| As both primary producers and degraders,marine microbes are highly diverse,widely spread in the ecosystem.Together with phytoplankton and zooplankton,microbes link all organisms by forming the?microbial loop?,and play indispensible roles in material cycling and energy transfer as well as biogeochemical cycling.Therefore,it is crucial to understand the diversity,structure and function of marine microbial communities.Previous studies mainly focused on microbial communities in natural sea waters,so little is known about the characteristics of microbial communities in aquaculture zones,especially in seaweed cultivation ecosystems.To study the structure and function of microbial communities in Gracilaria lemaneiformis zones?GZ?,water samples in different depths and growth times were collected from the seaweed cultivation zones and control zones?CZ?,respectively.DNA isolated from these water samples was then analyzed using 16S rRNA gene MiSeq sequencing and GeoChip 5.0 technologies to reveal the effects of G.lemaneiformis cultivation on water/sediment microbial communities and ascertain the dissimilarity of microbial communities between the two study zones.In addition,DNA isolated from the surface of fresh thalli of G.lemaneiformis was analyzed to understand the epiphytic microbial community.The main results are as follows.1.Effects of large-scale cultivation of G.lemaneiformis on the structure of microbial communities near the Nan'ao Island.The major objectives of this study were to test the hypotheses that?i?changes in environmental properties resulting from seaweed cultivation would shift the diversity,composition and structure of water and sediment microbial communities;and?ii?selective microbial groups would be enriched in the seaweed cultivation ecosystem.To test these hypotheses,water and sediment samples were collected in April 2014from a seaweed cultivation zone?the G.lemaneiformis cultivation zone?and a similar marine zone without seaweed for this study.DNA isolated from water and sediment samples was analyzed using Illumina 16S rRNA gene amplicon sequencing to examine the shift of microbial community structure under seaweed cultivation conditions.Over 533,000 sequences were obtained from all 18 samples and 3,454OTUs were generated at 97%sequence identity classified into 28 phyla,74 classes,134 orders,275 families and 618 genera.Significant dissimilarities?p<0.05?of microbial community structure were observed between GZ and CZ in both bottom water and sediment samples although the community structure in the surface water did not show obvious differences between the two zones.The dominant phyla or classes?relative abundance>1%?in both surface and bottom water were Bacteroidetes,?-Proteobacteria and?-Proteobacteria,but their proportions differed at the two study zones.In the sediment,the dominant phyla at both zones were Bacteroidetes,?-Proteobacteria and?-Proteobacteria.At the genus level,higher abundances of Aestuariibacter,Crocinitomix,Leucothrix and Tenacibaculum were observed in the surface water at GZ than those at CZ?p<0.05?.In the bottom water,Aestuariibacter,Bacillariophyta and Croceitalea showed higher percentages?p<0.05?at GZ in comparison with CZ.In the sediment,Arenibacter,Glaciecola,Halomonas,Leisingera,Maribacter,Peredibacter and Sulfitobacter exhibited a higher abundance at GZ than at CZ?p<0.05?.Most of the above genera were associated with seaweed,such as Arenibacter,Croceitalea,Glaciecola,Leucothrix and Maribacter were enriched at the cultivation zone.The response ratio analysis further indicated the effect of G.lemaneiformis cultivation on microbial community composition at the OTU level,revealing significant changes in the surface water,bottom water and sediment at GZ.In the surface and bottom water,the relative abundance of significantly changed OTUs increased at GZ by 89.9%and 72%,respectively.The relative abundance of significantly changed OTUs in sediment decreased by 84.5%at GZ.Canonical correspondence analysis?CCA?and Pearson correlation analysis were applied to detect the environmental factors on shaping the microbial community structure.The result of CCA showed the microbial community structure was strongly correlated with temperature,DO,pH and Chl-a at GZ,while it was most closely associated with salinity and TN at CZ.Pearson correlation analysis showed that significantly changed OTUs exhibited a generally positive correlation with temperature,pH and DO?p<0.01?,and a negative correlation with TN and salinity?p<0.01?in both surface and bottom water samples.In addition,the abundance of the most significantly changed OTUs in the sediment showed a strongly positive relationship with TN and TS concentrations?p<0.01?,and a negative correlation with TC?p<0.01?.Based on the above results,a conceptual model was proposed for future studies.G.lemaneiformis cultivation increased p H and DO,and decreased nutrients by photosynthesis and secreting specific compounds?e.g.,algal polysaccharides?and producing detritus in the ecosystem.Those changes might not only lead to improved water quality,but also alter the microbial community in terms of?-diversity,composition?e.g.,enriched Arenibacter,Croceitalea,Glaciecola,Leucothrix and Maribacter groups?and structure.This conceptual model can be used as a basic molecular mechanism to explain the interaction between seaweed and microbial communities.2.Temporal and spatial dynamics of microbial communities in G.lemaneiformis cultivation ecosystem.The major objectives of this study were to address the following questions:?i?how diversity and structure of microbial communities responded to the 5-months‘seaweed growth;?ii?whether the diversity of microbial communities decreased along with the water depths;and?iii?did seaweed have a selection effect on microbial communities assembly.To answer the above questions,a field investigation was conducted at G.lemaneiformis cultivation zones and natural sea zones near the Nan‘ao Island,South China Sea.Water samples were collected at different depths?surface,1-m depth and near the bottom?at the two study zones from December 2014 to July 2015.The dynamics of microbial communities responses to seaweed growth and different water depths was analyzed by sequencing 16S rRNA gene amplicons.Large-scale cultivation of G.lemaneiformis improved water quality at seaweed cultivation zones.Adonis tests revealed that the structure of microbial communities was significantly influenced by seaweed biomass changes,time and water depths?p<0.01?.Also,seaweed cultivation,time and the interaction between cultivation and time all affected the microbial community structure at all water depths?p<0.05?.Our results showed that significantly divergent community composition was detected during the seaweed cultivation period?January to May 2015?between cultivation zones and control zones,especially at 1-m depth and bottom water?Adonis test,p<0.05?,indicating that G.lemaneiformis cultivation did shift the microbial community assembly.Nonmetric multidimensional scaling?NMDS?analyses confirmed that seaweed cultivation time was the most important factor driving the microbial community structure.Only?-diversity of surface water in the cultivation zones showed a negative linear correlation with cultivation time,revealing that G.lemaneiformis may select certain microbial groups.Bray-Curtis distance based?-diversity of microbial communities in all water depths increased and it increased faster in cultivation zones compared to control zone in the surface and 1-m depth water by the linear regression.The results of CCA demonstrated that DO,NO3-,NO2-and TP showed a positive correlation with the structure of microbial communities during the primary stage of seaweed growth,whereas pH and TN were positively correlated with microbial communities when the biomass of G.lemaneiformis reached a high level.The major results on the temporal turnover of microbial communities implied that seaweed cultivation has a selection effect on microbial community assembly.3.The effects of Gracilaria cultivation on microbial functional potentialTo understand the temporal and spatial dynamics of functional microbial community strucuture and potential in response to seaweed cultivation,water samples at 1-m depth throughout the growth cycle of Gracilaria lemaneiformis?December 2014 to July 2015?and water samples at different depths?i.e.surface water,1-m depth,half of water depth,75%of water depth and near the bottom?were collected at the Gracilaria cultivation zones?GZ?and the control zones?CZ?,respectively.Samples were analyzed using a functional gene array?GeoCip 5.0?to test the following hypotheses that:?i?Gracilaria cultivation would alter the microbial functional structure with the increasing biomass of seaweed;and?ii?the overall structure or functional gene composition in various categories would shift at differernt water depths due to seaweed cultivation.Our results showed that significant variances of community functional potentials were observed between GZ and CZ along with the growth of seaweed,especially in April and May when seaweed biomass was high.Higher intensities of gene nrfA,nir and nasA and lower intensity of amoA were detected at GZ,indicating a acculation of NH4+,which is the favorable nutrient to Gracilaria.In addition,seaweed cultivation shifted the microbial functional structure between the two study zones at differernt depths in May,especially at 1-m depth and the bottom water.Most of genes involved in C degradation and organic remediation had lower signal intensities at GZ than at CZ.Envrionmental variables were also key factors shaping functional composition of microbial communities.The close relationships among functional gene changes,seaweed biomass and environmental factors could reflect the intricate interactions between seaweed-microbe-environment.These results indicate that micro-environment affected by Gracilaria cultivation may mediate the effects of seaweed cultivation and environmental changes on the functional community composition.The temporal and spatial dynamics of functional microbial community structure in seaweed cultivation ecosystems can shed light on the future work of microbial community functional potentials in mariculture ecosystems.4.Interactions between G.lemaneiformis and epiphytic microbesSeaweed surface provides suitable substrates and organic matters for epiphytes to live on.As one of the important roles in epiphytes,epiphytic microbes of G.lemaneiformis are rarely reported.Here,we hypothesized that:?i?the structure and function of epiphytic microbial communities would be significantly different from the free living planktonic microbial communities;and?ii?certain seaweed associated microbial groups and functional genes would be enriched in epiphytic microbial communities;and?iii?G.lemaneiformis would select epiphytic microbes to perform stable interactions between the host seaweed and their epiphytic microbes.To test the above hypotheses,fresh seaweed was collected in April 2015 and analyzed using 16S rRNA and ITS gene amplicon sequencing as well as GeoChip 5.0 to compare the epiphytic community with the free-living microbial community in the surrounding water.Significant divergences in the composition and function of microbial communities were observed between epiphytes and free-living groups according to a dissimilarity test.More similar structure of microbial communities was detected in the epiphytic community with a lower Shannon index and Simpson index,and higher Simpson evenness compared to the free living community.Some epiphytic genera,such as Croceitalea,Formosa,Granulosicoccus,Leucothrix,Polaribacter,Aureobasidium,Penicillium,in high relative abundances are known to specialize in an alga-associated lifestyle with agarolytic or algal polysaccharides degrading potential.Genes associated with adaptation or responses to the seaweed surface were enriched in the epiphytic microbial community.The abundances of most genes involved in the biogeochemical cycling decreased,while those affiliated with assimilatory N reduction,sulfide/sulfur oxidation,chlorinated solvents and herbicide related compounds were enriched in the epiphytic community compared to the free-living community.Therefore,the distinct structure and function of epiphytic microbial communities generally support our hypotheses that seaweed can select microbes especially those seaweed-associated microbial groups.Understanding the interactions between G.lemaneiformis and epiphytic microbes can help us to explore the community assembly mechanism between seaweed and microbes in the coastal environment.In summary,a large-scale cultivation of G.lemaneiformis has significant effects on the diversity,structure and function of water and sediment microbial communities in the G.lemaneiformis cultivation ecosystem through changing environmental factors such as increases in DO and pH,and decreases in nutrients.The?-diversity of microbial communities decreased in the seaweed cultivation zone,but the?-diversity increased with the growth of G.lemaneiformis.Seaweed-associated genera were enriched in the water column and sediment of cultivation zones,and epiphytes of G.lemaneiformis,indicating that a selection effect may occur in the G.lemaneiformis cultivation ecosystem.For example,higher abundances of seaweed-associated genera Croceitalea,Formosa,Leucothrix,Polaribacter,Arenibacter,Glaciecola,and Maribacter were observed in the G.lemaneiformis cultivation ecosystem.Also,genes cqsS involved in quorum sensing,amsG for biofilm formation,tre-arc associated with desiccation tolerance,and many genes in the stress response category were enriched in epiphytic communities.Thus,G.lemaneiformis cultivation altered the diversity,structure and function of microbial communities in seaweed cultivation zone by changing environmental factors and selecting seaweed-associated microbes.This study provides possible molecular mechanisms by which G.lemaneiformis cultivation influences microbial communities,revealing the role of microbes in the biogeochemical cycling of seaweed cultivation ecosystems and importance of epiphytes to the host seaweed.The above results provide basic data and scientific basis to uncover the structure and functional potential of microbial communities in mariculture ecosystems. |
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