Microbial Diversity Course Marine Biological Laboratory July 8 to August 22, 2018

Imaging the Microbial Community of the Marine prolifera

Monica E. McCallum, Ph.D. Department of Chemistry and Chemical Biology Harvard University

BACKGROUND Marine are prolific producers of biologically active compounds and are particularly valued as sources of novel antibiotics and chemotherapeutics. The sponges themselves provide housing and nutrients for a complex microbial community that accounts for approximately 40% of any sponge’s mass and likely produces these complex bioactive natural products.1 However, the cultivation of these microbial symbionts, which would facilitate the study of their biosynthetic capabilities, has proven to be immensely challenging. Understanding the niche of microbial within the sponge microbiome may help facilitate culturing of these organisms away from their sponge host for further study.

In the early 2000s, three successive MBL Microbial Diversity students attempted to interrogate the microbial populations of three different local sponge species.2 The first was conducted by Dovi Kelman in 2001 on the local sponge Suberites ficus. Two years later, Gil Zeidner profiled the microbial community of the local sponge Holichondria sp. and was able to detect archaea using FISH microscopy with the ARCH915 probe.2b Kou-San Ju constructed a small 16s rDNA library of Clathria prolifera using universal Eubacterial primers 9F and 1492.2c Subsequent cloning into E. coli and sequencing led to the identification of 42 unique species across 14 types of bacteria, but no mention was made of any archaea present.

Clathria prolifera, which is commonly known as the Red Beard Sponge, is available from the MBL MRC and has been studied extensively as a model organism for instruction in biology and environmental science, but its microbiome has been minimally surveyed.3 The microbiomes of other marine sponges have been extensively catalogued by the Sponge Micrbiome Project.4 However, little is known of the archaeal symbionts of marine sponges, though sequences corresponding to ammonia oxidizing archaea are frequently reported in sponge metagenomic studies. DeLong and coworkers described the first symbiotic association of a sponge (Axinella mexicana) and archaeon (Cenarchaeum symbiosum) in 1996.5 A decade later, DeLong and coworkers reported the first genomic analysis of C. symbiosum, but to date that organism remains uncultivated.6 The first marine crenarchaeon to be isolated in pure culture was a free- living autotrophic ammonia-oxidizer,7 but the subsequent cultivation of additional ammonia oxidizing archaea from marine environments has been limited.

Sponges are important to nutrient and nitrogen cycling in the ocean, but the bacteria and archaea within these sponges may be responsible for the majority of nitrogen flux. Ammonia oxidizing archaea (AOAs) and bacteria (AOBs) have been found in a diverse array of sponges throughout the Earth’s oceans via 16S sequencing and via PCR amplification of the ammonia monooxygenase subunit A (amoA).8,9,10 Cultivation of an AOA, or any archaeon, from a sponge has yet to be reported. This miniproject aimed to use a variety of microscopy techniques to determine where different microbes reside within the sponge host C. prolifera and to attempt to enrich ammonia oxidizing archaea from C. prolifera.

RESULTS AND DISCUSSION The marine sponge C. prolifera is a with a skeleton made of spongin spicules that grows in shallow fast-flowing water along the coast of Falmouth, MA. The branching structure of C. prolifera provided easily accessible portions of sponge tissue for repeated sampling with minimal trauma to the sponge (Fig. 1a). The sponge tissue was homogenized via bead beating (Fig. 1b-c, d-e) and used to inoculate enrichment cultures containing minimal medium tailored to cultivate chemolithoautotrophic ammonia oxidizing archaea and bacteria. Growth of ammonia oxidizers was monitored using the colorimetric Griess test to observe the nitrite produced by this metabolism. After two weeks, none of the enrichment cultures had ever tested positive for nitrite and only a single tube of this enrichment showed visible growth (Fig. 1f).

Figure 1. (a) the marine sponge Clathria prolifera (b) excised live sponge tissue (c) homogenized live sponge tissue (d) excised dead sponge tissue (e) homogenized dead sponge tissue (f) biofilm growing on enrichment culture after 14 days.

The sample of C. prolifera contained two distinct regions of tissue: bright orange, live sponge tissue (Fig. 1b) and brown, dead tissue (Fig. 1d). Under differential interference contrast (DIC) light microscopy, these two tissues had distinct morphological features (Fig. 2). The live tissue consisted primarily of round sponge tissue cells interspersed with the spongin spicules that provide structural support in the sponge. The dead tissue contained few intact sponge tissue cells and was dominated by large, fast-moving microbes with spiral morphology and a variety of amoeba and ciliates.

Figure 2. DIC images of live sponge tissue (left) and dead sponge tissue (right) at 40x magnification. After observing the sponge tissues under light microscopy, a series of observations were made using fluorescence microscopy. From this, it was determined that the sponge tissue and its microbial inhabitants exhibit bright autofluorescence under every filter cube set available on the Zeiss microscope (Fig. 3). Both the live sponge tissue and the dead sponge tissue exhibited autofluorescence, though it appeared to be qualitatively less in the dead sponge tissue.

Figure 3. Autofluorescence of (a) live sponge tissue homogenate (b) dead sponge tissue homogenate. Labels in top left indicate filter cube used to acquire the image – no fluorescent probes were added.

In hopes of overcoming the inherent autofluorescence of the sponge tissue to image its microbial symbionts, the samples were incubated with a series of fluorescent probes. Figure 4 shows the staining of lectins (top left) present in the biofilm from the enrichment culture that was inoculated with homogenized live sponge tissue. The aggregates of enriched microbes seen under brightfield light microscopy do not exhibit autofluorescence (Fig. 4, bottom left) but do stain with ConA-rhodamine. Microbes that are not aggregated do not stain with ConA-rhodamine. Conversely, attempts to employ mono fluorescence in situ hybridization (FISH) with EUB-Alexa488 and ARCH915-Cy3 to stain bacteria and archaea respectively and overcome the autofluorescence of the sponge failed (Fig. 4, right).

Figure 4. (a) lectin staining (top) of the culture containing the biofilm and control for autofluorescence (bottom); (b) attempted monoFISH with EUB-Alexa488 and ARCH915-Cy3 on fixed live sponge tissue (top) and fixed dead sponge tissue (bottom). Labels in top left indicate filter cube and fluorescent probe used to acquire the image.

Having failed to overcome the sponge autofluorescence with mono-FISH, microbial identification through passive clarity technique (MiPACT) was attempted.14 Samples of sponge tissue were embedded in resin, cleared with SDS, and cut into 1 mm3 pieces prior to incubation with probes for hybridization chain reaction FISH (HCR-FISH), which amplifies the fluorescence signal by an order of magnitude as compared to mono-FISH. An additional benefit of this technique is that embedding the tissue in resin and imaging using confocal microscopy allows for 3D reconstructions of the environment inside of the sponge. Samples of sponge tissue were prepared for MiPACT with no HCR-FISH probe, EUB B1 probe, or NON B1 probe. Sections of the sponge tissue were imaged in 10 x 80 x 80 um cubes. Without a HCR-FISH probe, the DAPI present in the mounting medium stained the nuclei of the sponge cells, which were visible as blue spheroids in Figure 5a. The autofluorescence of the sponge was still visible under confocal microscopy, particularly around the spicules (top right of Fig. 5a), but it appeared to be greatly diminished as compared to the standard fluorescence microscopy carried out earlier. For the sample containing EUB B1 probe, there is a strong signal from the Alexa-488 fluorophore that may be colocalized with bacterial symbionts (small spheroids and rods, Fig. 5b). The large green patches present in Figure 5b may correspond to non-specific probe binding or to thick biofilms coating the interior of the pores in the sponge tissue. However, the sample with the NON B1 probe, which is a nonsense rearrangement of the bases in the EUB B1 probe with an Alexa-488 fluorophore, does not show this pattern and looks similar to the tissue sample without HCR-probe treatment. With optimization, this technique could be used to visualize the 3D distribution of microbial populations on and within the sponge tissue, perhaps even intracellularly.

Figure 5. Stills from a 10 um z-stack of (a) DAPI-only stain of MiPACT–prepared live sponge tissue (b) DAPI, EUB B1, and Con-A stained MiPACT-prepared live sponge tissue

In an attempt to determine what microbes were present on the sponge tissue, samples of live and dead sponge tissue were homogenized and their metagenomic DNA was extracted. 16S rDNA was amplified using archaeal specific primers in hopes of obtaining sequences corresponding to ammonia oxidizing archaea. The crude amplification reactions were ligated into a pGEM-T vector system and clones containing inserts were identified via blue/white selection. These clones were cultured, miniprepped, and the plasmids containing the inserts were submitted for sequencing. The results of the single direction Sanger sequencing are presented in a rough unrooted phylogenetic tree in Figure 6. These trees were generated by BLASTing the sequencing reads against either the nt database via blastn (Fig. 6, right) or by BLASTing the reads against the archaeal subset of the nt database (Fig. 6, left). Unfortunately, it is difficult to draw any conclusions from these reads, other than to state that there may possibly be archaea present in the sponge as well as bacteria.

Figure 6. Unrooted tree showing tentative relationship between 16S sequences obtained from the dead sponge tissue and reference archaea (left) or bacteria (right) sequences which are denoted in bold.

CONCLUSIONS AND FUTURE DIRECTIONS The attempt to enrich for ammonia oxidizing archaea and bacteria from C. prolifera failed, but an unidentified biofilm forming consortium was obtained that stains with ConA. Experiments to observe the microbial population of C. prolifera using mono-FISH were also unsuccessful, as were attempts to ascertain the microbial population through 16S rDNA sequencing. Efforts to amplify the ammonia monooxygenase subunit A (amoA) gene as a proxy for the presence of ammonia oxidizers failed.

The application of the MiPACT technique, which was developed by the Newman lab for sputum samples from CF patients, to sponge tissue shows promise as a technique to identify the 3D distribution of microbial communities within sponges. Studies to improve the specificity of this approach, both to reduce non-specific binding of the probe and to target more specific classes of microbes within the sponge tissue, are ongoing and will be reported in due course.

MATERIALS AND METHODS Sample Collection. The specimens of Clathria prolifera were collected by the Marine Resource Center (MRC) of the Marine Biological Laboratory, Woods Hole, MA. The MRC acquired these specimens from Little River Shipyard at low tide on July 27th, 2018 where they were growing attached to the substrate about two feet below the surface of the water. After collection, the samples were stored in a holding tank at the MRC for less than 12h before being transferred to a container in a saltwater table with a constant supply of fast-flowing filtered seawater at ~10 ºC. Small pieces (~1 cm) of live sponge tissue were immediately removed from the exterior of the sponge and fixed for MiPACT or frozen for gDNA extraction. The sponge was then maintained under fast-flowing filtered seawater at ~10 ºC. After 7 days, sediment form Garbage Beach (~1 L) was added to in order to feed the sponge, which had begun to die. Additional ~1cm pieces were removed as the course progressed for light microscopy, enrichment culturing, and mono-FISH.

Light Microscopy. Pieces of untreated sponge tissue (cut from the live or dead sections of the sponge) were washed with sterile seawater (3x 5mL) and homogenized by vortexing in a 50 mL conical tube with ~10 sterile beads for ~2 min. 10 uL of the resulting suspension was placed on a glass slide, flattened under a cover slip and imaged on the Zeiss Imager.A2 with a Axiocam 503mono or Axiocam 503color.

Enrichment for Ammonia Oxidizers.7 5 test tubes were prepared containing 10 mL of marine ammonia oxidizing archaea medium, MAOM, [342.2 mM NaCl, 14.8 mM MgCl2 • 6 H2O, 1.0 mM CaCl2 • 6 H2O, 10 mM MOPS buffer (pH 7.2), 0.1 mM K2PO4, 1.0 mM Na2SO4, 1.0 mM NH4Cl, 2.0 mM NaHCO3, trace elements, vitamin solution]. Another 5 test tubes were prepared containing MAOM plus 50 ug/mL kanamycin/streptomycin. The first tube in each series was inoculated with 500 uL of the aforementioned live sponge homogenate. The tube was vortexed gently, and 1 mL was transferred to the next tube containing fresh media. These serial dilutions were repeated to the end of each set of 5 tubes. Another set of tubes containing MAOM and +/-50 ug/mL kanamycin/streptomycin were established with homogenized dead sponge tissue in the same manner.

Griess Test of Enrichment Cultures. The production of nitrite was measure by combining 100 uL of sulfanilamide solution and 50 uL of culture supernatant in a 96-well plate following the procedure in the Promega protocol.11 Each measurement was taken in duplicate with a new standard curve for each set of measurements.

Lectin Staining.12 1 mL of enrichment culture was transferred to a 1.5 mL microcentrifuge tube and centrifuged at 10k rpm for 5 min. The supernatant was decanted and the pellet was resuspended in 100 uL of 1x PBS with 18 ug/uL of ConA- rhodamine and incubated in the dark at room temperature for 5 min, diluted with 900 uL of fresh 1x PBS, then centrifuged for 5 min at 10k. The supernatant was removed and the pellet was resuspended in the residual liquid. 1 uL was plated on a glass slide and imaged using the Cy3 filter on the Zeiss microscope. mono-FISH. Approximately 200 uL of homogenized sponge tissue was combined with 500 uL of 4% paraformaldehyde in 1x PBS and allowed to fix for 16 h at 4 ºC. Fixative was washed via three cycles of centrifugation (10k, 5 min), decanting supernatant, and resuspending in 500 uL of fresh 1x PBS. Samples were stored in 1x PBS at 4 ºC. Samples were prepared for mono-FISH following the procedure described in the course lab manual.13

MiPACT of Sponge Tissue (adapted from DePas et al 201614 and the MiPACT-HCR protocol of Peter Jorth as edited by Nicki Limoli)

Fixing and Embedding. ~1cm long pieces of sponge tissue were removed from the C. prolifera and washed with sterile 1x PBS (3x 5mL), then fixed overnight (in 4% paraformaldehyde in 1x PBS) at 4 ºC. The fixed sponge pieces were washed with sterile 1x PBS (3x 5mL), then transferred to a 1.5 mL microcentrifuge tube and covered with 0.5 mL of embedding solution [500 uL of 40% 29:1 acrylamide:bisacrylamide; 100 uL of 12.5% VA-044; 4.4 mL of 1x PBS]. The embedding solution was set by cycling the tubes into the airlock of the Coy anaerobic chamber and left open to anaerobic atmosphere for 5 min, then removed from the anaerobic chamber, sealed, and placed in a 38 ºC water bath for 4 h to cure the resin. After curing, the embedded sponge tissue was cut into ~1mm cubes and transferred to 50 mL of 8% SDS in PBS and incubated in a shaker at 37 ºC and 250 rpm for 7 days.

Lysozyme/Proteinase K Treatment. After clearing with SDS, the cubes were retrieved via filtration through a cell strainer, transferred to 40 mL of 1x PBS, and returned to the shaker at 37 ºC and 250rpm for 30 min. The cubes were washed in this manner twice more for 30 min each, each time with fresh 1x PBS. The cubes were then incubated in 500 uL of lysozyme/proteinase K solution [5 uL of 1M Tris-HCl; 5 uL of 100 mg/mL lysozyme; 0.5 uL of 20 mg/mL proteinase K; 490 uL of mQ water] at 37 ºC for 1 h. The cubes were then retrieved from the solution, transferred to 40 mL of 1x PBS, and returned to the shaker at 37 ºC and 250rpm for 30 min. Repeated this wash.

HCR-FISH. While the cubes were being washed, prepared the buffer for HCR-FISH [100 mg dextran sulfate, 100 uL 20X SSC, 200 uL formamide, 600 uL mQ water]. HCR- Probe EUB B1 (15 uL of 1 uM stock) was added to 500 uL of HCR-FISH buffer and vortexed gently to mix. Three of the freshly washed sponge cubes were added to this solution of buffer and probe and incubated in the dark at 45 ºC overnight. The same process was repeated for the HCR-Probe NON B1. After hybridization, the cubes were transferred to a 50 mL conical containing 40mL of 84 mM FISH Wash and placed in a 52 ºC water bath for 6 h. Meanwhile, prepared HCR amplification paper [200 mg dextran sulfate, 200 uL 20X SSC, 1.6 mL mQ water]. Immediately before use, 12 uL of each amplification hairpin, B1H1 (Alexa 488) and B1H2 (Alexa 488), were placed in individual PCR tubes and heat-shocked at 95 ºC for 90 sec, then held at 21 ºC for 30 min in the dark. A 1.5 mL microcentrifuge tube was charged with 110 uL of HCR amplification buffer, 5 uL of each heatshocked hairpin, and three sponge cubes from the EUB B1 treatment. The same set up was repeated for the NON B1 treated cubes. These tubes were wrapped in foil and taped to a shaker at room temperature overnight. Cubes were removed from the HCR amplification buffer, transferred to 50 mL conical tubes containing 40 mL of 337.5 mM FISH was buffer and incubated in a 48 ºC water bath for 3 h. Cubes were retrieved from the wash buffer and placed into a microcentrifuge tube containing 1 mL of 1x PBS with 20 ug/mL of ConA-rhodamine, foiled, taped to the shaker at room temperature for 24 h. Cubes were transferred to fresh microcentrifuge tubes containing 1x PBS, foiled, returned to shaker at room temperature for 24h. Cubes were retrieved and placed in 100 uL of RIMS+DAPI in separate microcentrifuge tubes, foiled, and returned to the shaker at room temperature for 24h. Cubes were stored at 4 ºC wrapped in foil.

Confocal Microscopy. Cubes were removed from storage in RIMS+DAPI and placed into wells on a silicon-mat-covered microscope slide, each containing ~75 uL RIMS+DAPI. The wells were sealed with a size 1.5 coverslip and mounted onto a Nikon Eclipse Ti2 and imaged with lasers at 409.3 nm (DAPI, 3%), 488.7 nm (FITC, 1.35%), 562.1 nm (TRITC, 1.6%), 638.2 nm (Cy5, 1.6%). Galvano, 1/4, 1024 px, 1 px = 0.08. Pinhole = 1.2 (75.4 uM) AU calculated for 638.2 nm. Image Z-stack of 10 um in 0.175um slices with all lasers on. 8x line averaging.

Generation of Clone Libraries. Genomic DNA was extracted from the live and dead sponge tissue using the QiaAMP PowerFecal DNA Kit. 16S inserts were generated by amplifying with the 16S archaeal primers 4FA (5’-TCCGGTTGATCCTGCCRG-3’) /1391R (5’-GACGGGCGGTGTGTRCA-3’) or 333FA (5’-TCCAGGCCCTACGGG- 3’)/1391R. Inserts for the amoA gene were generated using the primers Arch_amoA- Fwd (5’-ATGGTYTGGBYWAGRMG-3’) and Arch_amoA-Rev (5’- GACCARGCGGCCATCCA-3’) or the primers Bact-amoA-1F (5’- GGGGTTTCTACTGGTGGT-3’) and Bact-amoA-2R (5’- CCCCTCKGSAAAGCCTTCTTC-3’). The PCR program used employed an initial denaturation at 95ºC for 5 min, followed by 35 cycles of 30 sec at 95 ºC, 1 min at 50 ºC, 1 min 30 sec at 72 ºC. The final extension was set for 5 min at 72 ºC. Crude amplified insert was ligated into the pGEM-T vector using the Promega pGEM-T Easy Vector System. Clones were selected using blue/white selection from the Promega kit and 24 clones were picked from each insert set. Clones were grown up overnight in 1 mL of LB+amp in a deep-well 96-well plate and plasmids were isolated using the Promega Wizard SV 96 Plasmid DNA Purification System. Plasmids were submitted to Quintara Biosciences for sequencing using the T7-Promoter forward primer.

Phylogenetic Tree Construction. Raw reads form the Quintara Biosciences Sanger sequencing data of the 96 well plate were trimmed to remove the portion of the read that contained the plasmid. Sequences that were <50% high quality bases (via Geneious analysis) after this initial trimming were discarded. The remaining 16S sequences were aligned and submitted to blastn. The top hit from each BLAST search was downloaded and the collection of reference sequence was aligned with the 16S reads. This alignment was used to create an unrooted neighbor-joining tree in Geneious.

ACKNOWLEDGEMENTS

I am grateful to all of the faculty, staff, TAs, CAs, scientific consultants and students in the Microbial Diversity 2018 course for all of their assistance and guidance throughout the summer. Prof. Rachel Whittaker and Prof. George O’Toole put together a phenomenal course and I am grateful to have been selected to participate. I am greatly indebted to Prof. Nicki Limoli, who guided me through every step of the MiPACT process and generously shared her knowledge and supplies. Special thanks to John Allen of Nikon for teaching me to use the confocal microscope and the Marine Resources Center of MBL for acquiring and providing samples of C. prolifera. Many thanks to Sarah G. for her help with sequencing preparations and numerous miscellaneous things. Thank you to Group 3 (Brittni, Sarah S., German and Jake) for your support in the early part of the course and throughout miniprojects.

I especially appreciate the support of Promega for providing almost all of the molecular biology tools used in this report, as well as the sponsorship of the course by the Simons Foundation, Agouron Institute, Gordon and Betty Moore Foundation, Howard Hughes Medical Institute, NASA, National Science Foundation, U.S. Department of Energy, Zeiss (particularly Jim Mcilvain), and Nikon. I also gratefully acknowledge my funding sources for attending this course: Damon Runyon Cancer Research Foundation Postdoctoral Fellowship, Eli Lilly, Course Funds, and the Stanley W. Watson Foundation Education Fund.

REFERENCES

1. Webster, N. S.; Torsten, T. The Sponge Hologenome. mBio 2016, 7, e00135-16.

2. (a) Ju, K.-S. Examining the Basis for Microbial Diversity in the Marine Sponge Microciona prolifera. MBL Microbial Diversity Course Report 2005

(b) Zeidner, G. Dynamics of microbial community in the marine sponge Holichondria sp. Microbial Diversity Course Report 2003

(c) Kelman, D. Phylogenetic Diversity of Bacteria and Archaea Associated with the Marine Sponge Suberites ficus. Microbial Diversity Course Report 2001

3. Isaacs, L.T.; et al. Comparison of the Bacterial Communities of Wild and Captive Sponge Clathria prolifera from the Chesapeake Bay. Mar. Biotechnol. 2009, 11, 758–770.

4. Thomas, T.; et al. The sponge microbiome project. GigaScience 2017, 1–7.

5. Preston, C. M.; Qu, K. Y.; Molinski, T. F.; DeLong, E. F. A psychrophilic crenarchaeon inhabits a marine sponge: Cenarchaeum sybiosum gen. nov., sp. nov. Proc. Natl. Acad. Sci. USA 1996, 93, 6241–6246.

6. Hallam, S.J.; Konstantinidis, K.T.; Putnam, N.; Schleper,C.; Watanabe, Y.-i.; Sugahara, J.; Preston, C.; de la Torre, J.; Richardson, P.M.; DeLong, E.F. Genomic analysis of the uncultivated marine crenarchaeote Cenarchaeum symbiosum Proc. Natl. Acad. Sci. USA 2006, 18296–18301.

7. Könneke, M.; Bernhard, A.E.; de la Torre, J. R.; Walker, C.B.; Waterbury, J.B.; Stahl, D.A. Isolation of an autotrophic ammonia-oxidizing marine archaeon. Nature 2005, 437, 543–546.

8. Wagner, M.; Taylor, M. W.; et al. Diversity and mode of transmission of ammonia- oxidizing archaea in marine sponges. Environmental Microbiology 2008, 10, 1087– 1094.

9. Feng, G.; Sung, W.; Zhang, F.; Orlić , S.; Li, Z. Functional Transcripts Indicate Phylogenetically Diverse Active Ammonia-Scavenging Microbiota in Sympatric Sponges. Marine Biotechnology 2018, 20, 131–143.

10. Hill, R. T.; et al. Symbiotic archaea in marine sponges shows stability and host specificity in community structure and ammonia oxidation functionality. FEMS Microbiol. Ecol. 2014, 90, 699–707.

11. Griess Reagent System Instructions for Use of Product G2930. Promega Corporation, 2800 Woods Hollow Road, Madison, WI 53711-5399 USA.

12. Chapter 17: Genetic Analyses. Microbial Diversity 2018 Lab Manual.

13. Chapter 14: Fluorescence in situ Hybridization with rRNA-targeted Oligonucleotide Probes. Microbial Diversity 2018 Lab Manual.

14. DePas, W.H*; Starwalt-Lee, R.*, Van Sambeek, L; Kumar, S.R; Gradinaru, V.; Newman, D.K. Exposing the three-dimensional biogeography and metabolic states of pathogens in cystic fibrosis sputum via hydrogel embedding, clearing, and rRNA labeling mBio 2016 7(5):e00796-16.