The Microbiome After Bail-out: Testing Individual Polyps from Pocillopora verrucosa as Models for Coral Microbiology Studies

doi:10.21203/rs.3.rs-4064631/v1

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The Microbiome After Bail-out: Testing Individual Polyps from Pocillopora verrucosa as Models for Coral Microbiology Studies

https://doi.org/10.21203/rs.3.rs-4064631/v1

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Micro-scale in vitro models are essential for the study of model organisms in highly reproducible and controlled environments. Coral research grapples with a substantial knowledge gap on micro-scale processes underlying symbiotic interactions and holobiont health, which could be addressed through the use of models adapted to microscopic studies. Individual coral polyps separated from their colonies through an acute stress-induced bail-out process have been suggested in the past as miniaturized models to study the coral holobiont. However, changes in the microbiome associated with these polyps after bail-out are still not completely understood. An aquarium experiment was therefore performed to monitor the diversity and composition of microbes of bailed-out polyps of Pocillopora verrucosa alongside their parental fragments over time. Our findings revealed no significant microbiome differences immediately following bail-out, with 80% of microbial members persisting for up to two weeks, decreasing slightly to 60% in the third week. Notably, despite a reduction in shared Amplicon Sequence Variants (ASVs), the dominant bacterial taxa's relative abundance stayed consistent across both the source fragments and bailed-out polyps for up to three weeks. This consistency underscores the potential of using micropropagates as valuable tools for microbiological research in corals. Further enhancements in polyp settlement techniques may refine these models, bolstering our capacity for comprehensive coral microbiome studies.

Biological sciences/Microbiology/Environmental microbiology

Earth and environmental sciences/Ecology/Microbial ecology

Coral tissues have highly abundant microbial communities associated with them, with members that are capable of positively or negatively influencing their host’s physiology ^1–7. Under stressful conditions, several microorganisms can negatively impact coral health and the stability of its microbiome, opening room for dysbiosis and disease ^2,4,8. Alternatively, beneficial symbionts play roles in several important mechanisms such as photosynthesis, nitrogen fixation, nutrient cycling and input, and protection against pathogens ^3,9,10. Coral probiotics, i.e. microorganisms that can provide advantageous characteristics ^3,9, have been employed to benefit coral health and growth ^11–18, and some microbial-derived mechanisms have been correlated with thermal protection with largely successful results ¹⁹.

The microscale effects of probiotics in corals have yet to be thoroughly explored ²⁰. The current knowledge gap related to the cellular and molecular mechanisms involved with the interactions in coral disease ^21–23 and probiotics ²⁴ demands studies that target microscale processes in corals, which are, in part, still hindered by the lack of established miniaturized in vitro study models. To overcome the limitations in culturing coral cells and to maintain a laboratory culture that preserves the overall tissue organization of corals, the use of isolated polyps has recently been proposed as a miniaturized model system for coral biology research ^25,26. The separation of live polyps occurs naturally by the process of polyp bail-out, in which acute stress causes the digestion of the coenosarc between the polyps, which can then detach from the colony's skeleton ^26–28.

Individual micropropagates have the potential to become a useful tool for coral studies at the molecular and cellular scales when they are detached from the coral skeleton, and are also an alternative to the use of other models, such as coral explants ²⁹, cell cultures ³⁰, or other cnidarians, such as the commonly used Exaiptasia pallida sea anemone ³¹. However, for microbiological studies, such as testing the effect of specific microorganisms on coral health, it is also essential to first characterize the microbiome of the polyps after the bail-out processes. For polyps to be a valid model for microbiological studies on corals, not only should the bailed-out polyps be healthy, but they should also retain a representative coral microbiome.

Cultivating coral micropropagates and using them as models could allow researchers to study in vivo processes at the tissue and cellular scales that cannot be visualized when analyzing whole coral colonies ²⁶. Recent studies have shown that individualized coral polyp morphology and physiology are highly similar to source coral colonies after recovery from bail-out ^25,32,33. Two of the commonly cited advantages of using coral micropropagates are the possibility of having replicates that are genetically identical to each other and a parent coral colony and their ability to be maintained in a highly controlled environment ^25,26. These two factors, together, can reduce the variability in coral physiological studies and become a useful tool to test treatments and approaches for coral conservation projects. Even though coral polyps have been used to study the inoculation of coral pathogenic bacteria ²⁵, only one recent study analyzed the microbiome at the moment of coral bail-out ²⁷, not tracking the subsequent changes to the microbiome.

Here, we study the microbiome dynamics and longer-term stability of individualized polyps belonging to the species Pocillopora verrucosa, in order to assess their utility as study models for coral microbiology and micro-scale studies. To do so, an aquarium experiment was performed to analyze the coral microbiome composition over 4 timepoints for three weeks after the induction of polyp bail-out by a high-salinity treatment.

Coral Collection and Maintenance

A colony belonging to the Pocillopora verrucosa coral species was collected from the Coral Probiotics Village, at the Al Fahal reef (22.305131095668266 N, 38.96457386841887 E) by SCUBA diving using a hammer and chisel to generate the polyp clones to be used as a model. The species of the collected organism was confirmed by sequencing of its cytochrome b gene amplified by the primer pair primers AcCytbF (5′-GCC GTC TCC TTC AAA TAT AAG-3′) or MCytbF, and AcCytbR (5′-AAA AGG CTC TTC TAC AAC-3′) and blasted against the NCBI database, as described in ³⁴. Al Fahal Reef is not part of an environmental protected area, and no special permits were needed for coral collection. The colony was maintained inside a 300 L aquarium kept at 26 ^oC with two aquarium heaters (Schego Heizer Titanrohr Heizstab SW MW 600 Watt), three pumps (Tunze Turbelle Nanostream 6025) and two light sources (RADION XR15 G5 PRO) maintaining a 12-hour light cycle with a constant income of filtered sea-water at a speed of 12 liters per minute. The temperature of the aquarium was controlled by connecting each one of the two heaters to a temperature controller (Schego TRD 112 thermostat). Light emission was programmed to start at 6 AM and finish at 6 PM, producing an irradiance curve that peaked at 12 PM with 230 µmol photons m^− 2s^− 1. After 33 days, the coral colony was cut with diagonal cutting pliers into 55 fragments of approximately 2 cm in height, which were maintained in the same aquarium for 13 other days before the beginning of the experiment.

Polyp Bail-out Induction, Aquarium Set-up, and Sample Collection

At the beginning of the experiment, five coral fragments were placed into cryotubes and flash-frozen in liquid nitrogen for future DNA extractions. On the same day, the other 50 fragments were divided into two equally numbered groups, a control group and a group for polyp bail-out induction. The 25 fragments of each group were put into sterile plastic trays filled with 12.8 liters of filtered seawater. Each trey was equipped with an aquarium heater connected to a thermostat set to 26 ^oC and was connected to an air pump to maintain water oxygenation. A peristaltic pump was then used to pump water at a speed of 9 mL per minute to the plastic trays. For the control group, filtered seawater at non-stressful salinity (41 PSU) was pumped, while in the bail-out induction group, NaCl-enriched seawater (74 PSU) was pumped in order to induce polyp bail-out through acute stress by salinity ³⁵. After 27 hours, polyps from the corals in the group treated with NaCl-enriched seawater had digested their interconnecting tissues, and a sterile pipette was used to collect the polyps from each fragment and place them in separate petri dishes, summing a total of 20 dishes with polyps from each fragment. More details and protocols for the coral bail-out process can be found at ³⁵. Once the polyps were collected, the pump was paused, and the water from each Petri dish was slowly exchanged with freshly filtered seawater (41 PSU) with a sterile pipette, until 50% of their volume was exchanged over a period of 10 minutes. The petri dishes containing coral polyps were then covered with a sterile plankton net (with 300 µm mesh size), and both the coral polyps and the coral fragments from the control group were returned to a new experimental aquarium. Pictures of the coral fragments and/or polyps were taken with an IC80 HD camera (Leica, Germany) attached to a Leica MDG33 stereomicroscope the day before bail-out induction (Ti), the day of bail-out induction (T0) and three days (T1), one week (T2), two weeks (T3) and three weeks (T4) after bail-out (Fig. 3). During each of these periods, five coral fragments from the control group and five pools of polyps from different Petri dishes (containing 15 polyps per pool) were placed in cryotubes and immediately frozen in liquid nitrogen for subsequent DNA extractions, with the exception of Ti, in which five fragments were collected before the separation of samples into two groups. Flash-frozen samples were transferred directly from liquid nitrogen to a -80 ^oC freezer before further analysis.

DNA extractions from Coral Samples

Frozen coral fragment samples were brought back to room temperature and inserted in 15 mL tubes. One mL of Buffer RLT Plus (Qiagen) was applied to the fragments, and the tissues were blasted away for one minute from the coral skeletons by pumping air through tubing attached to an air pump with a filtered pipette tip attached to its end. The polyps were suspended in 300 µL of Buffer RLT Plus, and their tissues were homogenized using a tissue grinder (Cole-Parmer, Vernon Hills, Illinois USA) for one minute. The resulting slurries were used for DNA extraction using the AllPrep DNA/RNA Micro Kit (Qiagen, Germany) following the instructions of the manufacturer, in parallel with extraction blanks that went through the same treatment without the addition of coral samples.

16s Gene sequencing, DNA Preparation and Analysis

The V3 and V4 regions of the 16s rRNA gene were amplified from the extracted DNA by Polymerase Chain Reaction (PCR) using the 341F and 785R primer pair with Illumina MiSeq adapter overhang sequences added to the gene-specific nucleotide sequences. The sequences of the primers were: 341F = 5' TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG, 785R = 5' GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC. The reaction was prepared with the KAPA HiFi HotStart ReadyMix (Roche, Switzerland), with 5 µM of each primer, 12.5 ng of DNA and 12.5 µL of the Mix, diluted with DNAse-free water to a volume of 25 µL. The temperature profile for amplification was as follows: one cycle of five minutes at 92 ^oC, followed by 30 cycles of 30 seconds at 92 ^oC, 30 seconds at 62 ^oC and 30 seconds at 72 ^oC, followed by one cycle at 72 ^oC. For each sample, reactions were done in triplicate and then combined in a single pool. All sequences were checked by agarose gel electrophoresis for correct amplification before pooling. The resulting PCR product was sent to Macrogen (Seoul, South Korea) for DNA sequencing using the MiSeq sequencing platform. Demultiplexed reads obtained by sequencing were filtered, trimmed, merged, denoised, and dereplicated using the DADA2 version 1.20 ³⁶ plugin in the RStudio software version 1.4.1717 ³⁷. Trimming and filtering were done using the default parameters with a truncation length of 255 bps for the forward reads and 200 bps for the reverse reads. The ASVs obtained were then classified taxonomically by the naive Bayesian classifier method using the Silva release 138 .1 database ³⁸. Sequences were then checked for contamination based on blank samples using the Decontam R package’s prevalence method ³⁹, and ASVs considered as possible contaminants were discarded. The statistical tests and figures related to the coral associated microbiota were performed using the ASV count and taxonomy tables then obtained, that were analyzed using the Phyloseq package ⁴⁰ and manipulated using the dplyr ⁴¹ package in the RStudio software. Dissimilarities in the taxonomic structure of the microbial communities found with the 16s rRNA gene amplicon data were analyzed by creating principal coordinate analyses based on Bray-Curtis dissimilarities and by using Permanova tests for each timepoint individually using the data normalized as relative abundance and the “ADONIS” function in R, as well as the “permutest” function from the vegan package to test for homogeneity between groups. To study the differences in the alpha diversity of coral polyp and fragment-associated prokaryotes, the ASV data was rarefied by sub-sampling to the number of reads of the smallest sample using the Phyloseq “rarefy_even_depth” function and then used for Shannon index calculation. The normality of the data of each timepoint was tested using Shapiro-Wilk tests. Differences between the two groups were tested through ANOVA (Analysis of Variance) tests for timepoints with normal data and through Wilcoxon tests for timepoints with data that did not follow normality. To test for significant differences between the two groups in each timepoint related to specific ASVs, the DESeq2 package ⁴² in the R software was used to perform Wald tests with the data without subsampling and filter the results through a False Discovery Rate (FDR) cutoff of 0.01. All images and statistical tests based on the 16s rRNA gene amplicon data were created and performed using the ggplot2 package ⁴³ in R. Sequencing data is available at NCBI under Bioproject PRJNA1082127 and Biosamples SAMN40199994- SAMN40200052.

Taxonomic Changes in the Coral Microbial Community after Bail-out

The group of fragments treated with high-salinity seawater completed the polyp bail-out process after 24 hours. Conversely, control fragments maintained intact tissue organization, as expected (Fig. 1). Bailed-out polyps maintained their original color (which is derived from the presence of their photosynthetic endosymbionts). Complete external morphology was observed in the polyps, including a visible basal region and an oral region with protruded tentacles up to three weeks after the bail-out, although contracted polyps with spherical shapes and polyp fusion could be found throughout the experiment (Figure S1).

A total of 6,260,676 sequence reads were obtained from the 16S rRNA gene amplicons from P. verrucosa fragments (n = 30) and 4,888,484 from the 15-polyps pools (n = 25) collected in different periods of time after bail-out, with a sum of 5896 ASVs being classified from the data, of which six were discarded as possible contaminants. The number of reads and ASVs per sample is shown in Table S1.

The beta diversity of microbial communities from the fragments and polyps was not significantly different immediately after or a week following polyp bail-out (Fig. 2; Permanova, p = 0.1575, Permutest, p = 0.637 and Permanova, p = 0.0572, Permutest, p = 0.76, respectively). However, significant differences were found between the fragments and individualized polyps in the intermediate time point, three days after bail-out (Permanova, p = 0.007, Permutest, p = 0.542). Significant differences were also found in the overall communities associated with each of these two groups two weeks and three weeks after the beginning of the experiment (Permanova, p = 0.0079, Permutest, p = 0.001 and Permanova, p = 0.0097, Permutest, p = 0.324, respectively). No significant differences were found between coral fragments or polyps regarding the Shannon diversity of their associated microbial communities in any of the time points of the experiment calculated by the Shannon index (Figure S2, ANOVA, p = 0.503; 0.259 and 0.931, for timepoints T0, T1 and T4, Wilcoxon, p = 0.8413 and 0.2222 for timepoints T2 and T3, respectively).

The abundance of individual ASVs of coral polyps and fragments was also compared, with no ASVs having significant differences in abundance immediately after polyp bail-out, while 37 (T1), 5 (T2), 37 (T3) and 81 (T4) ASVs out of 5887 showed significant differences in abundance during the other timepoints (Fig. 3). These ASVs belonged to 17 (T1), 5 (T2), 20 (T3), 43 (T4) different genera, and 2 (T1 and T2), 8 (T3) and 9 (T4) different phyla, having amplicon variants belonging to the Bacteroidota and Proteobacteria phyla representing the majority of significantly different ASVs in all time points.

In order to observe what proportion of ASVs was still found in both polyps and fragments, the ASVs from each time point were divided into common ASVs and variants specific to each group and later weighed by relative abundance (Fig. 4). High rates of common ASVs were found between fragments and polyps after bail-out, varying from 98.42% (immediately after bail-out) to 62.87% (three weeks after bail-out). Furthermore, the relative abundance of the dominant bacterial taxa remained consistent between the fragments and bailed-out polyps across the three-week experiment (Figure S3-S4), with Endozoicomonas and Simkania, together, composing at least 50% of the bacterial community in both groups across all timepoints (Figure S4).

Our research underscores the robustness of coral polyp microbiomes post bail-out, with their microbial signatures remaining nearly identical to the parent coral fragments immediately after the stress-induced separation. These findings reinforce the premise that recently detached micropropagates offer a viable model for examining coral biology not only at cellular and molecular levels, as previously demonstrated ^28,32 for delving into the complexities of microbiological processes. Remarkably, three weeks after bail-out, a significant portion (about 60%) of the coral-associated ASVs were preserved in detached polyps. Despite the onset of divergence from the microbiome of the whole coral fragments, the detached polyps present a microcosm retaining a substantial part of their original microbial community. This not only posits them as an effective model but also paves the way for future investigations aimed at refining the conditions to bolster ASV retention post bail-out. Experimentation in controlled environments simulating natural coral ecosystems could provide further insights into this phenomenon, particularly through standardizing the settlement of polyps after separation.

As previously reported, polyp bail-out did not significantly alter the microbial community of the polyp when compared to the whole fragment ²⁷, even under high salinity stress. However, the lack of difference found in the 16S rRNA gene data immediately after bail-out does not ensure the lack of difference between the microbial communities, since there could still be remaining DNA from dead microbial members. Nonetheless, here we show that bailed-out polyps kept in the same tank as fragments from the same coral colony were able to maintain microbial communities without significant differences in alpha and beta diversities, even one week after bail-out. These results are aligned with previous experiments using coral colonies of another species exposed to short-term high-salinity stress ⁴⁴, although polyp bail-out was not induced in that case. Additionally, our data also highlight that the DNA extraction methods used in this experiment (airbrushing in fragments versus tissue maceration in polyps) were able to recover similar microbial communities from the two types of samples without introducing contamination.

Even though the microbiome remained stable during the first week after the bail-out process, this pattern gradually changed in the second and third weeks. This result indicates that, although the bail-out induction did not bring significant changes to the microbiome in the first subsequent week, the long-term maintenance of the polyps in Petri dishes can lead to the development of a slightly altered polyp-specific microbiome. These changes may be due to the lack of settlement and/or skeletal structures in the bailed-out polyps, which house a highly diverse microbial community in adult corals ^45,46. Due to the limited time that coral polyps can survive without fixation, it is also likely that the changes in the microbiome in relation to fragments could be due to health-related issues of this free-living state, or simply caused by the water flow or other local environmental differences that accumulate over time. If this hypothesis is proven accurate, developing a system that can fix the polyps or keep them in a more adequate environment than Petri dishes (i.e., microfluidic chips) ²⁶ could prevent this shift in the microbiome and increase the survival rates and time of these individuals.

Only ASVs belonging to the Bactereidota and Proteobacteria phyla were significantly different at the first time points (immediately and one week after bail-out), and still accounted for over half of the significantly different ASVs two and three weeks after bail-out, when over eight different phyla also accounted for the significantly different variants. This pattern can partly be explained by the fact that those two phyla are between the three most abundant ones found in samples from coral fragments and polyps at most time points of the experiment. The genera the dissimilar ASVs belonged to, however, varied between most of the experimental time points, with no specific genus having the number of differential ASVs consistently increasing or decreasing throughout the experimental time.

Common members of the coral microbial community have been demonstrated to greatly vary within organisms of the same species, with environmental factors, developmental stages, and genotypes affecting their composition ^47–49. This natural potential of microbiome flexibility ⁵⁰ makes it challenging to draw the line on the amount of variation that is acceptable for different coral organisms to be comparable in microbiological studies. Due to the fact that even microorganisms in low abundance in an environment can have a profound physiological or ecological effect ⁵¹, it is important to consider the roles that the associated microorganisms have ^3,7,9,10,52 to determine the microbiome functionality and, ultimately, its effect on the host, in order to customize microbial-based solutions ⁵³. Taking this into account, to have a better understanding of the consequences of the changes in the microbiome caused by long-term maintenance of corals as isolated polyps, the functionality of the microbiome should also be studied in the future.

Even though significant differences in microbiome composition were found in the last weeks of the experiment, over 80% of the relative bacterial abundance was common between these two groups up to two weeks after bail-out. This similarity is an advantage in relation to alternative models to keep in vitro cultures of coral biological material, such as cell cultures, that would need to eliminate coral-associated bacteria in order to maintain a viable culture. The cnidarian symbiosis model of study, Aiptasia, has been suggested due to its overall similar microbiome taxonomic composition and surface topography to corals ³¹. The microbiome of Aiptasia has been demonstrated to mainly consist of fewer taxa than corals, with the anemones being referred to as a “non-complex” model of the microbiome ⁵⁴. While this is still highly effective as simplified models in proof-of-concept surveys, a less complex microbiome will have different responses to microbial inoculations of specific pathogens or probiotics in relation to corals. The use of isolated polyps of species that are capable of bail-out would allow for a more direct comparison to colonies or fragments of that same species of coral. Having isolated coral polyps as an alternative model with complex associated microbiomes similar to coral colonies could, therefore, be of great convenience for such studies.

Data Availability

Sequencing data generated and analysis in this study is available in the Sequence Read Archive (SRA) of NCBI (National Center for Biotechnology Information) under Bioproject PRJNA1082127 and Biosamples SAMN40199994- SAMN40200052.

Author Contribution

Experimental design: RSP, HV, PC; Experiments and analyses: PC, HV, AB, RDR; Supervision and resources: RSP; Original draft: PC; Editing and writing: All authors

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No competing interests reported.

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The Microbiome After Bail-out: Testing Individual Polyps from Pocillopora verrucosa as Models for Coral Microbiology Studies

Status:

Version 1

Abstract

Figures

Introduction

Methods

Coral Collection and Maintenance

Polyp Bail-out Induction, Aquarium Set-up, and Sample Collection

DNA extractions from Coral Samples

16s Gene sequencing, DNA Preparation and Analysis

Results

Taxonomic Changes in the Coral Microbial Community after Bail-out

Discussion

Declarations

Data Availability

Author Contribution

References

Additional Declarations

Supplementary Files

Status:

Version 1