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 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 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 bac- teria (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 intracellu- lar 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 immedi- ately 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 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 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 office 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 ho- mogenized 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.