Sea Squirt Symbionts! Or What I Did on My Summer Vacation… Leah Blasiak 2011 Microbial Diversity Course
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Sea Squirt Symbionts! Or what I did on my summer vacation… Leah Blasiak 2011 Microbial Diversity Course Abstract Microbial symbionts of tunicates (sea squirts) have been recognized for their capacity to produce novel bioactive compounds. However, little is known about most tunicate-associated microbial communities, even in the embryology model organism Ciona intestinalis. In this project I explored 3 local tunicate species (Ciona intestinalis, Molgula manhattensis, and Didemnum vexillum) to identify potential symbiotic bacteria. Tunicate-specific bacterial communities were observed for all three species and their tissue specific location was determined by CARD-FISH. Introduction Tunicates and other marine invertebrates are prolific sources of novel natural products for drug discovery (reviewed in Blunt, 2010). Many of these compounds are biosynthesized by a microbial symbiont of the animal, rather than produced by the animal itself (Schmidt, 2010). For example, the anti-cancer drug patellamide, originally isolated from the colonial ascidian Lissoclinum patella, is now known to be produced by an obligate cyanobacterial symbiont, Prochloron didemni (Schmidt, 2005). Research on such microbial symbionts has focused on their potential for overcoming the “supply problem.” Chemical synthesis of natural products is often challenging and expensive, and isolation of sufficient quantities of drug for clinical trials from wild sources may be impossible or environmentally costly. Culture of the microbial symbiont or heterologous expression of the biosynthetic genes offers a relatively economical solution. Although the microbial origin of many tunicate compounds is now well established, relatively little is known about the extent of such symbiotic associations in tunicates and their biological function. Tunicates (or sea squirts) present an interesting system in which to study bacterial/eukaryotic symbiosis as they are deep-branching members of the Phylum Chordata (Passamaneck, 2005 and Buchsbaum, 1948). They possess a notochord and dorsal neural tube in their larval stage but lose the notochord during metamorphosis. Most adult tunicates are sessile filter feeders. The name tunicate comes from the thick outer coating or “tunic” of adult animals, which contains cellulose. Tunicates group into two main morphotypes, solitary and colonial. Solitary animals, including Ciona and Molgula species, resemble a bag of jelly with an incurrent and excurrent siphon at the top. The colonial animals, such as Didemnum, grow in spreading mats with many tiny animals or “zooids” contained within the same tunic. For this project, I chose to focus on three locally available tunicate species with unique features and some indication in the literature of a possible bacterial symbiosis. Ciona intestinalis is a well-studied model organism in embryology and development and has a sequenced genome. Like many tunicates C. intestinalis accumulates mM vanadium in its blood as protein-bound V(IV) [VO2+] (Trivedi, 2003). There is one report in the literature of a potential symbiosis with a possible cyanobacterial symbiont in the C. intestinalis tunic (De Leo, 1980), as well as one electron microscopy study that mentions numerous bacterial cells in the “ground substance” of the tunic (De Leo, 1981). Molgula manhattensis and Didemnum vexillum were selected based on intriguing early results from 16S clone libraries produced in Stefan Sievert’s lab at Woods hole (Tait, 2007). The Sievert lab found only one phylotype of spiroplasma-like sequences in the 16S rDNA libraries of M. manhattensis gonad that had similarity to sequences from the colonial tunicate Ecteinascidia turbinata (Herdman) (Moss, 2003). Spiroplasma, which group within the Mollicutes, are often pathogens of insects and one strain is known to cause “trembling disease” in Chinese mitten crab, Eriocheir sinensis. In neither tunicate has the cellular location of this consistently found spiroplasma phylotype been determined, and its role as pathogen or commensal is unknown. The renal sac of M. manhattensis is also known to contain a very unusual apicomplexan symbiont , Nephromyces, which contains within it an intracellular bacterial endosymbiont (Saffo, 2010). D. vexillum is a highly invasive colonial tunicate that causes substantial damage by overgrowing marine surfaces and sessile animals. Several colonial tunicates are known to contain symbiotic cyanobacteria or alphaproteobacteria in their tunics (Martinez-Garcia, 2007 and Hirose, 2006). The Sievert lab constructed a 16S rDNA library from the tunic surface of D. vexillum that contained two dominant alpha-proteobacterial phylotypes with no close cultured relatives (Tait, 2007). One study (Yokobori, 2006) refers to D. vexillum (formerly Didemnum sp. A) as a “photsymbiotic” species, although no reference or data is given. The D. vexillum from Eel pond does not have the green color characteristic of a typical Prochloron –containing tunicate. Tunicates are a fascinating system in which to search for bacterial symbiosis and ask questions about microbial/eukaryotic interactions. As chordates, they represent a vertebrate outgroup and some representatives like C. intestinalis have well-studied development and immunology. The (unfortunate) invasiveness of D. vexillum presents the potential to examine the biogeography of symbiosis. Further, the available 16S rDNA data from D. vexillum and M. manhattensis indicates that these tunicates are a potential source of as-yet-uncultured bacterial diversity. The role of bacterial communities play in shaping eukaryotic biology is still largely unknown, and symbiosis is more widespread than many eukaryotic biologists appreciate. What interactions allow for selection of a particular bacterial symbiont or community? What do the participants gain and lose from the symbiosis? How are symbiotic relationships established and maintained, and how do they evolve? Methods Tunicate sampling Samples of D. vexillum were collected by the Marine Resource Center (MRC) from Eel Pond. All selected D. vexillum specimens were growing over another tunicate, Steyla clava. The first three specimens were collected on 7/6/11 with an associated water sample. Specimens 4 and 5 were collected on 7/11/11, again with an associated water sample. C. intestinalis specimens were obtained from an MRC tank with associated water samples. Individuals 1-3 were collected on 7/6/11 and individuals 4 and 5 were obtained from the same tank on 7/18/11. All M. manhattensis specimens with an associated water sample were collected by from East Falmouth Harbor on 7/13/11. All specimens were stored at 4 deg until processing and dissected the same day as collection. Dissections Dissections were greatly aided by the online reference http://webs.lander.edu/rsfox/invertebrates/ (Fox, 2004). Further help with Didemnum dissections and anatomy was obtained from Lauren Stefaniak, a Ph.D. student at the University of Connecticut Department of Marine Sciences. All tissue specimens were rinsed 3 times in sterile ASW in large petri dishes. Samples for DNA extraction were flash frozen in liquid N2 and stored at -20deg C until use. Samples for FISH were prepared using treatments described in the following section on fixation. Images of dissections are shown in Figures 2 and 3. Didemnum sample 1 (D1) Approximately 1cm2 sections of tunic and zooids were dissected away from the underlying Steleya and trimmed in a dish of sterile ASW. Two parts of the specimen were sampled- a flat portion of tunic and a long, thin hanging cylindrical growth. This specimen was relatively thin with generally flat growth of the tunic and small, closely spaced zooids. Didemnum sample 2 and 3 (D2 and D3) These specimens were larger and had thicker tunics with a deeply wrinkled surface. Approximately 1cm2 sections of tunic and zooids were dissected away from the underlying Steyela and trimmed in a dish of sterile ASW. Didemnum sample 4 and 5 (D4 and D5) These specimens were obtained to attempt a zooid-free dissection and dissection of the tunic cuticle surface. This turned out to be extremely difficult and would require much finer dissection scissors and forceps. The cuticle was pulled from the surface of the animal by holding the tunic with one pair of forceps and stripping away the cuticle with a second forceps. The remaining stuck zooids were then removed one by one with forceps under a dissecting scope. I was unable to remove all the attached zooids in this manner, so I decided not to try a “cuticle only” or “tunic only” clone library. Instead, cross sections through the entire colony were prepared for fixation as for D1-3. Ciona specimens 1 to 3 Ciona specimen 1 was used to learn the dissection technique. Ciona specimen 2 was dissected and preserved for FISH and DNA clone libraries. First, the tunic was cut open at the bottom with a scalpel and then opened with scissors from siphon to siphon. The tunic was dissected completely away from the intact mantle. A section of the tunic and cuticle from the middle, pigmented part of the animal was preserved for FISH and DNA (C2TC). A portion of the yellow upper tunic was also preserved (CYT). The mantle was removed and the gonad was dissected away and preserved for FISH (CG). A portion of the pharyngeal basket was dissected away, smashed under a cover slip, and moving cilia were observed by phase contrast microscopy. The gonad of specimen 3 was preserved for DNA extraction. Ciona specimens 4 and 5 These animals were obtained and dissected in order to 1) check for the presence of bacteria across multiple individuals, 2) obtain more sections for FISH with euk and other specific bacterial probes, and 3) obtain another gonad for FISH and to retry the 16S PCR from gonad tissue. The tunic was dissected completely away from the intact mantle. A section of the tunic and cuticle from the middle, pigmented part of the animal was preserved for FISH (C4 and C5). The gonad of specimen C5 was dissected away, cut in half and preserved for FISH and DNA extraction (C5G). Molgula specimens 1 to 3 Three gonads from 3 M. manhattensis tunicates were successfully dissected. The 2nd specimen (M2) was very small and the gonad dissection was not completely clean.