Structure Of Sponge Symbiont Microbial Communities During Different Life Stages And Preliminary Study Of Functions Of Stage-specific Microorganisms

Sponge contains a rich variety of commensal microbial communities as the oldest and most primitive multicellular organism.Microbes can account for 40-60%of sponge biomass.After six hundred million years of co-evolution,commensal microorganisms and sponges form a complex holobiont.Symbiotic microorganisms play an important role in the growth,development and metabolism of sponge hosts.However,there are less reports focusing on the interactions between symbiotic microorganisms and their sponge hosts during life cycle,mainly because of the difficulty of collecting sponge samples at different life stages and the lack of data about the key symbiotic microorganism groups associated with sponges.High-throughput sequencing of 16S rDNA amplicon was used to explore the structure and diversity of symbiotic microorganisms of Tedania sp.and Callyspongia sp.during life cycle(embryo-larvae-adult).The results showed that the abundance of sponge symbiotic microbes was significantly different from seawater microbes.The relative abundances of ?-Proteobacteria with Tedania sp.and Callyspongia sp.were over 3 times higher than that from seawater.Sponges at different growth stages have great microbial diversity.15098 operational taxonomic units(OTUs)were obtained from Tedania sp..Among them,13 OTUs existed exclusively in each stage of sponge Tedania sp.,but were absent in seawater,which could thus be identified as sponge-specific bacteria.14152 OTUs were obtained from sponge Callyspongia sp.,and 6 OTUs were present exclusively in all stages of life cycle.The composition of symbiotic microbial of embyro/larva sponge and adult sponge at the level of phylum were similar,but the diversity and abundance are not consistent.Sponges in different stages have their characteristic microbial communities,such as Clostridia(5.6%)and Bacteroidia(2%)in Tedania sp.were markedly more abundant in competent larvae than other growth stages(<1%)and even undetected.They also had the highest abundance in the embryonic stage of Callyspongia sp.,accounting for 3.6%and 1.6%,followed by competent larvae,while the abundances in other stages were less than 1%.At the stage of adult,Cyanobacteria was dominated in Tedania sp.and Callyspongia sp.,which was twice more than larval sponges and 10 times as much as seawater.Based on the conjoint analysis of the stage-specific microbial population and the growth stage characteristic of host,sponge-associated microbes that have been isolated in our lab previously were selected in studying the function of symbiotic microbes in the larvae and adult sponges.On the one hand,larval settlement of Tedania sp.was observed by adding different bacteria to the water in which sponge larvae lived.The experimental results showed that the number of settled larvae with Bacteroidetes additive(Bac group)was higher than those bacterial additives from seawater.The settlement rate of lavae added with Bac2 group and Bac3 group at the concentration of 104 cfu/mL repectively,were 22%which were significantly higher than that in seawater-bacterial group(only 10%).Moreover,in the experimental groups of mixed Bacteroidetes,the proportion of settled larvae in Bac 1+2+3 group at the total concentration of 104 cfu/mL was the highest(up to 48%)and the rate in Bac 1+2 group at the concentration of 103 cfu/mL was second(28%).Both of them had significant difference from normal seawater group.According to these results,it was speculated that Bacteroidetes group could promote the settlement of larvae of Tedania sp..On the other hand,Cyanobacteria plays an important role in marine C/N/P cycle.The content of polyphosphate was detected quantitatively by fluorescence in this study.As a result,the percentage of polyphosphate in adult Tedania sp.was as high as 0.372 ± 0.027%,which was much higher than that of adult Callyspongia sp.and adult Mycale sp.(0.001-0.005%),Massive polyP granules were observed in the tissue section of Tedania sp.under confocal microscopy.Comparing the abundances of Cyanobacteria of different sponges,it was found that the concentration of Synechococcophycideae in Tedania sp.was abundant,while the content in Callyspongia sp.and Mycale sp.were rare.Moreover,polyphosphate kinase(ppk)gene of Synechococcales was amplified from the total DNA of sponge holobiont,which proved that symbiotic Synechococcales was able to synthesize polyphosphates.Therefore,it was implied that during the growth of adult Tedania sp.,Synechococcales could synthesizes polyphosphate as energy reserve to supply the host sponge.