Characterization of the sampling sites
Notable differences were observed between the physicochemical characteristics of the surface water samples collected from the Pingtan Sea (PIN1-PIN5) and the Xiamen Bay (XIA1-XIA3) (Fig. S1, Table S1). The surface water temperature was higher at the Xiamen Bay sites by approximately 3 °C at the time of sampling, and the pH and salinity were slightly but significantly higher (p<0.05) at the Pingtan Sea sites. The dissolved oxygen (DO) concentration of the surface water ranged between 4.7 and 6.9 mg/L, but no significant difference was found between the two regions (p>0.05). The measured oxidation reduction potential (ORP) values were 43.9±2.5 mV and 22.1±6.5 mV at the Pingtan Sea and the Xiamen Bay sites, respectively (Fig. S1). Overall, Xiamen Bay was more nutrient-rich than the Pingtan Sea. The dissolved inorganic nitrogen (DIN) concentration was significantly higher (p<0.05) in the Xiamen Bay samples (1.5±0.3 mg-N/L ) than in the Pingtan Sea samples (0.76±0.2 mg-N/L). The NO3- and NO2- concentrations were both significantly higher (p<0.05) in Xiamen Bay with 1.1±0.2 mg NO3--N/L and 0.070±0.020 mg NO2--N/L, as compared to 0.53±0.08 mg-NO3--N/L and 0.010±0.001 mg NO2--N/L measured in Pingtan Sea. NH4+ concentrations were not significantly different (p>0.05) between Xiamen Bay (0.31±0.11 mg NH4+-N /L) and Pingtan Sea (0.23±0.10 mg NH4+-N /L) (Fig S1). The concentrations of soluble reactive phosphorus (SRP), dissolved total phosphorus (DTP), and total phosphorus (TP) were all significantly higher in Xiamen Bay than Pingtan Sea (Fig. S1). Depletion of SRP was particularly notable in the surface seawater of the Pingtan Sea, as the SRP concentrations measured at the Pingtan Sea sites were 26±18% of the concentrations measured at the Xiamen Bay sites. The DTN-to-DTP ratio was significantly higher (p<0.05) in Pingtan Sea.
According to the criterion based on the Carlson’s Trophic Indices (TSI), three of the five sampling locations in Pingtan Sea (PIN1, PIN4, and PIN5) and two of three in Xiamen Bay (XIA1 and XIA2) were classified as eutrophic (TSI>50). The total eukaryotic algal cell count was the highest at PIN4 (1.2 x 106 cells/L), and the algal cells morphologically identified as Prorocentrum spp. accounted for 91.0% of the total algal population (Table S2). The only other site with a substantial Prorocentrum spp. population was PIN5, where 3.6 x 105 cells/L, out of the total algal population of 5.2 x 105 cells/L, were identified as Prorocentrum spp. The total 16S rRNA gene copy numbers, as an indicator of prokaryotic population, ranged between 9.9±1.3 x107 and 4.2±0.2 x108 copies/L in Pingtan Sea and between 9.1±0.9 x107 and 2.9±0.9 x108 copies/L in Xiamen Bay (Fig 1b and Table S2).
Analysis of prokaryotic community and core microbiome designation
Analysis of the prokaryotic community structures of PIN1-5 and XIA1-3 identified Proteobacteria as the dominant phylum, with its relative abundance ranging from 76.4% to 88.2% (Fig. 2a). Alphaproteobacteria and Gammaproteobacteria constituted 18.0 - 34.6% and 41.5 - 70.0% of the prokaryotic communities, respectively. Bacteroidia were also abundant, constituting 6.3-17.0% of the total prokaryotic community. Although archaeal OTUs affiliated to Thermoplasmata and Nitrososphaeria were found in the metagenomes, the cumulative relative abundance of the archaeal OTUs was below 1.5% in any of the samples analyzed. The beta diversity analysis of the sea surface microbiomes clearly set the Pingtan sites apart from the Xiamen sites (Fig. 2c-e). The non-metric multidimension scaling (NMDS) plots constructed via taxonomic grouping of 16S rRNA gene sequences and functional assignments of translated enzyme sequences to KEGG Orthology (KO) groups and CAZymes groups invariably separated PIN1-5 from XIA1-3 (Fig. 2c-e), suggesting disparities between the microbiomes of these two regions, in terms of both taxonomic and functional make-ups.
The pairwise Mantel tests identified significant correlations between the taxonomic and functional compositions of the microbiomes and the states of the surface seawater at the Pingtan and Xiamen sites (Fig. 2b). Significant correlation was observed between algal biomass and 16S rRNA-based community composition (rm=0.39, p=0.04). Algal biomass was also significantly correlated with the functional profiles of the microbiomes (rm=0.55, p=0.03 and rm=0.55, p=0.03 for correlations with KO-based and CAZyme-based functional profiles, respectively). A stronger statistically significant correlation was observed between the physicochemical states of the seawater and the prokaryotic community compositions (rm=0.61, p=0.002) and the KO-based and CAZyme-based functional profiles (rm=0.57, p=0.001 and rm=0.71, p=0.002, respectively) of the microbiomes. The physicochemical states were not significantly correlated with the algal biomass (rm=0.35, p=0.093), suggesting that a snapshot of the physicochemical state of seawater is not sufficient as an indicator for HAB. Possibly, the significant correlation between the microbiome composition and the algal biomass may imply that the history of the recent biogeochemical events, missing in a snapshot characterization of physicochemical properties, is ingrained in the taxonomic and functional compositions of the microbiome . Another possible scenario that can be inferred from the Mantel test results is the microbiota shaped by environmental pressure playing a direct role in algal bloom development, in a sense analogous to the key principle underlying diet-microbiome-host interactions in human and animal guts .
The OTUs identified in the samples were assigned to 379 bacterial and 5 archaeal taxa (according to genus-level classification), and 47 of these taxa, shared by all 8 metagenomes, were further considered as the members of the core microbiome (Table S3). The core microbiome consisted of 46 bacterial taxa and one archaeal taxon, most of which are uncultured and understudied. The members of the core microbiome constituted vast majority of the total prokaryotic community in each of the analyzed samples, such that their cumulative relative abundance ranged between 70.4 and 76.9% at the Pingtan Sea sites and between 62.6 and 73.6% at the Xiamen Bay sites (Table S3). The core microbiome taxa with >1% relative abundance at all sites included the SAR11 clade, Pseudoalteromonas, Vibrio, Candidatus Actinomarina, the NS5 marine group, and an unidentified genus of Rhodobacteraceae. The high abundance of Vibrio spp. at PIN1, PIN4, PIN5, and XIA1 was especially notable. Vibrio spp. constituted 15.2%, 32.7%, 9.9%, and 19.3% of the prokaryotic populations at the respective sites, all of which were categorized as eutrophic according to their TSI values (>50). The relative abundance of the Vibrio was <4.5% at the other four sites, where, with sole exception of XIA2, the TSI values indicated mesotrophic conditions. Another core microbiome taxon affiliated to the Vibrionaceae family (unassignable to any distinct genus) was found at PIN2 and PIN3 sites with >10% relative abundance; however, its low abundance in PIN4 (1.3%) suggests relevance of this taxon to eutrophication or algal bloom unlikely.
Metagenomic analysis of nitrogen and phosphorus metabolism
The metagenomic profiles of nitrogen metabolism-related genes in the microbiomes suggest possible involvement of the prokaryotic microbiome in altering the availability of labile nitrogen to Prorocentrum-dominated algal populations (Fig. 3 and Table S4). Apart from the genes involved with urea uptake and mineralization (ureABC and urtABCDE), all of the most abundant nitrogen functional genes were relevant to dissimilatory and assimilatory NO3--to-NO2- reduction (napAB and nasA) and dissimilatory nitrite reduction to ammonium (DNRA; nrfA and nirB). The genes encoding for the periplasmic nitrate reductase, napAB, had substantially higher relative abundance at the Pingtan Sea sites (10.2-39.8 RPKM for napA and 6.0-27.3 RPKM for napB) than in the Xiamen Bay sites (6.9-17.1 RPKM for napA and 8.0-14.2 RPKM for napB), although the differences were not statistically significant (p>0.05). The nirB and nrfA genes, encoding the catalytic subunits of the two distinct forms of nitrite reductases central to the DNRA pathway, showed high relative abundance at the Pingtan Sea and Xiamen Bay sites . The relative abundance of nirB ranged between 26.6 and 87.5 RPKM in the Pingtan Sea metagenomes, and 17.5 and 50.4 RPKM in the Xiamen Bay metagenomes. The nrfA genes, recovered in lower abundance than nirB in all metagenomes, were significantly more abundant in the Pingtan Sea metagenomes (12.7±8.9 RPKM) than in Xiamen Bay metagenomes (3.1±3.5 RPKM). Disproportionately large shares of napA, nirB, and nrfA genes were phylogenetically affiliated with Vibrio spp., considering that the relative abundance of Vibrio spp. did not exceed 33% of the total prokaryotic population according to the 16S rRNA-based community composition analyses. At the Pingtan Sea sites where this tendency was more pronounced, >93.2%, >95.8% and >56.1% of the reads mapped onto napA, napB, and nirB, respectively, were phylogenetically affiliated with the genus Vibrio. The gene-coding sequences identified as nrfA were invariably phylogenetically affiliated with Vibrio spp. in all metagenomes, and the relative abundance of Vibrio spp. exhibited significant correlation with that of nrfA (p<0.05 according to the Spearman’s rank correlation).
Compared to the functional genes involved with NO3--to-NO2- reduction and DNRA, the functional genes encoding key enzymes for nitrification and denitrification were, in general, recovered in relatively low abundance (Fig. 3c). The gene abundance of amoA was below 3.1 RPKM in all metagenomes (1.8±0.7 and 1.9±1.7 RPKM at the Pingtan Sea and Xiamen Bay sites, respectively), and all recovered amoA sequences were affiliated with ammonia oxidizing archaea (AOA) of the Candidatus genus Nitrosomarinus. Despite relatively high NH4+-N concentrations measured at the Pingtan and Xiamen Bay sites (0.23±0.10 and 0.31±0.11 mg/L, respectively), no bacterial amoA was recovered in any of the metagenomes. The collective relative abundance of nxrB, narG, and narZ were 5.1±2.7 and 9.1±2.8 RPKM at the Pingtan Sea and Xiamen Bay sites, respectively. No nirS gene, encoding cytochrome cd1 nitrite reductase, was found in any metagenome, and the relative abundance of the nirK genes, encoding the copper-dependent nitrite reductase, was below 1.5 RPKM. Notably, the nrfA-to-nirK and nirB-to-nirK ratios were significantly higher in the Pingtan Sea metagenomes (80.1±68.0 and 352.5±291.2, respectively) than in the Xiamen Bay metagenomes (2.6±2.2 and 34.4±19.8, respectively), and the PIN4 site, with by far the highest algal biomass, exhibited the highest nrfA-to-nirK and nirB-to-nirK ratio (163.1 and 532.3, respectively). This selective enrichment of nrfA in the Pingtan Sea metagenomes appear too heavily biased to be solely attributable to the high relative abundance of Vibrio spp. (12.5±12.5%, versus 5.8±9.1% of Xiamen Bay metagenomes). None of the signature genes for nitrogen fixation (nifDHK) or anammox (hzs and hdh) was recovered at a significant abundance (<1.7 RPKM).
Metagenomic interrogation of phosphorus uptake and metabolism also identified several notable differences between the Pingtan Sea and Xiamen Bay microbiomes (Fig. 3a and Fig. 3d). The ugpABCE genes, encoding ATP-binding cassette (ABC) transporters for uptake of glycerol-3-phosphate and glycerophocholine, were found in significantly higher relative abundance in the Pingtan Sea metagenomes than in the Xiamen Bay metagenomes. The phosphonate transporter gene phnD and the high-affinity phosphate binding protein pstS, were both also significantly more abundant in the Pingtan Sea microbiomes (51.7±15.0 RPKM and 75.5±21.6 RPKM, respectively) than in the Xiamen Bay microbiomes (27.4±5.7 RPKM and 50.9±11.8 RPKM, respectively). As these phosphorus uptake genes are known to be expressed under Pi-deficient conditions to cope for phosphorus starvation, their abundance in Pingtan Sea metagenomes was consistent with the low concentrations of soluble reactive phosphorous (7.9±4.3x10-3 mg/L) at the Pingtan Sea sites [40-42]. Substantial proportions of phnD (35.5±11.5%), pstS (43.3±15.0%), and ugpB (34.8±15.8%) sequences recovered from the Pingtan Sea metagenomes were affiliated with Vibrio spp., suggesting that possession of these diverse means of alternative phosphorus uptake may have contributed to the disproportionate proliferation of Vibrio spp. observed in Pingtan Sea.
The genes encoding three distinct forms of alkaline phosphatases, phoA, phoX, and phoD, were found in all metagenomes, and phoA and phoD, both with RPKM values above 50, were among the most abundant nitrogen- or phosphorus-related functional genes in the metagenomes (Fig. 3d). The phoA genes were significantly more abundant in the Pingtan metagenomes (68.7±8.4 RPKM) than in the Xiamen Bay metagenomes (49.5±2.6 RPKM; p<0.05); however, the abundances of phoX and phoD were not significantly different between the two regions (p>0.05). In the Pingtan metagenomes, Vibrio-affiliated sequences accounted for the majority of phoX (62.3±13.4%), while only 13.6±7.0% and 0.8±0.5% of phoA and phoD, both at least 3.4-fold more abundant than phoX, were affiliated with Vibrio spp. Therefore, the abundances of alkaline phosphatase genes appear to have little relevance to Vibrio proliferation or Prorocentrum bloom.
Vibrio-dinoflagellate relationship as inferred from the microbial network analysis of the core microbiome
Microbial network analysis of the core microbiome further substantiated the alleged association between abundance of Vibrio spp. and dinoflagellate bloom (Fig. 4). The relative abundances of two core microbiome taxa, including Pseudoalteromonas and an unidentified genus of the family Cryomorphaceae, showed significant positive correlation with the algal biomass, and the relative abundances of four other core microbiome taxa exhibited significant negative correlation (Spearman's |r| >0.7, p<0.05; Fig. 4). The core microbiome taxon affiliated to the genus Vibrio was positively but not significantly correlated with algal biomass (r=0.26, p=0.53); however, its relative abundance had strong significant positive correlation (r=0.81, p=0.015) with the relative abundance of Pseudoalteromonas, and significant negative correlations with those of three core microbiome taxa negatively correlated with algal biomass, including Marine Group II of the phylum Euryarchaeota, an unidentified genus of the family Parvibaculaceae, and the IS-44 subgroup of the family Nitrosomonadaceae (r=-0.71, p=0.047; r=-0.71, p=0.047; r=-0.79, p=0.021, respectively). Of note, co-abundance of Pseudoalteromonas spp. with eukaryotic algae has been previously observed and mechanistically explained with basis on the capability of the microorganisms belonging to this taxon to prey on eukaryotic algae . Alongside the observation that Vibrio spp. was the single most abundant taxon in the PIN4 microbiome most severely affected by HAB, these co-occurrence patterns surrounding Vibrio in the core microbiome network corroborate the possibility of a causal association of Vibrio spp. with HAB, despite the lack of statistically significant correlation between the relative abundance of the Vibrio and the algal biomass.
Ecological association of Vibrio spp. with dinoflagellate algal bloom
As these metagenome analyses suggest, association of Vibrio spp. to the Prorocentrum-dominated algal blooms was apparent in the examined segment of coastal East China Sea. Strong significant correlations between the abundance of Vibrio spp. and the environmental metrics for eutrophication (TSI; r=0.833, p=0.011) as well as the chlorophyll a concentration (r=0.762, p=0.028) also support that the Vibrio and algal proliferation may be mechanistically correlated (Table S5). These observations are in line with a number of previous studies that have reported occurrences of Vibrio blooms in coastal seas simultaneous with blooms of phytoplanktons, e.g., cyanobacteria, diatoms, and dinoflagellates, or immediately following their demise [44-48]. In a study that monitored microbial population dynamics over a six-year period off the coast of Plymouth, UK, the time point at which a single Vibrio sp. dominated the prokaryotic population, representing 54% of the entire pool of 16S rRNA sequences, coincided with a peak in the population of a diatom species Chaetoceros compressus . Another study performed during raphidophyte blooms in Delaware’s inland bays identified significant correlation between the abundances of particle-associated Vibrio spp. and the raphidophyte population .
These previous studies have attributed the Vibrio-algae association to protection from zooplankton predation and/or provision of algal exudates or detritus as organic substrates to opportunistic fast-growing Vibrio spp. [45-47]. Although Vibrio spp. may form biofilms attached to microalgal cells and detritus, physically and chemically protected from grazing, recent culture-independent studies have repeatedly identified particle-associated fractions as minor fractions of Vibrio population in seawater . Thus, supply of labile organics, photosynthetically produced and released by algal counterparts, is deemed more likely as the primary mechanism via which algal blooms accommodate disproportionate proliferation of Vibrio spp. . Further, several Vibrio species have been physiologically characterized as alginolytic, and thus may be able to utilize dead algal biomass or even be algicidal. In this study, core microbiome analysis identified a strong significant positive correlation between Vibrio and Pseudoalteromonas, a taxon widely-known to include algicidal bacteria . This correlation may be interpreted as two groups of specialists sharing a habitat abound with their common substrates, i.e., live or dead algal biomass and/or algae-derived organic materials. It may also be possible that smaller organic compounds produced from Pseudoalteromanas-mediated algal biomass digestion may serve as more labile organic substrates for fast-growing Vibrio spp. . Either hypothesized mechanism would be consistent with the co-occurrence of Vibrio proliferation and Prorocentrum-dominant algal bloom observed in this study.
Whether the presumed Vibrio-Prorocentrum association may be a commensal relationship, benefitting only Vibrio spp., or a mutually-beneficial symbiotic relationship, has not been previously discussed. The comparative metagenomic analysis of the functional genes related to nitrogen metabolism suggest possible scenarios how Vibrio proliferation may provide positive feedback to growth of Prorocentrum spp. and other eukaryotic microalgae. The proportion of Vibrio-associated functional genes were, in general, higher at the Pingtan Sea sites than at the Xiamen Bay sites, probably due merely to the higher Vibrio abundance; however, Vibrio’s exclusive ownership of DNRA-related functional genes, i.e., napAB and nrfA, and the near complete absence of denitrification-related functional genes (nirK and nirS) that are often found in genomes of marine heterotrophic bacteria (e.g., algicidal Pseudoalteromonas spp.) are not explicable merely with the high Vibrio abundance. The DO concentrations of the bulk seawater at the Pingtan Sea sites, including the PIN4 site with the highest Prorocentrum population and algal biomass, indicated that the bulk seawater was oxic at the time of sampling, which, due to logistical reasons, took place during the daytime. Under oxic conditions, DNRA is not likely to occur, as it is known as a strictly anaerobic process; however considering the high populations of the fast-metabolizing Vibrio and Prorocentrum that may be potent O2 sinks at dark hours, it is plausible that periodic oxic-anoxic shifts may occur in the region, especially at highly eutrophicated locales such as the PIN4 and PIN5 sites [52, 53]. Spatial microaerobic or anoxic niches may also exist, such as in biofilms attached to particle surfaces, microbial aggregates, and intracellular spaces within zooplankton or phytoplankton where Vibrio spp. may be able to colonize [52, 54-56]. Rapid decay of algal carcass, and/or upwelling of hypoxic bottom water may also cause temporal anoxia that may not be captured by snapshots of DO measurement [57, 58]. Further, the redox potentials measured in situ at the Pingtan Sea (43.9±2.5 mV) sites were in fact, substantially lower than the standard redox potentials (at pH 7.0) of NO3--to-NO2- (420 mV) and NO2--to-NH4+ reduction (440 mV), despite the DO concentrations ranging between 4.73 – 6.89 mg L-1. Thus, it is highly likely that at least some of the napAB- and nrfA-possessing Vibrio spp. are DNRA-active, producing NH4+ from NO3- and possibly supplying Prorocentrum spp. with NH4+, generally known as a more efficient nitrogen source for fast-growing dinoflagellates than NO3- . Although any decisive conclusion cannot be reached solely based on the metagenome analysis, the findings from this study are certainly compelling indications that NH4+-production via DNRA may be key to the hypothesized symbiotic association between Vibrio spp. and Prorocentrum spp. and warrant further attention to the role that DNRA may play in Prorocentrum-dominated HAB.