Honors Thesis

Chemical Effects of Vermetid Snails on Coral Microflora By Noah Hackney: [email protected] Department of Microbiology and Cell Science Thesis advisor: Tom Frazer CALS Honors Program Noah Hackney Chemical Effects of Vermetid Snails on Coral Microflora Noah Hackney Abstract Coral reefs benefit many different marine organisms and coastal populations by providing habitats, shelter, and sustenance. Corals, however, are threatened by a variety of stressors (including vermetid snails) that reduce growth and survivorship. Ceraesignum maximum is a common vermetid on the shallow reefs of Mo’orea, French Polynesia that casts a mucus net used for feeding. This net leads to reductions in growth and survival of corals. The mechanism underlying the deleterious effects of the mucus net, however, is not entirely clear. We hypothesized that vermetids negatively affect coral colonies by altering Surface Mucus Layer (SML) microbial communities (the corals’ first line of defense) via selective inhibition by chemicals in the snail’s mucus nets. We isolated nine unique cellular morphologies primarily comprised of Firmicutes, Bacteriodetes, and Proteobacteria, and assessed antibacterial activity of each group using crude chemical extracts from vermetid mucus nets. Our data showed that chemicals in the mucus net did not inhibit growth of the isolated microbes, which suggests chemicals from Ceraesignum maximum do not have an inhibitive effect on isolated massive Porites microflora. Introduction Coral reefs are among the most productive and bio-diverse ecosystems in the world and are estimated to support 25% of all marine life (Spalding, 2001). Coral reefs provide a broad suite of ecosystem and other services including, for example, protection of shorelines from storm generated waves, production of fisheries related resources, and tourism related revenues. Healthy coral reefs can, in fact, provide a net benefit of $29.8 billion per year globally as a result of such services (Cesar et al., 2003), and often serve as the basis of coastal, tropical economies. A variety of natural and anthropogenic factors threaten coral reefs and their associated fauna. Humans alter reef habitats through unsustainable fishing practices (Reed et al., 2007), pollution (Koop et al., 2001), and coastal construction (Rajasuriya et al., 1995). Additionally, increases in temperature and ocean acidification due to global climate change lead to reduced coral growth (Anlauf et al., 2011). Such anthropogenic changes to reefs are likely to have a profound influence on the microbial communities of corals (Meron et al., 2011), which can make corals more susceptible to disease. Many coral reef stressors can also impact the coral’s interactions with their symbionts. Corals are characterized as having multiple symbiotic interactions. In addition to harboring photosynthetic microalgae, Symbiondinium, in their soft tissues, corals host complex microbial (i.e. bacterial) communities including, for example, those found in the Surface Mucus Layer (SML). Coral microbiota assist in sulfur cycling, nitrogen fixation, and production of anti-microbial compounds 3 (Krediet et al., 2013). Importantly, bacterial communities in the surface microbial layer serve as a coral’s first line of defense against invading pathogens. For example, mucus from healthy Acropora palmata inhibited the growth of potentially invasive microbes (Ritchie, 2006). Stressful events (i.e. bleaching) can lead to the loss of the protective function provided by SML associated bacteria and cause an increase in the numbers of coral pathogens such as Vibrio spp. (Bourne et al., 2008). Microbial community shifts can also occur when corals are in contact with other organisms. Macroalgae, for example, are able to concurrently alter coral microbial communities and coral growth rate (Thurber et al., 2012). Shifts in microbial communities are a potential mechanism by which macroalgae reduce coral health and increase coral loss (Thurber et al., 2012). Another group of organisms that threaten corals are vermetid gastropods, the largest of which is Ceraesignum maximum (formerly known as Dendropoma maximum). Vermetids, commonly known as worm snails, are sessile gastropods that are common on coral reefs and rocky shores and have larvae that settle onto hard surfaces. Vermetids have tube-like shells that affix or burrow onto hard sub- strata. Vermetid snails feed by releasing a mucus net into the water column which traps phytoplankton, zooplankton, and other particles. On average, the mucus net, when protracted, covers the coral substrate within 10 cm of the vermetid (Kappner, 2000). Vermetid nets are periodically retracted back into the animal’s shell using its lateral jaws (Hughes et al., 2009). C. maximum nets are also known to contain at least two bioactive compounds, one of which showed to reduce V. fisheri bioluminescence (Köppel, 2013). Vermetids present a variety of problems to coral reefs. Cerasignum maximum significantly reduce the skeletal growth and survivorship of corals by up to 81% and 52% respectively (Shima et al., 2010). Cerasignum maximum presence is shown to affect the formation of corals. Zvuloni (2008) found that Stylophora pistillata, in the presence of C. maximum, was 52% shorter and 35% more slender. Although the negative effects of vermetid mucus nets on corals are established, the underlying mechanisms have yet to be fully resolved. Several hypotheses that could explain this interaction include reduction of water flow to the coral, decreased sunlight penetrating to the coral zooxanthelle, and abrasion of the coral tissue (Klöppel et al., 2013; Zvuloni et al., 2008). We hypothesize that the bioactive compounds from the mucus nets of C. maximum are able to inhibit the growth of coral mucus associated bacteria. To test our hypothesis, we isolated groups of bacteria from massive Porites surface mucus, and sequenced these groups to determine which groups might be amenable to isolation. We then conducted antimicrobial assays on microbes isolated from the surface mucus layer of massive Porites with chemicals extracted from C. maximum mucus nets. 4 Materials and Methods Mucus net collection: We collected C. maximum mucus from two different back reef locations on the north coast of Mo’orea, French Polynesia, amongst the reefs located near (17.480062 S, 149.832010 W) and (17.475986 S, 149.812320 W) from May to July 2014. Using snorkeling gear, we used tweezers to twirl the mucus into clumps of roughly 100 vermetid nets and collected these clumps in 15-ml centrifuge tubes. Each centrifuge tube held 500-1500 nets. We transported the collected nets back to our research lab, Gump Station, and stored them in a -20° C freezer after removing excess water from the tubes. We were careful not to collect C. maximum nets in the field that were fouled or covered by algae or sediment. Coral mucus collection and microbial isolation: We collected coral mucus from 15 different massive Porites corals located within the reef located around (17.480062 S, 149.832010 W) from June to July 2014. We used 5-ml luer tip sterile syringes to agitate a 5-cm long strip of coral surface then suctioned the mucus into the syringe. We brought the syringes back to lab in for plating. In the lab, we drained excess seawater from the syringes, and moved the ball of coral mucus into 1.5-mL centrifuge tubes with sterilized seawater (SSW). We vortexed the samples to break up the coral mucus. After vortexing, we added 2 ml of the SSW + mucus mixture onto agar plates. We used Glycerol Added Sea-Water Agar (GASWA) as the agar for both microbe isolation and antimicrobial assays (Ritchie, 2006). After 48 hours, we inoculated new plates with unique colony morphologies observed in the plates inoculated with the massive Porites surface mucus. We allowed the microbes to grow on plates for 48-72 hours before we continued to streak for isolation. We performed streaks for isolation until we obtained pure cultures of the unique morphologies. We defined unique morphologies as colonies that differed in color, thickness, and shape compared to other colonies. All microbes were incubated at 24° C. Mucus net extraction and antimicrobial assays: To make crude extracts, we soaked 10 mL (displacement volume) of the collected vermetid nets in 100% methanol for 5 hours, filtered the extract and then removed the solvent with rotary evaporation. We kept dried extracts at - 20°C for between 1-5 days until use. We then resolved the extracts in 5 mL of 100% methanol. We soaked sterile paper disks (7-mm diameter) in methanol and net extract + methanol to serve as our control and experimental groups, respectively. We inoculated new GASWA plates with our pure unique colonies to create bacterial lawns. We placed two paper disks, one control and one experimental, onto 10 freshly inoculated microbial lawn plates and repeated this for each unique colony morphology we isolated (Figure 1). The lawns we allowed to grow along with the experimental and control disks for 48 hours before we took 5 pictures of the agar plates. We used ImageJ to measure the diameter of the inhibition zones. We measured diameters from 3 randomly selected transects across the affected zone of the microbial lawn and averaged these three values. We calculated our inhibition zones as the average of the three transects minus the diameter of the paper disc. Paired t-tests were used to compare the diameters of the inhibition zones created by experimental (extract) and control (methanol only) groups for each microbial isolate (α = 0.05). Also, we sent samples of our bacterial cultures to Mr. DNA, a genetic sequencing company in Shallowater, Texas, USA. They created DNA libraries of the V4 variable region of the 16S rRNA genes of our bacterial samples and used a nucleotide BLAST from a database derived from GreenGenes, RDPII, and NCBI (DeSantis et al., 2007) to find the most probable taxonomic families each sample belonged to. Results We isolated 9 unique colony morphologies of bacteria collected from massive Porites. Analysis of the 16S rRNA showed that one isolate was from the phylum Bacteriodetes, three isolates from the phylum Firmicutes, and five from the phylum Proteobacteria.

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