Chemosynthetic symbioses model based on Solemya velum endosymbiont

Mini-project reportMicrobial 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

Supervisor:Colleen Cavanaugh, Ph.D.

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 finalreport represents a description of the project carried out during the second part of thecourse. It consists of 7 chapters, starting with a theoretical background introducing thereader to the topic of science of this study. The introduction is followed with descriptionof materials, methods and obtained results with a discussion. Finally a summarization ofwork is presented in conclusions followed by future perspectives. The report is finishedwith a bibliography and enclosures. References are made according to Harvard referringsystem, and are presented in the references section at the end of this report. In the textall references are marked as follows: (authors surname, year of publication). I would liketo thank the course directors Steve and Dan, all of the Teacher Assistants and coursemembers for their help and support.

Piotr Starnawski

Contents

1 Theoretical background 1

1.1 Chemosynthetic Symbioses . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.2 Solemya velum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21.3 Endosymbiont characterization . . . . . . . . . . . . . . . . . . . . . . . . 2

2 Materials and methods 3

2.1 Specimens collection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32.2 Symbiont cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32.3 Scanning Electron Microscopy of the gill tissue . . . . . . . . . . . . . . . . 62.4 Fluorescent In-Situ Hybridization of gill tissue . . . . . . . . . . . . . . . . 72.5 BLAST search of local databases for the symbiont . . . . . . . . . . . . . . 82.6 454 gill microbiome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

3 Results 11

3.1 Specimens collection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113.2 Symbiont cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113.3 Scanning Electron Microscopy of the gill tissue . . . . . . . . . . . . . . . . 153.4 Fluorescent In-Situ Hybridization of gill tissue . . . . . . . . . . . . . . . . 163.5 BLAST search of local databases for the symbiont . . . . . . . . . . . . . . 163.6 454 gill microbiome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

4 Discussion 21

4.1 Symbiont cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214.2 Symbiont imaging in the gill tissue . . . . . . . . . . . . . . . . . . . . . . 224.3 454 gill microbiome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

5 Conclusions 23

6 Perspective 25

Bibliography 27

7 Enclosures 297.1 Primers and probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 297.2 Wizard PCR Preps DNA Purification System . . . . . . . . . . . . . . . . 297.3 Mo-Bio fast soil DNA extraction kit . . . . . . . . . . . . . . . . . . . . . . 317.4 Media compositions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

7.4.1 Sea Water Base . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 337.4.2 Thiosulfate medium . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

Abstract

The aim of this project was first of all to isolate and attempt to cultivate the intracellularsulfide-oxidizing symbiont of Solemya velum. This was done by dissecting the gills andcultivating the extract in solid and liquid media in dilution series. Cultures were monitoredwith symbiont-specific PCR for the growth of desired organism and all obtained isolateshad their 16S gene sequences. Further work aimed at characterizing the overall bacterialcommunity found in the bivalves gills using 454 pyrosequencing. DNA form gills from 6specimens was prepared and additional controls of 2 other clams living in the area weresequenced and the resulting community was analyzed. In order to better characterizethe symbiosis, Fluorescent In-Situ Hybridization and Scanning Electron microscopy andtechniques were applied for visualization of the symbiont and possible other bacteria livingon the gill tissue. All this information was supplemented by analysis of existing data onthis symbiosis model which comprised of symbiont partial genome and 454 datasets fromsediment known to be occupied by Solemya velum.

1Theoretical background

1.1 Chemosynthetic Symbioses

The word ”symbiosis” originates from greek and means ”living together” and is used todescribe two di↵erent organisms which live together, regardless if the outcomes are positiveor negative. Chemosynthesis refers to the process of energy conservation using chemicals(Madigan et. al. 2012). This report focuses on symbioses between bacteria and marineinvertebrate, where the host provides the conditions for the bacterium to grow and providefixed 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 environmentsare methanotrophy and chemoautotrophy - see Table 1.1.

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

Host (genus or class) Common name Habitat Symbiont typePorifera (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 inorganiccompounds for energy and use it to fix CO

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for biomass, like in case of chemolitotrophsusing sulfide as the receded compound or (ii) the ability to use singe carbon compounds asboth 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 fortheir 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 435and 500 million years ago, and its members maintain a primitive form even today. Theclam digs very characteristic Y-shaped burrow (Figure 1.1 a), to place itself lower in thesediment in the sulfide-rich region, while still having access to fresh, oxygenated water.The relatively recent discovery of symbionts provided the information about the reasonfor such behavior. The bacterial endosymbionts supply the animal nutrition derived fromthe chemoautotrophic fixation of CO

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fueled by the oxidation of reduced inorganic sulfurcompounds 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 (FromStanley 1970)

(b) Transverse section of gill filamentsof Solemya borealis, showing intracellu-lar rod-shaped bacteria (arrows, rectan-gle) b: bacteriocyte nucleus; c: ciliatedcell 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 bacterialsymbionts to new generations, and that bacteria derived from this seed population colonizethe gills as filaments are formed in juvenile clams. The functional role of the symbiontsin young Solemya velum hosts remains unknown, however. The symbionts are probablynot metabolically active before and just after spawning, but they colonize the gill budsand appear to be actively growing while still within the larval egg capsule (Krueger et. al.1996).

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2Materials 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 Naushonisland (Figure 2.1) in two distinct sampling sites (Figure 2.2). Sediment was collected andsieved for Solemya velum. Simultaneously other clams were collected to serve as a controland sediment was collected for keeping the animals in the lab. Organisms were placed ina plastic container with salt water and transferred to lab. Dissections were done immedi-ately after collection (1-2h) and remaining clams were placed in sediment containers keptin continuous fresh sea water flow aquarium.

Dissections of organisms were performed using sterile forceps and scissors for eachseparate organism. After separating the gill from the rest of the body it was washed inautoclaved 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 °C and used later forDNA extractions. For the cultivation purposes the gills were not frozen but homogenizedimmediately.

2.2 Symbiont cultures

In order to try to obtain an isolate of the gill symbiont, numerous liquid and plate cul-tures 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 Eppendorftube with 100 µl of the 80 % SWB. Homogenization was done by hand using a plastictissue homogenizer for around 2 minutes. The volume of homogenate was then adjustedto 1 ml with the same solution and used for incubations according to Figure 2.3. A totalnumber of 5 di↵erent gills were used for starting these cultures. All tubes and plates wereheld in dark (cardboard box for the plates and o�ce 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

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Materials and methods

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

The monitoring of cultures was done by the use of symbiont-specific primers providedby Colleen Cavanaugh’s lab in Harvard (see Enclosures for sequences). The monitoringwas performed by PCR method where the colony or 1 µl of liquid culture was added tothe 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 thecolonies/liquid cultures

Volume [µl] Compound6.25 Promega Go-Taq Green 2X Mix1.00 Forward primer 120F (15 pmol)1.00 Reverse primer 1612R (15 pmol)3.25 Nuclease-free water11.50 Total volume per sample

The product was then ran on a 1 % agarose gel to check for a product of around 1.5kbp. For a positive control, purified DNA from gill extracts from Solemya velum usedfor 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

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Materials and methods

Sanger sequencing to establish the identity of the colony. This was done using the universal8F/1492R primer pair (see Enclosures for sequence) according to the mix presented in Table2.2

Table 2.2: PCR mix used to check for bacterial 16S product from gill extracts

Volume [µl] Compound12.5 Promega Go-Taq Green 2X Mix2.0 Forward primer Univ8F (15 pmol)2.0 Reverse primer Univ1492R (15 pmol)6.5 Nuclease-free water23.0 Total volume per sample

Similarly the product was checked on 1 % agarose gel. Purification of the product wasdone using the PromegaWizard PCR Preps DNA Purification System according to protocol(see Enclosures). Purified colony 16S products were then quantified on a NanoDrop systemand 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 cutunder the dissecting microscope. After they were opened, two were placed in 80 % SWB4 % formaldehyde fixative and two in 1X Phosphate Bu↵ered Saline (PBS), 4 % formalde-hyde, 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 withtheir 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 piecesof the gill were placed (under submersion) in plastic containers and remained there for therest 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 % EtOH3) 3 times 10 minutes in 50 % EtOH4) 3 times 10 minutes in 75 % EtOH5) 3 times 10 minutes in 100 % EtOH

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

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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-SituHybridization (FISH) was applied. A specimen of Solemya velum was dissected under adissecting scope and the gill tissue was separated from the rest of the body. Then it wasplaced in 4 % formaldehyde solution in 80 % SWB for 1 h 30 min. After that a thoroughwas with 80 % SWB was performed to remove all formaldehyde and such fixed gill wasstored at 4 °C. The sample for FISH was prepared in a 10-well glass slide. Gill was dissectedfurther until single ”pages” of the ”book gill” tissue could be seen. These were collectedby forceps and placed in a water drop inside of the slide well. A total number of 10 tissuepieces were isolated in such way. Slide was then dried at 46 °C to firmly place the gilltissue inside the well. After that a dehydration step was performed using a series of 3ethanol baths for the slide: 50 % EtOH, 80 % EtOH and 96 % EtOH, in all cases 2 minutewash each. After slide has dried o↵ the hybridization was performed by dropping 15 µl ofbu↵er-probe solution to each well. The solution was prepared by mixing the hybridizationbu↵er (see Table 2.4) and the desired probe (EubI-III and Gam42a + Bac42 a competitorprobe in case of this study) in 300:1 ratio (300 µl of bu↵er and 1 µl of 50 ng/µl probe).

Table 2.3: Hybridization and Washing bu↵ers composition used for the FISH of the gilltissue

Hybridization bu↵er volume [µl] Washing bu↵er volume [µl] Compound700 - Formaldehyde- 500 0.5 M EDTA360 700 5 M NaCl40 1000 1 M Tris-HCl1 25 20 % SDS900 fill-up to 50 ml MQ water

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

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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 befound in the free-living state a search of existing datasets was performed to find, if sequencesbelonging to the symbiont were previously found in the area surrounding Woods Hole. 454datasets from 2010, 2011 and 2012 were selected for that, where 2010 was a metagenome of aLittle Sipperwisset Salt Marsh microbial mat and the remaining two were large 16S surveysof various environments. Using the blast+ package ”makeblastdb” command databasesfor 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’slab in Harvard, of the symbiont was also tested (68 contigs genome, unpublished). Thesewere are checked against the database on the nucleotide level (”blastn” command) and theresults were summarized using the circos imaging software package. For the 16S sequencea percentage-identity cuto↵ of 97 % was used (in order to look for the same operationaltaxonomic units) and for the partial genome 93 % percentage-identity and 1e-100 e-valuecuto↵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, two Tagelus plebeius

and two Yoldia limatula were chosen (the latter two serving as controls not containing thesymbiont). Frozen gills were thawed on ice and then DNA was extracted using the Mo-BioPowerSoil DNA Isolation Kit according to protocol (See Enclosures). The only change tothe standard flow was shortening the homogenization step to 1 min due to using a morethorough homogenizer. Extracted DNA was then checked by performing a PCR reactionusing regular 8F and 1492R (see Enclosures for sequences) primers to see if a product isobtained. PCR mix is presented in Table 2.2. For the reaction, 2 µl of gill extract was used.30 cycles of PCR were performed and 5 µl of the product was run on a 1 % agarose gelwith a 1 kb DNA ladder. After checking the result the DNA extract was used to performa new PCR with barcoded primers. For each sample, one distinct barcode was selected.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] Compound15 Phusion 2x HF MasterMix2.4 DMSO, 100 %0.6 Forward primer 515F (6.25 uM)2.4 Reverse primer 907R (25 uM)4.6 Nuclease-free water25.0 Total volume per sample

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Materials and methods

To each tube 5 µl of gill extract was added and the PCR was run for 30 cycles. Obtainedproduct was checked on a 1 % agarose gel with a 1 kbp marker. Barcoded productswere pooled, purified and send for sequencing on a 454 machine. Sequences were thenanalyzed using Qiime software according to the standard 454 operating procedure. Sampleswere separated from the whole plate according to the barcode using the split libraries.pycommand. Sequences of quality scores below 25 were discarded, and analysis was performedon sequences between 400 and 600 bp long. Next step comprised of starting the workflow forOperational Taxonomic Units (OTU) picking with pick de novo otus.py command whichalso assigned RDP taxonomy to picked OTUs. Finally, the results were summarized usingsummarize taxa through plots.py command. To check, if the 454 data contained the gillsymbiont sequences an additional analysis was done, where a blast database was preparedfrom the 454 data and the 16S gill symbiont sequence (GenBank: M90415.1) was used asa query against it. Sequences were filtered for 99 %, 98 % and 97 % identity to the queryto look for the exact same sequence or one belonging to the same OTU.

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3Results

3.1 Specimens collection

After the collection of organisms they were stored in 1 l plastic containers filled withsulfidic sediment collected at the same place where organisms were collected.

Figure 3.1: Container usedfor storing the organisms,kept in an aquarium withconstant seawater flow

These containers were covered with a net to avoid organ-isms from escaping and kept in an aquarium under constantseawater flow (See Figure 3.1). Dissections were preformed inorder to separate gills that contain the symbiont bacterium.Organism size was noted and gills were washed in autoclaved80% SWB and weighted on a laboratory balance. Some of theextracts were used for starting cultures and some for DNA ex-tractions for 454 pyrosequencing - the summary of dissectionsand which samples were used for DNA extracts is presentedin Table 3.1. Other specimens were used for starting cultureswith them in order to cultivate the symbiont. All extractswere kept at -20°C after flash-freezing in liquid CO

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3.2 Symbiont cultures

During the three-week culturing of solid, liquid and gradient cultures of the gill extractvery little growth has been observed. The most prominent changes in the medium havebeen served for the gill extract from organism Sv2 where the medium became very milkyin comparison to the starting, transparent solution (Figure 3.4). This was for the firstdilution of the series, 10�1. This dilution was used for inoculation of two new tubes (withand 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 nosignificant darkening of the liquid cultures was observed, however the plate cultures yieldedgrowth. 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 toform on the Sv13 and Sv14 plates with YE using the 10�1 dilution. These changed with timefrom transparent ones to light-green in the inside indicating a pH change (bromothymolblue 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 werechosen to be screened with a PCR reaction using symbiont-specific primers. The resultinggel is presented in Figure 3.3 a.

Table 3.1: Summary of dissections performed on all collected organisms. NA = data notcollected. Starred (⇤) samples were used for 454 pyrosequencing

Sample name Organism Size [mm] Gill wet mass [mg] CommentSampling site ASv1

Solemya velum

NA 40.4 Shared dissecting toolsSv2 14 20.3 Shared dissecting toolsSv3 13 36.4 Shared dissecting toolsSv4 14 18.0 Shared dissecting toolsSv5 NA 19.4 Shared dissecting toolsSv6 NA 16.9 Shared dissecting toolsSv7 ⇤ 18 105.0Sv8 ⇤ 18 38.2Sv9 ⇤ 18 47.2Sv10 ⇤ 14 38.6Sv11 ⇤ 14 48.3Sv12 ⇤ 12 28.8Sv13 12 21.6 Shared dissecting toolsSv14 13 17.4 Shared dissecting toolsTag1 ⇤

Tagelus plebeius

24 33.7 Not rinsed in SWBTag2 ⇤ 24 9.5 Not rinsed in SWBSampling site BYl1

Yoldia limatula

NA 23.6 Gills from two specimensYl2 ⇤ NA 17.8Yl3 NA 16.0 Part of gut attachedYl4 NA 10.6 Only one gillYl5 ⇤ NA 11.4

The PCR test with the symbiont specific primers did not gave any result indicatingthat the obtained colony or liquid culture can be the symbiont. Nevertheless single colonieswere picked again and the 16S was amplified using the 16S universal primers (see Figure 3.3b). Samples 3, 8 and 11 from Figure 3.3 b did not give any product and thus were droppedfrom further analysis. The positive results were purified and send for Sanger sequencingto check what kind of organism has been isolated from the gill extracts.

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Results

(a) Sv2 culturewith a reference

(b) Sv13 10�1 plate streak with coloredcolonies

(c) Sv13 10�1 plate zoomon the colored colonies

Figure 3.2: Positive results observed during the symbiont cultivation attempt

(a) Symbiont-specific PCR test on platecolonies and liquid cultures. Numbersare following samples: 1: Sv7 DNA ex-tract (positive control), 2: Sv13 10�1

+YE plate, 3: Sv14 10�1 +YE plate, 4:Sv2 10�1 -YE re-streak 2, 5: Sv2 10�1

+YE re-streak 1, 6: Sv2 10�1 -YE re-streak 1, 7: Sv2 10�1 +YE liquid trans-fer 1, 8: Sv2 10�1 -YE liquid transfer1, 9: Sv2 10�1 -YE liquid single colony,10: Sv13 10�1 -YE liquid, 11: Sv14 10�1

-YE liquid, 12: negative-controll

(b) Colony PCR using universal 16S primers forSanger sequencing. Numbers correspond to follow-ing samples: 1&9: Sv13 10�1 +YE green colony,2&10: Sv13 10�1 +YE white colony, 3&11: Sv1410�1 +YE green colony, 4&12: Sv14 10�1 +YEwhite colony, 5&13: Sv2 10�1 -YE re-streak 1colony, 6&14: Sv2 10�1 +YE re-streak 1 colony,7&15: Sv2 10�1 -YE re-streak 2 colony

Figure 3.3: Positive results observed during the symbiont cultivation attempt

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Results

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

Figure 3.4: UPGMA tree of the isolates with their most similar sequences. Isolates arenamed 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 fromthe plates can be classified in the Gammaproteobacteria phylum. Specifically, two maingenus are observed - Endozoicomonas and Pseudoalteromonas. Green-colored coloniesobtained on YE plates from Sv13 and Sv14 gill extracts are most closely related to theEndozoicomonas, while being most closely related to each other. The strakes transparentcolonies and white colonies from the same plate as the green-colored ones can be consideredto be closely related to Pseudoalteromonas, although here a variation can be observed.although still, the duplicate samples cluster together.

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Results

(a) An intact book gill, EHT = 2.5 kV, Mag =61 X

(b) Gill structure, EHT = 2.5 kV, Mag = 160X

(c) A zoom out on a sliced gill fragment, EHT= 2.5 kV, Mag = 281 X

(d) A zoom in on section presented in c), show-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

A total number of four gill tissue specimens were investigated using a Scanning ElectronMicroscope (SEM). Imaging enabled to investigate the structure of the gill, by localizingand imaging the ciliated and microvillar part of the gill tissue (see Figure 3.5 a-d). Imagesb-d were obtained by slicing the intact gills (image a) by a two-sided razor blade, re-coatingand re-imaging. This way a sliced section of the inside of the gill (Figure 3.5 b) could havebeen obtained, where the boundary between the two regions of the gill is easily visible. Inthe microvillar part of the gill (image c) the symbionts were found which can be seen onimage d.

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Results

3.4 Fluorescent In-Situ Hybridization of gill tissue

Figure 3.6: DAPI stain-ing of the fixed gill tissue,1000X magnification

The hybridization on the gill tissue and further imaging ona fluorescent microscope provided images to locate the sym-biont in the gill tissue. First experiment comprised of simpleDAPI staining of the fixed gill tissue - see Figure 3.6. Hereone can see that the large nucleus is stained, but numeroussmaller positive signals can be observed. These are expectedto be the symbionts living inside of the tissue. To confirmthat to the extend possible, two FISH probes were used - onetargeting all bacteria (to confirm symbiont is a bacterium,EUBI-III probe) and one targeting Gammaproteobacteria (toconfirm that symbiont belongs to that phylum, Gam42a probewith a Bet42 competitor). In both cases the presence of thesymbiont in the tissue and its belonging to these groups wasconfirmed - see Figure 3.7 a-f. Blue color on the figures showsthe eukaryotic nuclei (from the DAPI staining) and the greencoloring 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 bacteriasignals 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 datafrom 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 16Swere found in both of the amplicon data sets. Moreover, the partial genome 16-23S operonhas also been receiving a number of hits. Apart from the 16S, the metagenomic data from2010 has also yielded numerous hits on the 23S region. The second config of the partialgenome which has received a few hits from the amplicon data set has received them ina region where no protein is annotated. However, because the data was exclusively 16Sfragments, it can be stated with a high degree of probability, that there is an unannotated16S open reading frame in that region. Summarizing, one could observe, that organismsfalling to the same OTU as the symbiont can be found free-living in locations like SaltPond water column, School St. Marsh and the sediment near the Wild harbor. Otherorganisms very highly similar to the symbiont can be found in various sediments (EeelPond, Martha’s Vineyard, Wild harbor) and microbial mats (Great and Little Sipperwissetmarsh) all around the Woods Hole area.

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Results

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

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

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

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

(e) Gam42a + Bet42a competitor probe, DAPI+ Fluorescence, 630X

(f) Gam42a + Bet42a competitor probe, DAPI+ Fluorescence + DIC, 630X

Figure 3.7: Merged channels pictures of the gill tissue taken on a fluorescent microscope andprocessed using ImageJ software. Captions describe the probe, channels and magnificationused. Blue color indicates DAPI staining, Green is the specific FISH probe and grey thetransmitted-light DIC images

17

Results

Figure 3.8: Graphical summarization of the BLAST study. Lines connecting parts ofcircle 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 universal16S primer pair 8F/1492R. Product was obtained for all extracts (See Figure 3.9 a) andthus a specific PCR was performed with barcodes used for 454 sequencing. In this case a515F/907R primer pair was used producing a shorter fragment. This was again checkedon 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 highersize than expected (expected being around 400bp) which may be the eukaryotic sequenceproduct.

18

Results

(a) Gill extract PCR using 8F/1492R universalprimers

(b) Gill extract PCR using 515F/907Rbarcoded 454 primers

Figure 3.9: 1 % agarose gel pictures. Sample names are noted at top of well, ’N’ standsfor a PCR negative control containing water

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

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

All Solemya velum gill samples have been reported to contain over 99.9 % of oneorganism, that could have been classified only to the class level - they belong in theGammaproteobacteria. Interestingly, a very similar result has been obtained for one of theTagelus 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 sequencesof unknown origin. Because the extend of information provided by Qiime was insu�cientto 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 adatabase 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 99.30Sv8 23,983 53.96 99.83 99.85Sv9 42,164 48.34 99.58 99.63Sv10 55,938 48.69 99.72 99.77Sv11 51,653 46.37 98.82 98.95Sv12 45,971 46.46 99.92 99.94Tag1 746 35.66 93.57 93.83Tag2 72 1.39 4.17 4.17Yl2 2,296 0.13 0.17 0.17Yl5 248 0.40 0.81 0.81Total reads 244,891

The results clearly show, that the Solemya velum gill is populated specifically by thepreviously reported symbiont, and not by any other bacterium. Already at 98 % similaritynearly all sequenced fragments map on the published symbiont 16S sequence. What isvery interesting, Tagelus plebeius sample 1 appears to also have the majority of sequencesidentical to the symbiont found in Solemya velum. The second sample of this clam has asignificantly lower percentage of hits, but one has to consider the overall low amount ofobtained sequences.

20

4Discussion

4.1 Symbiont cultures

The culturing of the symbiont attempts yielded a few colonies, which were then screenedwith 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, theobtained 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 ofstones, marine algae, marine invertebrates, and salted foods. They are chemotrophs pos-sessing strictly aerobic metabolism with oxygen as a terminal electron acceptor (Bowmanand McMeekin, 1995). This is in line with the growth conditions used in the study, wherethe symbiont cultures were kept aerobically and the chemical compounds that would benecessary for growth could have came from either the gill inoculate (growth was observedfrom 10�1 cultures, where there is still organic matter from the grinder gill) or from thelow amount of yeast extract added to some of the media. Inoculum was also washed insterile sea-water, but the washing was possibly not rough enough to remove all organismsthat could reside on the gill tissue.

The second group are colored colonies that grew from inoculates from organisms Sv13and Sv14 on the yeast extract plates. These organisms were found to classify very wellinside of Endozoicomonas genus. These organisms were previously reported to be isolatedfrom sponges, sea slugs, corals and starfishes. They are characterized as aerobic or fac-ultative anaerobes performing carbohydrate fermentation (Nishijima et. al., 2013). Someof the species inside of the genus have been reported to be capable to reduce nitrate tonitrite (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 ofSolemya velum was also removed and grinder with the gill. The other possibility would bethat these organisms are found also free living in the environment, as the 454 sequencingof 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 ofconfirming the presence of of the organism inside the tissue and nowhere else. FluorescentIn-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 sureone would have to design a specific probe targeting the symbiont 16S. Scanning ElectronMicroscopy proved to be a very useful technique to image the location and orientation ofthe cells inside of the gill tissue, however the full structure of the bacteriosomes could notbe easily visualized. Based on previous studies, the cells should be oriented perpendicularto the plane of the gill (Cavanaugh et. al. 2006) whereas in this study only a few imagesof co-oriented cells were obtained. One of the reasons may be the time of fixation, whichcould have been too long. Also the method of sectioning the gill with a razor-blade mayhave 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 strongmanner the presence and prevalence of the symbiont is the gill tissue. The usage of Qiimeworkflow did not provide however conclusive results, as the taxonomic classification of thesequences was not deep enough. Confirmation of the sequences belonging to the symbiontOTU done by blast yielded also some interesting results in regard to the control razor clamTagelus plebeius, where the symbiont sequences were found in large numbers in one of thesamples. Specimens of this clam were collected at the same place as Solemya velum used inthis study and moreover it is reported to also burrow inside of the sediment (up to around10cm) and use the siphon holes (Holland et.al., 1977). The fact that they both clams burrowinside of the sediment and that the sediment is highly sulfidic could indicate the possibilityof the Tagelus plebeius also possessing the symbionts in its gills. Moreover, the partialgenome 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) andthus it could be picked-up by similar organisms in the environment. Arguments against thistheory would be the possibility of cross-contamination of the samples during the laboratoryprocedures, the low number of 454 reads in comparison to symbiont-positive samples andthe lack of clear product when regular 16S PCR was performed on the gill extracts. One alsohas to remember, that because the symbiont is free-living (which has been also confirmedby the blast-screen of datasets obtained form local 454 surveys) it can also reside on thegill and be picked-up if the washing was not thorough enough.

22

5Conclusions

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. Culturingattempts resulted in pure cultures, obtained from agar plates, of a typical marine bacteriumand a symbiont, which has been associated with the gut but not with the gill. Liquidand gradient tubes did not result in any substantial results. The Fluorescent In-SituHybridization and Scanning Electron Microscopy provided a solid proof of the presenceof the symbiont in the gill tissue and of the size and orientation of its colonies. Finallythe 454 sequencing and data mining have proven the presence of organisms highly similarto the symbiont (same Operational Taxonomic Unit) in sediments and sea waters aroundWoods Hole area, proving the concept of symbol being a free-living organism. Moreoverthe 454 sequencing of the gill micro biome also confirmed the identity of the symbiont andthat it is the only bacterium found inside of the gill tissue. A very interesting outcome wasthe finding of symbiont sequences inside of Tagelus plebeius gill tissue, however this wouldhave to be confirmed by other techniques.

Overall, the project was a very successful study which opens grounds for new researchto be conducted, and provides data which can be analyzed further and in parallel withother studies.

6Perspective

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 obtainedfrom the gill extracts on yeast-extract containing plates. Although they are closely relatedto gut associated symbionts, one could try to characterize them further, as they wereobtain from gills. Cultivation on media without yeast extract would be a good control, tocheck if they utilize organic matter or are they fixing carbon dioxide. Second suggestionwould 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 makea culture from the gut separately and separately from the gill. This could provide moreinformation on whether they are in fact obtained from gills or not. The second mainsubject worth pursuing would be the Tagelus plebeius clam and the possibility of it hostingthe symbiont in the gill. A more broad screening of the clams would be one way to pursuethis goal, as well as applying imaging techniques used in this study to the gill tissue of thisclam. In case of the hybridization imaging techniques, a symbiont specific probe would besuggested, after applying simple approaches like DNA-staining just to see, if any bacterialcells can be found in the tissue.

Bibliography

Kurahashi M. and Yokota A., (2006). Endozoicomonas elysicola gen. nov., sp. nov., a gamma-proteobacterium 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 ple-

beius : 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, andemended 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). MarineChemosynthetic Symbioses, The Prokaryotes 1:475-507

Krueger D. M., Gustafson R. G. and Cavanaugh C. M. (1996). ?Vertical Transmission ofChemoautotrophic 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 ofChemoautotrophic 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 ofMicroorganisms, 13th Edition, Pearson Education, Inc.

7Enclosures

7.1 Primers and probes

Name Sequence8F 5’-AGAGTTTGATCCTGGCTCAG-3’

1492R 5’-GGTTACCTTGTTACGACTT-3’

515F 5’-CGTATCGCCTCCCTCGCGCCATCAGkNNNNNNNNNkGAkGTGYCAGCMGCCGCGGTAA-3’

907R 5’-CTATGCGCCTTGCCAGCCCGCTCAGkGGkCCGYCAATTCMTTTRAGTTT-3’

120F 5’-ACCTAGTAGTGGGGGACAACTACCG-3’

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 µl of Direct Purification Bu↵er into a 1.5 ml Eppendorf tube and add30-300 µl of the PCR product

2. Vortex briefly to mix and add 1 ml of Resin3. 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 manifold4. Pipet the DNA/Resin mix to the syringe barrel and apply vacuum to draw the

DNA/Resin into the mini column5. 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 seconds6. Remove the syringe barrel and transfer the mini column to a new 1.5 tube7. Centrifuge the mini column at 10,000 g for 2 minutes to remove isopropanol8. Transfer the mini column to a new 1.5 ml Eppendorf tube and apply 50 µl of

Nuclease-free water or TE bu↵er to the mini column and wait for 1 minute9. 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 sample2. Gently vortex to mix3. Check Solution C1. If Solution C1 is precipitated, heat solution to 60 °C until

dissolved before use4. Add 60 µl of Solution C1 and invert several times or vortex briefly5. Secure PowerBead Tubes horizontally using the MO BIO Vortex Adapter tube holder

for the vortex6. Make sure the PowerBead Tubes rotate freely in your centrifuge without rubbing.

Centrifuge tubes at 10,000 g for 30 seconds at room temperature7. Transfer the supernatant to a clean 2 ml Collection Tube (provided)8. Add 250 µl of Solution C2 and vortex for 5 seconds. Incubate at 4 °C for 5 minutes9. Centrifuge the tubes at room temperature for 1 minute at 10,000 g10. 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 µl of Solution C3 and vortex briefly. Incubate at 4 °C for 5 minutes12. Centrifuge the tubes at room temperature for 1 minute at 10,000 g13. Avoiding the pellet, transfer up to, but no more than, 750 µl of supernatant into a

clean 2 ml Collection Tube (provided)14. Shake to mix Solution C4 before use. Add 1200 µl of Solution C4 to the supernatant

and vortex for 5 seconds15. 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 µl ofsupernatant 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 minuteat room temperature

16. Add 500 µl of Solution C5 and centrifuge at room temperature for 30 seconds at10,000 g

17. Discard the flow through18. Centrifuge again at room temperature for 1 minute at 10,000 g19. Carefully place spin filter in a clean 2 ml Collection Tube (provided). Avoid

splashing any Solution C5 onto the Spin Filter20. 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 Filtermembrane at this step

21. Centrifuge at room temperature for 30 seconds at 10,000 g22. Discard the Spin Filter

31

Enclosures

7.4 Media compositions

7.4.1 Sea Water Base

Component Final Concentration [mM]NaCl 342.20MgCl

2

6H2

O 14.80CaCl

2

2H2

O 1.00KCl 6.71

7.4.2 Thiosulfate medium

Component AmountBefore autoclaving

NaSO4

30 mMNH

4

Cl100X 1ml/lK

2

HPO4

100X 0.1 ml/lSWB up to 1l

For solid mediumBromothymol blue 0.02 g/lAgar 15 g/l

After autoclavingNa

2

S2

O3

5 mMNaHCO

3

10 mMMOPS bu↵er pH 7.2 10 mMVitamin solution 1000X 1ml/lTrace element solution 1000X 1ml/l

33