Occurrence and Distribution of a Diatom-Diazotrophic Cyanobacteria Association during a Trichodesmium Bloom in the Southeastern Arabian Sea

Symbiotic diatom-diazotrophic cyanobacteria association (DDA) of Rhizosolenia hebetata and Rhizosolenia formosa with endosymbiotic cyanobacteria Richelia intracellularis was noticed and documented for the first time during a bloom of the cyanobacterium Trichodesmium erythraeum in the oligotrophic shelf waters along Kochi and Mangalore transects, southeastern Arabian Sea (SEAS), during spring intermonsoon (April 2012). Although the host is frequently seen, the symbiont is rarely reported in the Indian EEZ. The presence of nitrogen-fixing symbiotic association of Rhizosolenia-Richelia along with Trichodesmium erythraeum highlights the significance of DDAs on the nutrient and energy budgets of phytoplankton in the oligotrophic environments of the Arabian Sea during spring intermonsoon.


Introduction
Planktonic organisms that fix atmospheric nitrogen (diazotrophs) have a critical role in oceanic production and in the marine nitrogen cycle [1].Abundance and growth rates of such organisms depend on their ability to assimilate various sources of nitrogen [2].The availability of fixed nitrogen (such as nitrate and ammonium) can limit the productivity of the sea [3].Diatom-diazotroph associations (DDAs) are widely reported in oligotrophic waters and have the capacity to form episodic, largely monospecific blooms that exhibit very high rates of carbon and nitrogen fixation worldwide [4][5][6][7].Annually, Trichodesmium contributes 1-5 mmol N m −2 d −1 ; while diazotrophic diatoms, contribute 0.4-2.4mmol N m −2 d −1 , which forms one quarter of the total input of nitrogen to the sea [8].Globally, DDAs fix 4.79 Tg N y −1 [9], which forms almost 25% of total input of nitrogen to the sea [8].
One of the most conspicuous free-living, diazotrophic species is the colony-forming cyanobacteria, a species of the Trichodesmium that is found throughout tropical and subtropical oceans and forms large-scale surface blooms [10].A unique group of open ocean diazotrophs is the heterocystous cyanobacteria that live symbiotically with other members of phytoplankton, primarily diatoms.Such endosymbiotic associations help most of the diatoms to fix atmospheric nitrogen in oligotrophic waters with the help of such endosymbiotic associations [11][12][13].The cyanobacteria, Richelia intracellularis and Calothrix rhizosoleniae, were found in association with diatom genera such as Rhizosolenia, Hemiaulus, Bacteriastrum, and Chaetoceros.They are also seen as epiphyte or endosymbiont in Guinardia cylindrus in warm tropical and subtropical oligotrophic waters [6,12,14].Only a few workers have reported Rhizosolenia-Richelia associations from the Indian Exclusive Economic Zone (EEZ) [15][16][17][18].Several reports [15][16][17] described the occurrence of this species from the southeast coast of India, while there is only one report [18] from the northeastern Arabian Sea (NEAS).There are no reports on this aspect from the southeastern Arabian Sea (SEAS).This report forms the first in this regard.

Materials and Methods
The study was a part of monthly observations made in the southeastern Arabian Sea (SEAS), and observations made along the Kochi and Mangalore transects during April 2012 (spring intermonsoon) are reported in this paper (Figure 1).Surface water samples were collected from both of these transects onboard Fisheries and Oceanographic Research Vessel (FORV) Sagar Sampada.Surface samples were collected from six stations based on depth (13,20,30,40,50, and 100 m).A surface sample was also collected from a bloom of Trichodesmium erythraeum located between the 50 and 100 m stations along Kochi transect (9 ∘ 57  52  N; 75 ∘ 50  38  E, Figure 2).Temperature and salinity data were obtained using a CTD rosette system (Sea-Bird SBE 911 plus).Dissolved oxygen (Winkler's method) and nutrients in the sea water were determined using standard procedures [19].Surface sea water samples were collected using a bucket sampling method.Twenty-five liters of sample were filtered through 20 m bolting silk net and preserved in 3% formalin and Lugol's

Results and Discussion
The endophytic cyanobacteria were identified as Richelia intracellularis J. Distribution of major groups in the phytoplankton showed that diatoms were dominant along both the Kochi and Mangalore transects, constituting 83% and 58%, respectively, of the total phytoplankton population (Figures 5(a) and 5(b)), followed by cyanobacteria and dinoflagellates.Trichodesmium erythraeum constituted 45-85% and 40-60% of the total cyanobacteria from the Kochi and Mangalore transects respectively.Rhizosolenia constituted 0.4% and 0.6% of the total diatoms at the two transects, respectively.Numerical abundance of cells containing diazotrophs was less than 5 cells L −1 at both of the transects.The occurrence was more widespread along the Mangalore transect where it was observed at all stations except at 13 m depth station.The bloom sample taken from deeper stations along the Kochi transect farther waters was dominated by Trichodesmium erythraeum (over 85%), while diatoms constituted only 6.4%.Rhizosolenia hebetata with endosymbiotic Richelia intracellularis abundance in the bloom sample was found to be 6 cells L −1 .
The blooming of the cyanobacterium Trichodesmium erythraeum is commonly seen in the SEAS during peak SIM (April-May) when favourable conditions like very warm, calm, and nitrogen depleted conditions prevail [18].During the present observation period, there were near nitrate depleted conditions at most of the stations (Table 1), which is sufficient enough for Trichodesmium erythraeum to proliferate and bloom.Interestingly, the Rhizosolenia-Richelia association was seen only with Trichodesmium erythraeum, which means that the environmental requirements for these to occur are the same.The diazotrophs release fixed nitrogen as ammonium or dissolved organic nitrogen, which is then available to the nutrient starved nondiazotrophic community [8].Nitrate and phosphate concentrations were comparatively low in the study area (Table 1).
Richelia intracellularis has been reported to be among the most important and widespread nitrogen-fixing, endosymbiotic cyanobacteria in marine pelagic tropical and subtropical waters [22,23].However, reports of such studies in the Arabian Sea are scant and not previously documented from the SEAS.The heterocystous R. intracellularis is an extracellular endosymbiont, which locates itself in the periplasmic space between the plasmalemma and silica cell wall in the diatoms [22].It was found as an endosymbiont in five species of Rhizosolenia, Hemiaulus membranaceus, H. hauckii, and Guinardia cylindrus and rarely as an epiphyte on Chaetoceros species, or exists freely in water [22].It is assumed that the abundance of endosymbiotic cyanobacteria was due to their capacity to fix atmospheric nitrogen in oligotrophic waters.This is the first reported observation on the occurrence of the nitrogen-fixing, endosymbiotic cyanobacterium, Richelia intracellularis in Rhizosolenia hebetata and R. formosa in the SEAS along with Trichodesmium erythraeum blooms, which occurred off most parts of Kochi and Mangalore transects and appeared like a saw-dust mat in the surface waters (Figure 2).These waters were characterised by stratification during the spring intermonsoon (SIM) and strong upwelling during the summer monsoon [24].The Arabian Sea during the SIM (March-May) is renowned for its stratified condition, which results in nutrient poor oligotrophic surface waters (Table 1).Such conditions favour high abundance of nitrogen-fixing International Journal of Oceanography  cyanobacteria in both coastal and open ocean waters.It was observed that the occurrence of Rhizosolenia species with Richelia intracellularis was coincided with Trichodesmium erythraeum where nitrate levels were very low (0.04-1.96 M) and ammonium concentrations were sufficiently high (0.37-1.94 M).This indicates that the diazotrophs along with cyanophyceans not only support the production of a nondiazotrophic phytoplankton community through regenerated ammonium produced from these nitrogen fixers [17], but also play a significant role in nitrogen cycling and the budget of oligotrophic SEAS during the SIM.
Observations on diatom-diazotroph symbioses are lacking in the eastern Arabian Sea as the importance of DDAs in the nutrient budget of these oligotrophic waters is unknown.The nutrient profile of the study area reflects the oligotrophic conditions, which may have resulted in the Rhizosolenia-Richelia symbiosis.Forming a symbiotic association might then be considered as an ecological adaptation to life in the oligotrophic ocean.Compared to their terrestrial counterparts [25], marine symbiotic systems are greatly understudied, and thus many intricacies of these relationships remain largely unresolved.This paper reports the first observation of occurrence of DDAs and is evidence for the presence of symbiotic association of nitrogen-fixing organisms in SEAS.This study is based on the visual examination of  diatom assemblages using light microscopy.It highlights the presence of potentially significant DDAs in the highly productive SEAS, which directly affect the oceanic nutrient inventory through the addition of new nitrogen to the ocean ecosystem.Although there have been observations on blooms of Trichodesmium species in the Arabian Sea, there have been relatively few reports on the contribution of nitrogen fixation by cyanobacteria to the marine nitrogen budget [26].According to Gandhi et al. [27], nitrogen fixation by Trichodesmium species occurs mainly in the upper 10 m of the ocean surface and their fluxes to the Arabian Sea were estimated to be at 15.4 ± 1.5 Tg N y −1 which is equivalent to ∼92% of total "new" nitrogen supply to the Arabian Sea and ∼11% of the global nitrogen fixation.DDAs, although found less in number in the SEAS during the present study, symbiotically support noncyanophyceans (diatoms) in fixing nitrogen for their metabolic activity; thereby their role in the nitrogen cycling of the region is very important.

Conclusion
The present study highlights the presence of diatomdiazotroph symbiotic associations, which, along with Trichodesmium erythraeum, influence the nutrient and energy budgets of phytoplankton in the oligotrophic environment of the SEAS.

Figure 2 :
Figure 2: Trichodesmium erythraeum bloom observed in the coastal waters along Kochi transect during April 2012.

Figure 4 :
Figure 4: Cell density of Rhizosolenia with and without Richelia intracellularis along (a) Kochi and (b) Mangalore transects.

Figure 5 :
Figure 5: Distribution of major groups in the phytoplankton along (a) Kochi and (b) Mangalore transects.

Table 1 :
Hydrographical variables observed along the Kochi and Mangalore transects during April 2012.
[20,21]Live and preserved samples were analysed using a Nikon Eclipse microscope with a Nikon DN 100 series digital camera, and photomicrographs of the samples were taken.Identification of species was done using standard references[20,21]