Institute of Oceanography (NIO) in Goa, India.Total bacterial count was determined using flow cytometry.Briefly, the samples were stained with SYBR Green I at 1:10,000 final concentrations (Marie et al. 1996) and incubated for 15 min in the dark at room temperature.After incubation, samples were analyzed using a BD FACSAria TM II flow cytometer equipped with a nuclear blue laser 488 nm, which can differentiate green fluorescence excited by blue light.Emitted light was collected through following filters sets 488/10 band pass (BP) for right angle light scatter (SSC) and 530/30 band pass (BP) for green fluorescence.Fluorescent beads (1μm, Polysciences) were used as internal standards.Gating was done against SSC versus green fluorescence.Flow cytometry data were processed using BD FACSDIVA software.
One-liter water samples were collected in sterilized Nalgene bottles at near surface (1 m below the surface) and near-bottom (1 m above the seafloor) in stations deeper than 10 m using a 5 L Niskin water sampler.Twenty microliters (20 µL) of water from the Nalgene bottles were spread plated in prepared Marine Agar (MA; Pronadisa, Spain).The inoculated plates were inverted and incubated at 24 ± 2°C in the dark at the Harmful Algal Bloom Laboratory, The Marine Science Institute, University of the Philippines, Diliman, Quezon City.Growth of the bacteria was observed for one week.The colonies were separated and purified through subsequent streaking/transferring into prepared MA plates.

Genetic identification of bacterial isolates
The bacterial isolates were genetically identified via amplifying and sequencing their 16S rDNA genes.DNA from bacterial culture broths was extracted using ZR Fungal/Bacterial DNA Mini Prep (Zymo Research TM ) following the instructions of the manufacturer.Amplification was done using these 16s rDNA primers: 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 534R (5'-AT-TACCGCGGCTGCTGG-3').Amplicons were then sent to First Base (Malaysia) for single pass reaction sequencing.The generated sequences were then compared by searching the nucleotide (nt) database of the National Center for Biotechnology Information (NCBI) using the nucleotide-to-nucleotide Basic Local Alignment Search Tool (BLASTN) (Altschul et al. 1990).

Phytoplankton assemblage and composition
Comprising a big fraction of the total phytoplankton assemblage, diatoms showed relative abundance of more than 98% cover in July and August 2012, and over 95% in April and May 2013 in South Harbour Manila (Figure 2).Skeletonema costatum comprised 90% of the total phytoplankton assemblage in July and August 2012; whereas, Chaetoceros spp.constituted over 85% among all stations on April and May 2013.

Bacterial abundance and identification
Mean bacterial abundance in July 2012 ranged from 9.53 x 10 2 -3.20 x 10 4 cells/mL and 3.30 x 10 3 -3.18x 10 5 cells/mL in surface and bottom waters, respectively (Figure 3).In   Table 2. List of bacterial species from six stations along South Harbour, Manila Bay, Philippines in August 2012 using 16S rDNA identification.Note: (-) absent, (+) present equivalent to 1 isolate, (*) reported human pathogen, same colored signs -one isolate has the same percent similarity (considering same length of base pairs) for two bacterial species.

Table 2. (continued)
List of bacterial species from six stations along South Harbour, Manila Bay, Philippines in August 2012 using 16S rDNA identification.Note: (-) absent, (+) present equivalent to 1 isolate, (*) reported human pathogen, same colored signs -one isolate has the same percent similarity (considering same length of base pairs) for two bacterial species (continued).general, higher mean abundance readings were observed in bottom waters than surface waters.Highest mean reading was recorded for station 2 (Pier 13).

DISCUSSION
This paper reports for the first time the abundance of potentially HAB forming algae, as well as other phytoplankton and bacterial assemblages in South Harbour, Manila.The phytoplankton assemblages were dominated mostly by diatoms in 2012 (July 6 and August 8) and also in 2013 (April 10 and May 10) (Figure 2).Previous studies by Azanza and Miranda (2001) also showed diatoms were the dominant taxon in Manila Bay in all seasons from 1997 to 1999.Furthermore, the abundance of Skeletonema costatum in July and August 2012, and Chaetoceros spp. in April and May 2013, in South Harbour were observed by Azanza and Miranda (2001) when both species were found to occur in high densities in Manila Bay.Other studies elsewhere, such as in Visakhapatnam Harbour in India, Rao and Mohanchand (1988) found S. costatum as the dominant species.Similarly, Huo and Shu (2005) showed S. costatum to be a cosmopolitan species in the coastal and estuarine areas in China, which could be associated with heavy rainfall (Liu et al. 2005).Five diatom taxa have also been observed dominating in South Harbour during separate sampling activities between 2011 and 2013, which included Rhizosolenia spp., Dytillum spp., and Thalassiosira spp.
The abundance of diatoms in the area may be associated with the high organic and nutrient loading in Manila Bay that could contribute to the increased frequency of microalgal blooms (Jacinto et al. 2006).Thornton and Thake (1998) found out that increasing temperature enhanced aggregation of S. costatum; whereas, certain species of Chaetoceros (e.g., C. gracilis, C. simplex, and C. wighamii) recorded optimal growth at higher temperature and salinity (Mortensen et al. 1988;Araujo and Garcia 2005;Hemalatha et al. 2012).In contrast, S. costatum population in South Harbour declined with increasing surface water temperatures from July to August in 2012 with sea surface temperature from 28°-31°C respectively and further dropped during the months of April and May in 2013 coinciding with 29°-33°C sea surface temperatures.These contrasting results may be an indication that other factors were affecting microalgal population growth.Heavy rainfall and tropical storms that flooded metro Manila between the months of June and September in 2012 may have also influenced the decreasing phytoplankton abundance from July to August in the same year, where salinity ranged between 14 psu and 30 psu.Moreover, the obvious increased densities of Chaetoceros spp. that was evident in the succeeding year during the months of April and May, may also indicate salinity-driven aggregation in some species (Araujo and Garcia 2005) where a higher salinity range was recorded in South Harbour from 30 to 34 psu in April and May 2013.
It is important to note that the abundance of these organisms maybe influenced by a combination of factors e.g., grazing, nutrient loading and physiological aspects of the dominating organisms.Manila Bay is known to be highly eutrophicated, with higher nitrogen concentrations particularly ammonium compounds (Chang et al. 2009), which may be linked with the concurrent microalgal growths including HABs in the area (Azanza et al. 2004).It is suggested that in the case in South Harbour, phytoplankton blooms may have been eutrophication driven, causing phytoplankton like Chaetoceros spp.and S. costatum to proliferate at a higher rate.Potentially harmful algal bloom-causing diatoms (e.g., Chaetoceros spp., Coscinodiscus spp., and Pseudo-nitszchia spp.) and dinoflagellates (e.g., Akashiwo sanguineum, Alexandrium spp., Ceratium furca, C. fusus, Dinophysis caudata, D. miles, Gymnodimium spp., Noctiluca scintillans, Prorocentrum micans, P. rhathymum, and P. sigmoides) were also identified in this study.Certain species have previously been reportedly problematic in some regions causing negative environmental and economic impacts (Mahoney and Steimi 1980;Albright et al. 1993;Kent et al. 1985;Volkman et al. 1999;d'Ippolito et al. 2004;Jessup et al. 2009;Fernandes and Frassao-Santos 2011;Nishikawa et al. 2010;Vale 2011;Tahira and Siddiqui 2012;Trainer et al. 2012).
On the other hand, the majority of bacterial isolates identified belonged to Phylum Proteobacteria, Class Alphaproteobacteria (Table 2).Bacteria from Order Rhodobacterales of this class were reported as the dominant primary surface-colonizers in temperate coastal marine waters (Dang et al. 2008).Three isolates were reported as pathogenic bacteria, namely: Gordonia bronchialis, Kytococcus sedentarius, and Microbacterium oleivorans.G. bronchialis is a human pathogen associated with pulmonary disease that has been isolated in various human tissues (Ivanova et al. 2010).It was assigned a new genus Gordona by Tsukamura (1971) using descriptions from sputum samples from Japanese patients with cavitary pulmonary tuberculosis and/or bronchiectasis and soil samples.K. sedentarius is a species of interest due to its capability to produce oligoketides, a natural antibiotic.It is also considered an opportunistic pathogen that causes valve endocarditis, hemorrhagic pneumonia and pitted keratolysis (Sims et al. 2009).M. oleivorans was proposed as a novel crude-oil degrading Gram-positive bacterium that was isolated from oil storage cavern 126 near Etzel, Germany and previously characterized by Bock et al. (1994) (see Schippers et al. 2005).In 2012, Kim and Lee reported a case of bacteremia in a 4-year old Korean boy caused by the said bacterium.
The presence of potentially harmful algae and pathogenic bacteria along with the increasing vessel traffic in South Harbour would implicate high risks in transporting them into new waters.Results of this study will serve as a baseline for phytoplankton particularly HAB species and marine bacteria in the area.Whether South Harbour serves as the receiver or donor habitat for the identified species may not be concluded from the acquired data; hence, further studies focused on determining critical factors that may contribute to proliferation of phytoplankton and bacterial species, possible risks/negative impacts and potential management schemes should follow.

Figure 1 .
Figure 1.Established sampling stations in South Harbour, Manila Bay, Luzon, The Philippines (Map Source: Re-drawn from Google Earth Pro and Google Maps).

Figure 2 .
Figure 2. Comparative logged abundance of diatoms and dinoflagellates sampled at six stations (1-6) in South Harbour Manila in 2012 (July 6 and August 8) and 2013 (April 10 and May 10).Note: Open circles represent values/abundance above 75% quantiles or most specifically represent bloom forming taxa.

Figure 3 .
Figure 3. Mean bacterial abundance (average of 3 readings) from surface and bottom waters in South Harbour in July 2012 per station.