Chemosynthetic Symbioses Model Based on Solemya Velum Endosymbiont

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Chemosynthetic symbioses model based on Solemya velum endosymbiont

Mini-project report Microbial Diversity Course Author: Piotr Starnawski Preface

Microbial Diversity mini project title: Chemosynthetic symbioses model based on Solemya velum endosymbiont

Project period: 01.07.2013-25.07.2013

Author: Piotr Starnawski

This report summarizes a mini-project, which was performed in the Marine Biologi- cal Laboratory, Woods Hole, during a summer course on Microbial Diversity. The ﬁnal report represents a description of the project carried out during the second part of the course. It consists of 7 chapters, starting with a theoretical background introducing the reader to the topic of science of this study. The introduction is followed with description of materials, methods and obtained results with a discussion. Finally a summarization of work is presented in conclusions followed by future perspectives. The report is ﬁnished with a bibliography and enclosures. References are made according to Harvard referring system, and are presented in the references section at the end of this report. In the text all references are marked as follows: (authors surname, year of publication). I would like to thank the course directors Steve and Dan, all of the Teacher Assistants and course members for their help and support.

Piotr Starnawski Contents

1 Theoretical background 1 1.1 Chemosynthetic Symbioses ...... 1 1.2 Solemya velum ...... 2 1.3 Endosymbiont characterization ...... 2

2 Materials and methods 3 2.1 Specimens collection ...... 3 2.2 Symbiont cultures ...... 3 2.3 Scanning Electron Microscopy of the gill tissue ...... 6 2.4 Fluorescent In-Situ Hybridization of gill tissue ...... 7 2.5 BLAST search of local databases for the symbiont ...... 8 2.6 454 gill microbiome ...... 8

3 Results 11 3.1 Specimens collection ...... 11 3.2 Symbiont cultures ...... 11 3.3 Scanning Electron Microscopy of the gill tissue ...... 15 3.4 Fluorescent In-Situ Hybridization of gill tissue ...... 16 3.5 BLAST search of local databases for the symbiont ...... 16 3.6 454 gill microbiome ...... 18

4 Discussion 21 4.1 Symbiont cultures ...... 21 4.2 Symbiont imaging in the gill tissue ...... 22 4.3 454 gill microbiome ...... 22

5 Conclusions 23 6 Perspective 25

Bibliography 27

7 Enclosures 29 7.1 Primers and probes ...... 29 7.2 Wizard PCR Preps DNA Puriﬁcation System ...... 29 7.3 Mo-Bio fast soil DNA extraction kit ...... 31 7.4 Media compositions ...... 33 7.4.1 Sea Water Base ...... 33 7.4.2 Thiosulfate medium ...... 33 Abstract

The aim of this project was first of all to isolate and attempt to cultivate the intracellular sulfide-oxidizing symbiont of Solemya velum.Thiswasdonebydissectingthegillsand cultivating the extract in solid and liquid media in dilution series. Cultures were monitored with symbiont-specific PCR for the growth of desired organism and all obtained isolates had their 16S gene sequences. Further work aimed at characterizing the overall bacterial community found in the bivalves gills using 454 pyrosequencing. DNA form gills from 6 specimens was prepared and additional controls of 2 other clams living in the area were sequenced and the resulting community was analyzed. In order to better characterize the symbiosis, Fluorescent In-Situ Hybridization and Scanning Electron microscopy and techniques were applied for visualization of the symbiont and possible other bacteria living on the gill tissue. All this information was supplemented by analysis of existing data on this symbiosis model which comprised of symbiont partial genome and 454 datasets from sediment known to be occupied by Solemya velum. 1 Theoretical background

1.1 Chemosynthetic Symbioses

The word ”symbiosis” originates from greek and means ”living together” and is used to describe two di↵erent organisms which live together, regardless if the outcomes are positive or negative. Chemosynthesis refers to the process of energy conservation using chemicals (Madigan et. al. 2012). This report focuses on symbioses between bacteria and marine invertebrate, where the host provides the conditions for the bacterium to grow and provide ﬁxed carbon which is then used directly or indirectly by the host (Cavanaugh et. al. 2006). The two main types of chemotrophic symbionts observed in marine environments are methanotrophy and chemoautotrophy - see Table 1.1.

Table 1.1: Marine animals with chemolithotrophic or methanotrophic endosymbiotic bacteria (Madigan et.al.,2012)

Host (genus or class) Common name Habitat Symbiont type Porifera (Demospongiae) Sponge Seeps Methanotrophs Platyhelminthes (Catenulida) Flatworm Shallow water Sulfur chemolithotrophs Nematoda (Monhysterida) Mouthless nematode Clam Shallow water Sulfur chemolithotrophs Mollusca (Solemya, Lucina) Clam Vents, seeps, shallow water Sulfur chemolithotrophs Mollusca (Calyptogena) Clam Vents, seeps, whale falls Sulfur chemolithotrophs Mollusca (Bathymodiolus) Mussel Vents, seeps, whale falls Methanotrophs Mollusca (Alviniconcha) Snail Vents Sulfur chemolithotrophs Annelida (Riftia) Tube worm Vents, seeps, whale falls Sulfur chemolithotrophs

Chemosynthesis refers to (i) the ability of an organism to oxidize reduced inorganic compounds for energy and use it to fix CO2 for biomass, like in case of chemolitotrophs using sulfide as the receded compound or (ii) the ability to use singe carbon compounds as both energy and carbon source, like in case of the methanotrophs (Stewart et. al. 2005). In this study, a focus is placed on the highly sulfidic environment, where the chemoli- totrophic endosymbionts are mostly encountered, utilizing the sulfide-rich environment for their energy metabolism. Theoretical background

1.2 Solemya velum

Solemyid clams are found throughout the worlds oceans, in shallow water and deep- sea habitats, and in temperate and tropical regions. The family diverged between 435 and 500 million years ago, and its members maintain a primitive form even today. The clam digs very characteristic Y-shaped burrow (Figure 1.1 a), to place itself lower in the sediment in the sulﬁde-rich region, while still having access to fresh, oxygenated water. The relatively recent discovery of symbionts provided the information about the reason for such behavior. The bacterial endosymbionts supply the animal nutrition derived from the chemoautotrophic ﬁxation of CO2 fueled by the oxidation of reduced inorganic sulfur compounds from the Solemya habitat. Indeed, as other Solemya species are reexamined, they have all been found to harbor bacteria in their gill cells (see Figure 1.1 b).

(a) Y-shaped burrows dug by Solemya velum (From (b) Transverse section of gill ﬁlaments Stanley 1970) of Solemya borealis, showing intracellular rod-shaped bacteria (arrows, rectan- gle) b: bacteriocyte nucleus; c: ciliated cell nucleus; i: intercalary cell nucleus; bl: blood space; ci: cilia; mv: microvilli Figure 1.1: Solemyid clams characteristic features (Cavanaugh et. al. 2006

1.3 Endosymbiont characterization

Symbionts are believed to be integratedgrated into the entire life cycle of Solemya velum clam, given their presence in reproductive tissue, larvae, juveniles, and adults (Cavanaugh, 1983, Krueger et. al. 1996). It is hypothesized that female hosts transmit the bacterial symbionts to new generations, and that bacteria derived from this seed population colonize the gills as ﬁlaments are formed in juvenile clams. The functional role of the symbionts in young Solemya velum hosts remains unknown, however. The symbionts are probably not metabolically active before and just after spawning, but they colonize the gill buds and appear to be actively growing while still within the larval egg capsule (Krueger et. al. 1996).

2 2 Materials and methods

2.1 Specimens collection

Specimens for all the studies performed during tho project were collected in the Eliz- abeths Islands area on the eastern coast of the USA. Sampling was done near Naushon island (Figure 2.1) in two distinct sampling sites (Figure 2.2). Sediment was collected and sieved for Solemya velum.Simultaneouslyotherclamswerecollectedtoserveasacontrol and sediment was collected for keeping the animals in the lab. Organisms were placed in a plastic container with salt water and transferred to lab. Dissections were done immediately after collection (1-2h) and remaining clams were placed in sediment containers kept in continuous fresh sea water ﬂow aquarium. Dissections of organisms were performed using sterile forceps and scissors for each separate organism. After separating the gill from the rest of the body it was washed in autoclaved 80 % Sea Water Base (SWB, see Enclosures), weighted and placed in an Eppen- dorf tube on dry ice for snap-freezing. Frozen gills were stored in -20 °Candusedlaterfor DNA extractions. For the cultivation purposes the gills were not frozen but homogenized immediately.

2.2 Symbiont cultures

In order to try to obtain an isolate of the gill symbiont, numerous liquid and plate cultures were prepared using the thiosulfate medium with or without yeast extract addition (0.02 g/l) and gradient tubes with sodium sulﬁde plug (see Enclosures for media descrip- tions). The dissected gills were washed in 80 % SWB and then placed in an Eppendorf tube with 100 µl of the 80 % SWB. Homogenization was done by hand using a plastic tissue homogenizer for around 2 minutes. The volume of homogenate was then adjusted to 1 ml with the same solution and used for incubations according to Figure 2.3. A total number of 5 di↵erent gills were used for starting these cultures. All tubes and plates were held in dark (cardboard box for the plates and oce drawer for gradient and regular tubes) and monitored daily for growth. Materials and methods

Figure 2.1: Location of the sampling sites in the Buzzards Bay

Figure 2.2: Precise situation of the two sampling sites for the specimens used in this study

4 Materials and methods

Figure 2.3: Schematic of the inoculations of liquid, solid and gradient cultures with homogenized gill tissue

The monitoring of cultures was done by the use of symbiont-speciﬁc primers provided by Colleen Cavanaugh’s lab in Harvard (see Enclosures for sequences). The monitoring was performed by PCR method where the colony or 1 µlofliquidculturewasaddedto the PCR reaction tube. For the reaction mix see Table 2.1.

Table 2.1: PCR mix used to check for symbiont-speciﬁc 16S product from the colonies/liquid cultures Volume [µl] Compound 6.25 Promega Go-Taq Green 2X Mix 1.00 Forward primer 120F (15 pmol) 1.00 Reverse primer 1612R (15 pmol) 3.25 Nuclease-free water 11.50 Total volume per sample

The product was then ran on a 1 % agarose gel to check for a product of around 1.5 kbp. For a positive control, puriﬁed DNA from gill extracts from Solemya velum used for 454 preparations were used. As a negative-control samples with MQ water were used. Selected isolate colonies obtained by re-streaking on solid medium were prepared for 16S

5 Materials and methods

Sanger sequencing to establish the identity of the colony. This was done using the universal 8F/1492R primer pair (see Enclosures for sequence) according to the mix presented in Table 2.2

Table 2.2: PCR mix used to check for bacterial 16S product from gill extracts Volume [µl] Compound 12.5 Promega Go-Taq Green 2X Mix 2.0 Forward primer Univ8F (15 pmol) 2.0 Reverse primer Univ1492R (15 pmol) 6.5 Nuclease-free water 23.0 Total volume per sample

Similarly the product was checked on 1 % agarose gel. Purification of the product was done using the Promega Wizard PCR Preps DNA Purification System according to protocol (see Enclosures). Purified colony 16S products were then quantified on a NanoDrop system and send for Sanger sequencing.

2.3 Scanning Electron Microscopy of the gill tissue

Further visualization of the symbiont organism in the gill tissue was done with Scan- ning Electron Microscopy (SEM). Four organisms were selected and their mantles were cut under the dissecting microscope. After they were opened, two were placed in 80 % SWB 4 % formaldehyde fixative and two in 1X Phosphate Bu↵ered Saline (PBS), 4 % formaldehyde, 0.5 % glutaraldehyde fixative solution (referred to later as SWB and PBS fixations). Fixation was carried out for 2 h and after that organisms were washed thoroughly with their respective bu↵ers (80 % SWB and 1X PBS). In the bu↵er the dissections were done, where the gill was separated from the body and cut in half with a razor blade. Half pieces of the gill were placed (under submersion) in plastic containers and remained there for the rest of the washing steps. The washings were as follows: 1) 3 times 10 minutes in the respective bu↵er (80 % SWB or 1X PBS)

2) 2 times 5 minutes in 25 % EtOH 3) 3 times 10 minutes in 50 % EtOH 4) 3 times 10 minutes in 75 % EtOH 5) 3 times 10 minutes in 100 % EtOH

After that samples were kept in fresh 100 % EtOH and transferred to the critical point drying machine - Samdri-780A. After drying, samples were placed on metal stages with carbon surface and coated with 10 nm layer of platinum in vacuum. Afterwards they were stored overnight in a desicator at room temperature and placed in the SEM for imaging.

6 Materials and methods

2.4 Fluorescent In-Situ Hybridization of gill tissue

In order to visualize the symbiont and it’s location in the gill tissue, Fluorescent In-Situ Hybridization (FISH) was applied. A specimen of Solemya velum was dissected under a dissecting scope and the gill tissue was separated from the rest of the body. Then it was placed in 4 % formaldehyde solution in 80 % SWB for 1 h 30 min. After that a thorough was with 80 % SWB was performed to remove all formaldehyde and such ﬁxed gill was stored at 4 °C. The sample for FISH was prepared in a 10-well glass slide. Gill was dissected further until single ”pages” of the ”book gill” tissue could be seen. These were collected by forceps and placed in a water drop inside of the slide well. A total number of 10 tissue pieces were isolated in such way. Slide was then dried at 46 °Ctoﬁrmlyplacethegill tissue inside the well. After that a dehydration step was performed using a series of 3 ethanol baths for the slide: 50 % EtOH, 80 % EtOH and 96 % EtOH, in all cases 2 minute wash each. After slide has dried o↵ the hybridization was performed by dropping 15 µlof bu↵er-probe solution to each well. The solution was prepared by mixing the hybridization bu↵er (see Table 2.4) and the desired probe (EubI-III and Gam42a + Bac42 a competitor probe in case of this study) in 300:1 ratio (300 µlofbu↵erand1µlof50ng/µlprobe).

Table 2.3: Hybridization and Washing bu↵ers composition used for the FISH of the gill tissue Hybridization bu↵er volume [µl] Washing bu↵er volume [µl] Compound 700 - Formaldehyde -5000.5MEDTA 360 700 5 M NaCl 40 1000 1 M Tris-HCl 12520%SDS 900 ﬁll-up to 50 ml MQ water

The slide with the bu↵er-probe solution was placed in a horizontally-situated 50 ml Falcon tube with a tissue paper soaked in the hybridization bu↵er situated at the end of the tube. Hybridization was performed for 4 h at 46 °C. After that slide was removed from the chamber and the wells were washed using the pre-heated to 48 °Cwashingbu↵er(see Table 2.4). Then the whole slide was placed vertically in a 50 ml Falcon tube ﬁlled with the hybridization bu↵er and left at a water bath at 48 °C for 15 min. After the washing slide was removed from the Falcon tube and washed again with MQ water. After the slide was dried a DAPI staining was done by placing 15 µl of the 1X DAPI stain in each well and incubating the slide for 10 min in the dark at room temperature. Finally the slide was washed with MQ water and then with 80 % EtOH and air-dried. During the microscopy Citiﬂuor solution was applied to enhance the signal.

7 Materials and methods

2.5 BLAST search of local databases for the symbiont

Due to the long history of research in the area and the fact, that the symbiont can be found in the free-living state a search of existing datasets was performed to ﬁnd, if sequences belonging to the symbiont were previously found in the area surrounding Woods Hole. 454 datasets from 2010, 2011 and 2012 were selected for that, where 2010 was a metagenome of a Little Sipperwisset Salt Marsh microbial mat and the remaining two were large 16S surveys of various environments. Using the blast+ package ”makeblastdb” command databases for all thee sets were made. As a query the 16S sequence of symbiont bacterium was used (GenBank: M90415.1) and additionally a partial genome, provided by Colleen Cavanaugh’s lab in Harvard, of the symbiont was also tested (68 contigs genome, unpublished). These were are checked against the database on the nucleotide level (”blastn” command) and the results were summarized using the circos imaging software package. For the 16S sequence apercentage-identitycuto↵of97%wasused(inordertolookforthesameoperational taxonomic units) and for the partial genome 93 % percentage-identity and 1e-100 e-value cuto↵s were used to target highly similar and signiﬁcant hits.

2.6 454 gill microbiome

For the preparation of the gill microbiome, six Solemya velum,twoTagelus plebeius and two Yoldia limatula were chosen (the latter two serving as controls not containing the symbiont). Frozen gills were thawed on ice and then DNA was extracted using the Mo-Bio PowerSoil DNA Isolation Kit according to protocol (See Enclosures). The only change to the standard ﬂow was shortening the homogenization step to 1 min due to using a more thorough homogenizer. Extracted DNA was then checked by performing a PCR reaction using regular 8F and 1492R (see Enclosures for sequences) primers to see if a product is obtained. PCR mix is presented in Table 2.2. For the reaction, 2 µlofgillextractwasused. 30 cycles of PCR were performed and 5 µloftheproductwasrunona1%agarosegel with a 1 kb DNA ladder. After checking the result the DNA extract was used to perform anewPCRwithbarcodedprimers.Foreachsample,onedistinctbarcodewasselected. The mixture was prepared according to volumes presented in Table 2.4.

Table 2.4: PCR mix used to obtain a barcoded 16S product from gill extracts Volume [µl] Compound 15 Phusion 2x HF MasterMix 2.4 DMSO, 100 % 0.6 Forward primer 515F (6.25 uM) 2.4 Reverse primer 907R (25 uM) 4.6 Nuclease-free water 25.0 Total volume per sample

8 Materials and methods

To each tube 5 µl of gill extract was added and the PCR was run for 30 cycles. Obtained product was checked on a 1 % agarose gel with a 1 kbp marker. Barcoded products were pooled, purified and send for sequencing on a 454 machine. Sequences were then analyzed using Qiime software according to the standard 454 operating procedure. Samples were separated from the whole plate according to the barcode using the split libraries.py command. Sequences of quality scores below 25 were discarded, and analysis was performed on sequences between 400 and 600 bp long. Next step comprised of starting the workflow for Operational Taxonomic Units (OTU) picking with pick de novo otus.py command which also assigned RDP taxonomy to picked OTUs. Finally, the results were summarized using summarize taxa through plots.py command. To check, if the 454 data contained the gill symbiont sequences an additional analysis was done, where a blast database was prepared from the 454 data and the 16S gill symbiont sequence (GenBank: M90415.1) was used as aqueryagainstit.Sequenceswerefilteredfor99%,98%and97%identitytothequery to look for the exact same sequence or one belonging to the same OTU.

3 Results

3.1 Specimens collection

After the collection of organisms they were stored in 1 l plastic containers filled with sulfidic sediment collected at the same place where organisms were collected. These containers were covered with a net to avoid organisms from escaping and kept in an aquarium under constant seawater flow (See Figure 3.1). Dissections were preformed in order to separate gills that contain the symbiont bacterium. Organism size was noted and gills were washed in autoclaved 80% SWB and weighted on a laboratory balance. Some of the extracts were used for starting cultures and some for DNA extractions for 454 pyrosequencing - the summary of dissections and which samples were used for DNA extracts is presented in Table 3.1. Other specimens were used for starting cultures with them in order to cultivate the symbiont. All extracts Figure 3.1: Container used were kept at -20°C after flash-freezing in liquid CO2 for storing the organisms, kept in an aquarium with 3.2 Symbiont cultures constant seawater flow

During the three-week culturing of solid, liquid and gradient cultures of the gill extract very little growth has been observed. The most prominent changes in the medium have been served for the gill extract from organism Sv2 where the medium became very milky in comparison to the starting, transparent solution (Figure 3.4). This was for the ﬁrst 1 dilution of the series, 10 .Thisdilutionwasusedforinoculationoftwonewtubes(with and without 0.02 g/l yeast extract - YE), to see if further enrichment can be obtained. Moreover solid plates were also streaked (also with and without YE). After a week no signiﬁcant darkening of the liquid cultures was observed, however the plate cultures yielded growth. These were re-streaked in order to obtain single colonies. It has been observed, that if a growth occurred on plates containing YE it would still occur, after re-streaking, on Results

a plate without YE (and vice-versa). During the last week colonies were also observed to 1 form on the Sv13 and Sv14 plates with YE using the 10 dilution. These changed with time from transparent ones to light-green in the inside indicating a pH change (bromothymol blue turns from blue color at basic pH to green around neutral and to yellow in acidic), which can be seen in Figure 3.2 b and c. Colonies from all plates and liquid cultures were chosen to be screened with a PCR reaction using symbiont-speciﬁc primers. The resulting gel is presented in Figure 3.3 a.

Table 3.1: Summary of dissections performed on all collected organisms. NA = data not collected. Starred ( )sampleswereusedfor454pyrosequencing ⇤ Sample name Organism Size [mm] Gill wet mass [mg] Comment Sampling site A Sv1 NA 40.4 Shared dissecting tools Sv2 14 20.3 Shared dissecting tools Sv3 13 36.4 Shared dissecting tools Sv4 14 18.0 Shared dissecting tools Sv5 NA 19.4 Shared dissecting tools Sv6 NA 16.9 Shared dissecting tools Sv7 18 105.0 ⇤ Solemya velum Sv8 18 38.2 ⇤ Sv9 18 47.2 ⇤ Sv10 14 38.6 ⇤ Sv11 14 48.3 ⇤ Sv12 12 28.8 ⇤ Sv13 12 21.6 Shared dissecting tools Sv14 13 17.4 Shared dissecting tools Tag1 24 33.7 Not rinsed in SWB ⇤ Tagelus plebeius Tag2 24 9.5 Not rinsed in SWB ⇤ Sampling site B Yl1 NA 23.6 Gills from two specimens Yl2 NA 17.8 ⇤ Yl3 Yoldia limatula NA 16.0 Part of gut attached Yl4 NA 10.6 Only one gill Yl5 NA 11.4 ⇤ The PCR test with the symbiont specific primers did not gave any result indicating that the obtained colony or liquid culture can be the symbiont. Nevertheless single colonies were picked again and the 16S was amplified using the 16S universal primers (see Figure 3.3 b). Samples 3, 8 and 11 from Figure 3.3 b did not give any product and thus were dropped from further analysis. The positive results were purified and send for Sanger sequencing to check what kind of organism has been isolated from the gill extracts.

12 Results

1 1 (a) Sv2 culture (b) Sv13 10 plate streak with colored (c) Sv13 10 plate zoom with a reference colonies on the colored colonies Figure 3.2: Positive results observed during the symbiont cultivation attempt

(a) Symbiont-speciﬁc PCR test on plate (b) Colony PCR using universal 16S primers for colonies and liquid cultures. Numbers Sanger sequencing. Numbers correspond to follow- 1 are following samples: 1: Sv7 DNA ex- ing samples: 1&9: Sv13 10 +YE green colony, 1 1 tract (positive control), 2: Sv13 10 2&10: Sv13 10 +YE white colony, 3&11: Sv14 1 1 1 +YE plate, 3: Sv14 10 +YE plate, 4: 10 +YE green colony, 4&12: Sv14 10 +YE 1 1 1 Sv2 10 -YE re-streak 2, 5: Sv2 10 white colony, 5&13: Sv2 10 -YE re-streak 1 1 1 +YE re-streak 1, 6: Sv2 10 -YE re- colony, 6&14: Sv2 10 +YE re-streak 1 colony, 1 1 streak 1, 7: Sv2 10 +YE liquid trans- 7&15: Sv2 10 -YE re-streak 2 colony 1 fer 1, 8: Sv2 10 -YE liquid transfer 1 1, 9: Sv2 10 -YE liquid single colony, 1 1 10: Sv13 10 -YE liquid, 11: Sv14 10 -YE liquid, 12: negative-controll Figure 3.3: Positive results observed during the symbiont cultivation attempt

13 Results

Sanger sequencing results were aligned with the most similar hits in the online Silva service and the tree was built using the CLC Main workbench. Resulting phylogenetic tree is presented in Figure 3.4.

Figure 3.4: UPGMA tree of the isolates with their most similar sequences. Isolates are named in the following fashion: gill isolate medium variation colony type where: str - streaked transparent colony, white - white milky colony and col - green colored colony

The analysis of the phylogenetic tree shows, that all of the isolates obtained from the plates can be classiﬁed in the Gammaproteobacteria phylum. Speciﬁcally, two main genus are observed - Endozoicomonas and Pseudoalteromonas.Green-coloredcolonies obtained on YE plates from Sv13 and Sv14 gill extracts are most closely related to the Endozoicomonas, while being most closely related to each other. The strakes transparent colonies and white colonies from the same plate as the green-colored ones can be considered to be closely related to Pseudoalteromonas, although here a variation can be observed. although still, the duplicate samples cluster together.

14 Results

(a) An intact book gill, EHT = 2.5 kV, Mag = (b) Gill structure, EHT = 2.5 kV, Mag = 160 61 X X

(c) A zoom out on a sliced gill fragment, EHT (d) A zoom in on section presented in c), show- = 2.5 kV, Mag = 281 X ing the symbionts in the tissue, EHT = 2.5 kV, Mag = 3720 X Figure 3.5: SEM pictures of the gill tissue and the symbionts inside of it

3.3 Scanning Electron Microscopy of the gill tissue

AtotalnumberoffourgilltissuespecimenswereinvestigatedusingaScanningElectron Microscope (SEM). Imaging enabled to investigate the structure of the gill, by localizing and imaging the ciliated and microvillar part of the gill tissue (see Figure 3.5 a-d). Images b-d were obtained by slicing the intact gills (image a) by a two-sided razor blade, re-coating and re-imaging. This way a sliced section of the inside of the gill (Figure 3.5 b) could have been obtained, where the boundary between the two regions of the gill is easily visible. In the microvillar part of the gill (image c) the symbionts were found which can be seen on image d.

15 Results

3.4 Fluorescent In-Situ Hybridization of gill tissue

The hybridization on the gill tissue and further imaging on afluorescentmicroscopeprovidedimagestolocatethesym- biont in the gill tissue. First experiment comprised of simple DAPI staining of the fixed gill tissue - see Figure 3.6. Here one can see that the large nucleus is stained, but numerous smaller positive signals can be observed. These are expected to be the symbionts living inside of the tissue. To confirm that to the extend possible, two FISH probes were used - one targeting all bacteria (to confirm symbiont is a bacterium, EUBI-III probe) and one targeting Gammaproteobacteria (to confirm that symbiont belongs to that phylum, Gam42a probe with a Bet42 competitor). In both cases the presence of the symbiont in the tissue and its belonging to these groups was Figure 3.6: DAPI stain- confirmed - see Figure 3.7 a-f. Blue color on the figures shows ing of the fixed gill tissue, the eukaryotic nuclei (from the DAPI staining) and the green 1000X magnification coloring indicates where the FISH probe annealed. Overlaid in FIgures 3.7 b, d and f are the DIC images under transmitted light to show, that bacteria signals are all inside of the tissue.

3.5 BLAST search of local databases for the symbiont

The screening of the local datasets from previous courses (2010 454 metagenomic data from a microbial mat, 2011 and 2012 454 16S amplicon data from various student projects) is summarized in Figure 3.8. Very highly similar hits (97 % identity) to the symbiont 16S were found in both of the amplicon data sets. Moreover, the partial genome 16-23S operon has also been receiving a number of hits. Apart from the 16S, the metagenomic data from 2010 has also yielded numerous hits on the 23S region. The second conﬁg of the partial genome which has received a few hits from the amplicon data set has received them in a region where no protein is annotated. However, because the data was exclusively 16S fragments, it can be stated with a high degree of probability, that there is an unannotated 16S open reading frame in that region. Summarizing, one could observe, that organisms falling to the same OTU as the symbiont can be found free-living in locations like Salt Pond water column, School St. Marsh and the sediment near the Wild harbor. Other organisms very highly similar to the symbiont can be found in various sediments (Eeel Pond, Martha’s Vineyard, Wild harbor) and microbial mats (Great and Little Sipperwisset marsh) all around the Woods Hole area.

16 Results

(a) EUBI-III probe, DAPI + Fluorescence, (b) EUBI-III probe, DAPI + Fluorescence + 400X DIC, 400X

(c) Gam42a + Bet42a competitor probe, DAPI (d) Gam42a + Bet42a competitor probe, DAPI + Fluorescence, 400X + Fluorescence + DIC, 400X

(e) Gam42a + Bet42a competitor probe, DAPI (f) Gam42a + Bet42a competitor probe, DAPI + Fluorescence, 630X + Fluorescence + DIC, 630X Figure 3.7: Merged channels pictures of the gill tissue taken on a fluorescent microscope and processed using ImageJ software. Captions describe the probe, channels and magnification used. Blue color indicates DAPI staining, Green is the specific FISH probe and grey the transmitted-light DIC images 17 Results

Figure 3.8: Graphical summarization of the BLAST study. Lines connecting parts of circle represent hits between the two databases, and their location the location of the hit. Di↵erent datasets are represented with di↵erent colors

3.6 454 gill microbiome

After preparing the gill extracts, the bacterial DNA presence was conﬁrmed by universal 16S primer pair 8F/1492R. Product was obtained for all extracts (See Figure 3.9 a) and thus a speciﬁc PCR was performed with barcodes used for 454 sequencing. In this case a 515F/907R primer pair was used producing a shorter fragment. This was again checked on a agarose gel and a product of desired size was observed for all Solemya velum samples (see Figure 3.9 b). The control samples Tag and Yl produced a band of a slightly higher size than expected (expected being around 400bp) which may be the eukaryotic sequence product.

18 Results

(a) Gill extract PCR using 8F/1492R universal (b) Gill extract PCR using 515F/907R primers barcoded 454 primers Figure 3.9: 1 % agarose gel pictures. Sample names are noted at top of well, ’N’ stands for a PCR negative control containing water

Puriﬁed products were sequenced on a 454 machine and sequences were analyzed using Qiime software. OTU clustering and further classiﬁcation using the RDP taxonomy results are summarized in Figure 3.10.

Figure 3.10: Graphical representation of the 454 data taxonomical classiﬁcation done by Qiime using RDP database

All Solemya velum gill samples have been reported to contain over 99.9 % of one organism, that could have been classiﬁed only to the class level - they belong in the Gammaproteobacteria.Interestingly,averysimilarresulthasbeenobtainedforoneofthe Tagelus plebeius clam samples, whereas the other sample has a much more diverse micro

19 Results

biome. The Yoldia limatula samples on the other hand were found to contain sequences of unknown origin. Because the extend of information provided by Qiime was insucient to conﬁrm, that the gills contain only the symbiont, additional blast check was performed, which results are presented in Table 3.2.

Table 3.2: Summary of the blast result, where 454 sequencing results were used as a database and 16S of the gill symbiont as a query Sample name Total reads 99 % identity [%] 99 % identity [%] 99 % identity [%] Sv7 22,723 45.69 99.19 99.30 Sv8 23,983 53.96 99.83 99.85 Sv9 42,164 48.34 99.58 99.63 Sv10 55,938 48.69 99.72 99.77 Sv11 51,653 46.37 98.82 98.95 Sv12 45,971 46.46 99.92 99.94 Tag1 746 35.66 93.57 93.83 Tag2 72 1.39 4.17 4.17 Yl2 2,296 0.13 0.17 0.17 Yl5 248 0.40 0.81 0.81 Total reads 244,891

The results clearly show, that the Solemya velum gill is populated speciﬁcally by the previously reported symbiont, and not by any other bacterium. Already at 98 % similarity nearly all sequenced fragments map on the published symbiont 16S sequence. What is very interesting, Tagelus plebeius sample 1 appears to also have the majority of sequences identical to the symbiont found in Solemya velum.Thesecondsampleofthisclamhasa signiﬁcantly lower percentage of hits, but one has to consider the overall low amount of obtained sequences.

20 4 Discussion

4.1 Symbiont cultures

The culturing of the symbiont attempts yielded a few colonies, which were then screened with the symbiont-specific 16S PCR and this was followed by by sequencing their 16S gene. Results from this part suggest, that the colonies that grew on the plates are not necessar- ily the symbiont (because of the lack of the specific product during PCR). However, the obtained colonies can be classified into two groups: Transparent, small colonies which were classified to cluster inside Pseudoalteromonas genus. These organism are very commonly found in seawater, sediment, sea ice, surfaces of stones, marine algae, marine invertebrates, and salted foods. They are chemotrophs possessing strictly aerobic metabolism with oxygen as a terminal electron acceptor (Bowman and McMeekin, 1995). This is in line with the growth conditions used in the study, where the symbiont cultures were kept aerobically and the chemical compounds that would be necessary for growth could have came from either the gill inoculate (growth was observed 1 from 10 cultures, where there is still organic matter from the grinder gill) or from the low amount of yeast extract added to some of the media. Inoculum was also washed in sterile sea-water, but the washing was possibly not rough enough to remove all organisms that could reside on the gill tissue. The second group are colored colonies that grew from inoculates from organisms Sv13 and Sv14 on the yeast extract plates. These organisms were found to classify very well inside of Endozoicomonas genus. These organisms were previously reported to be isolated from sponges, sea slugs, corals and starfishes. They are characterized as aerobic or fac- ultative anaerobes performing carbohydrate fermentation (Nishijima et. al.,2013).Some of the species inside of the genus have been reported to be capable to reduce nitrate to nitrite (Kurahashi and Yokota, 2007). In all studies they were isolated from gastrointesti- nal tracks, which could suggest, that during the dissection of the gills a part of the gut of Solemya velum was also removed and grinder with the gill. The other possibility would be that these organisms are found also free living in the environment, as the 454 sequencing of the gill tissue did no yield their sequences inside of the gill. Discussion

4.2 Symbiont imaging in the gill tissue

The imaging of the bacteria inside of the gill tissue was successful to the extend of confirming the presence of of the organism inside the tissue and nowhere else. Fluorescent In-Situ Hybridization was able to confirm the belonging of the symbiont to Gammapro- teobacteria and the location inside of the tissue, however in order to be absolutely sure one would have to design a specific probe targeting the symbiont 16S. Scanning Electron Microscopy proved to be a very useful technique to image the location and orientation of the cells inside of the gill tissue, however the full structure of the bacteriosomes could not be easily visualized. Based on previous studies, the cells should be oriented perpendicular to the plane of the gill (Cavanaugh et. al. 2006) whereas in this study only a few images of co-oriented cells were obtained. One of the reasons may be the time of fixation, which could have been too long. Also the method of sectioning the gill with a razor-blade may have been to invasive, destroying the sliced surface.

4.3 454 gill microbiome

The data obtained from the gill micro biome 454 sequencing confirmed in a very strong manner the presence and prevalence of the symbiont is the gill tissue. The usage of Qiime workflow did not provide however conclusive results, as the taxonomic classification of the sequences was not deep enough. Confirmation of the sequences belonging to the symbiont OTU done by blast yielded also some interesting results in regard to the control razor clam Tagelus plebeius,wherethesymbiontsequenceswerefoundinlargenumbersinoneofthe samples. Specimens of this clam were collected at the same place as Solemya velum used in this study and moreover it is reported to also burrow inside of the sediment (up to around 10cm) and use the siphon holes (Holland et.al., 1977). The fact that they both clams burrow inside of the sediment and that the sediment is highly sulfidic could indicate the possibility of the Tagelus plebeius also possessing the symbionts in its gills. Moreover, the partial genome of the symbiont (courtesy of Colleen Cavanaugh group in Harvard) suggests a free- living organism (due to large genome size and transposable elements in the genome) and thus it could be picked-up by similar organisms in the environment. Arguments against this theory would be the possibility of cross-contamination of the samples during the laboratory procedures, the low number of 454 reads in comparison to symbiont-positive samples and the lack of clear product when regular 16S PCR was performed on the gill extracts. One also has to remember, that because the symbiont is free-living (which has been also confirmed by the blast-screen of datasets obtained form local 454 surveys) it can also reside on the gill and be picked-up if the washing was not thorough enough.

22 5 Conclusions

The outcomes from this project have proven to be very interesting and insightful, giving a better understanding of the symbioses between the host and bacterium. Culturing attempts resulted in pure cultures, obtained from agar plates, of a typical marine bacterium and a symbiont, which has been associated with the gut but not with the gill. Liquid and gradient tubes did not result in any substantial results. The Fluorescent In-Situ Hybridization and Scanning Electron Microscopy provided a solid proof of the presence of the symbiont in the gill tissue and of the size and orientation of its colonies. Finally the 454 sequencing and data mining have proven the presence of organisms highly similar to the symbiont (same Operational Taxonomic Unit) in sediments and sea waters around Woods Hole area, proving the concept of symbol being a free-living organism. Moreover the 454 sequencing of the gill micro biome also confirmed the identity of the symbiont and that it is the only bacterium found inside of the gill tissue. A very interesting outcome was the finding of symbiont sequences inside of Tagelus plebeius gill tissue, however this would have to be confirmed by other techniques. Overall, the project was a very successful study which opens grounds for new research to be conducted, and provides data which can be analyzed further and in parallel with other studies.

6 Perspective

The information obtained from this study opens grounds for numerous follow-up studies. One of the two main subjects to be investigated would be the colored colonies obtained from the gill extracts on yeast-extract containing plates. Although they are closely related to gut associated symbionts, one could try to characterize them further, as they were obtain from gills. Cultivation on media without yeast extract would be a good control, to check if they utilize organic matter or are they ﬁxing carbon dioxide. Second suggestion would be to check their ability to reduce nitrate to nitrite, which was reported in literature (and because the cultivation media contains nitrate). Finally one could also try to make aculturefromthegutseparatelyandseparatelyfromthegill.Thiscouldprovidemore information on whether they are in fact obtained from gills or not. The second main subject worth pursuing would be the Tagelus plebeius clam and the possibility of it hosting the symbiont in the gill. A more broad screening of the clams would be one way to pursue this goal, as well as applying imaging techniques used in this study to the gill tissue of this clam. In case of the hybridization imaging techniques, a symbiont speciﬁc probe would be suggested, after applying simple approaches like DNA-staining just to see, if any bacterial cells can be found in the tissue.

Bibliography

Kurahashi M. and Yokota A., (2006). Endozoicomonas elysicola gen. nov., sp. nov., a gammaproteobacterium isolated from the sea slug Elysia ornata, Systematic and Applied Micro- biology 30:202-206

Holland A. F. and Dean J. M. (1977). The Biology of the Stout Razor Clam Tagelus plebeius: I. Animal-Sediment Relationships, Feeding Mechanism, and Community Biology, Chesapeake Science 18:58-66

Nishijima M., Adachi K., Katsuta A., Shizuri Y. and Yamasato K. (2013). Endozoicomonas numazuensis sp. nov., a gammaproteobacterium isolated from marine sponges, and emended description of the genus Endozoicomonas Kurahashi and Yokota 2007 Inter- national Journal of Systematic and Evolutionary Microbiology 63:709-714

Bowman J. P. and McMeekin T. A. (1995). Pseudoalteromonas description in Bergeys man- ual of systematic bacteriology, volume 2

Cavanaugh C. M., McKiness Z. P., Newton I. L. G. and Stewart F. J. (2006). Marine Chemosynthetic Symbioses, The Prokaryotes 1:475-507

Krueger D. M., Gustafson R. G. and Cavanaugh C. M. (1996). ?Vertical Transmission of Chemoautotrophic Symbionts in the Bivalve Solemya velum (Bivalvia: Protobranchia) The Biological Bulletin 190:195-202

Eisen J. A., Smith S. W. and Cavanaugh C. M. (1992). Phylogenetic Relationships of Chemoautotrophic Bacterial Symbionts of Solemya velum Say (Mollusca: Bivalvia) De- termined by 16S rRNA Gene Sequence Analysis Journal of Bacteriology 3416-3421

Madigan M. T., Martinko J. M., Stahl D. A. and Clark D. P. (2012). Brock Biology of Microorganisms, 13th Edition, Pearson Education, Inc.

7 Enclosures

7.1 Primers and probes

Name Sequence 8F 5’-AGAGTTTGATCCTGGCTCAG-3’ 1492R 5’-GGTTACCTTGTTACGACTT-3’ 515F 5’-CGTATCGCCTCCCTCGCGCCATCAG NNNNNNNNN GA GTGYCAGCMGCCGCGGTAA-3’ 907R 5’-CTATGCGCCTTGCCAGCCCGCTCAGkGG CCGYCAATTCMTTTRAGTTT-3’k k 120F 5’-ACCTAGTAGTGGGGGACAACTACCG-3’k k 1216R 5’-CGAATCTGAACGCGAGACCCGT-3’ EUBI-III 5’-GCWGCCWCCCGTAGGWGT-3’ Gam42a 5’-GCCTTCCCACATCGTTT-3’ Bet42a 5’-GCCTTCCCACTTCGTTT-3’

7.2 Wizard PCR Preps DNA Puriﬁcation System

1. Aliquote 100 µlofDirectPuriﬁcationBu↵erintoa1.5mlEppendorftubeandadd 30-300 µl of the PCR product 2. Vortex brieﬂy to mix and add 1 ml of Resin 3. Vortex 3 times over 1 minute period, place the mini column on the tip of syringe barrel and place the whole assembly into a vacuum manifold 4. Pipet the DNA/Resin mix to the syringe barrel and apply vacuum to draw the DNA/Resin into the mini column 5. Add 2 ml of 80 % isopropanol to syringe barrel and apply vacuum to wash the column. Dry the resin by continuing to draw the vacuum for 30 seconds 6. Remove the syringe barrel and transfer the mini column to a new 1.5 tube 7. Centrifuge the mini column at 10,000 g for 2 minutes to remove isopropanol 8. Transfer the mini column to a new 1.5 ml Eppendorf tube and apply 50 µlof Nuclease-free water or TE bu↵er to the mini column and wait for 1 minute 9. Centrifuge the mini column at 10,000 g for 30 seconds and then remove and discard the mini column

Enclosures

7.3 Mo-Bio fast soil DNA extraction kit

1. To the PowerBead Tubes provided, 0.25 grams of soil sample 2. Gently vortex to mix 3. Check Solution C1. If Solution C1 is precipitated, heat solution to 60 °C until dissolved before use 4. Add 60 µl of Solution C1 and invert several times or vortex briefly 5. Secure PowerBead Tubes horizontally using the MO BIO Vortex Adapter tube holder for the vortex 6. Make sure the PowerBead Tubes rotate freely in your centrifuge without rubbing. Centrifuge tubes at 10,000 g for 30 seconds at room temperature 7. Transfer the supernatant to a clean 2 ml Collection Tube (provided) 8. Add 250 µlofSolutionC2andvortexfor5seconds.Incubateat4°Cfor5minutes 9. Centrifuge the tubes at room temperature for 1 minute at 10,000 g 10. Avoiding the pellet, transfer up to, but no more than, 600 µl of supernatant to a clean 2 ml Collection Tube (provided) 11. Add 200 µlofSolutionC3andvortexbriefly.Incubateat4°Cfor5minutes 12. Centrifuge the tubes at room temperature for 1 minute at 10,000 g 13. Avoiding the pellet, transfer up to, but no more than, 750 µlofsupernatantintoa clean 2 ml Collection Tube (provided) 14. Shake to mix Solution C4 before use. Add 1200 µlofSolutionC4tothesupernatant and vortex for 5 seconds 15. Load approximately 675 µl onto a Spin Filter and centrifuge at 10,000 g for 1 minute at room temperature. Discard the flow through and add an additional 675 µlof supernatant to the Spin Filter and centrifuge at 10,000 g for 1 minute at room temperature. Load the remaining supernatant onto the Spin Filter and centrifuge at 10,000 g for 1 minute at room temperature 16. Add 500 µl of Solution C5 and centrifuge at room temperature for 30 seconds at 10,000 g 17. Discard the flow through 18. Centrifuge again at room temperature for 1 minute at 10,000 g 19. Carefully place spin filter in a clean 2 ml Collection Tube (provided). Avoid splashing any Solution C5 onto the Spin Filter 20. Add 100 µl of Solution C6 to the centre of the white filter membrane. Alternatively, sterile DNA-Free PCR Grade Water may be used for elution from the silica Spin Filter membrane at this step 21. Centrifuge at room temperature for 30 seconds at 10,000 g 22. Discard the Spin Filter

Enclosures

7.4 Media compositions

7.4.1 Sea Water Base

Component Final Concentration [mM] NaCl 342.20

MgCl26H2O14.80 CaCl22H2O1.00 KCl 6.71

7.4.2 Thiosulfate medium

Component Amount Before autoclaving

NaSO4 30 mM NH4Cl100X 1ml/l K2HPO4 100X 0.1 ml/l SWB up to 1l For solid medium Bromothymol blue 0.02 g/l Agar 15 g/l After autoclaving

Na2S2O3 5mM NaHCO3 10 mM MOPS bu↵er pH 7.2 10 mM Vitamin solution 1000X 1ml/l Trace element solution 1000X 1ml/l