Efforts Towards a Pure Culture of a Vorticella Sp. from Cedar Swamp Elaina Graham1 1 University of Southern California
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Efforts towards a pure culture of a Vorticella sp. from Cedar Swamp Elaina Graham1 1 University of Southern California Abstract Microbial eukaryotes are extremely morphologically diverse. Vorticella, which are ciliates are a unique group of sessile peritrichs that form a contractile stalk. Unlike other organisms with contractile stalks like Zoothamnium and Carchesium, Vorticella has a single stalk and single zooid. Despite previous descriptions of the group few large imaging efforts have been done to capture cellular and nuclei structure. Here I use DAPI to show the macro- and micro- nuclei structure in in depth 3-dimensions using a Z-stack. The microtubule network is also visualized similarly in 3-dimensions to show the structural difference in the telotroch and trophont life stages, as well as the unique microtubulin centers along the length of the contractile tail. Introduction Within marine and freshwater ecosystem, a diverse array of microbial life exists. Microbes, which include bacteria, archaea, and eukaryotes plat important roles in global biogeochemical cycling, particularly with respect to carbon 1. In recent years much effort has been placed in understanding the metabolic diversity, biogeography, and ecology of bacteria and archaea through massive sequencing initiatives2–4. Microbial eukaryotes are extremely underrepresented in genomics though. If you opened up NCBI today you’d find that 152,040 genomes representing bacteria and archaea (Date Collected 07/26/2018). In contrast NCBI contains only 6,262 eukaryotic genomes, of which 3,329 are fungi, 1646 are animals, 622 are plants, and 665 are protists. This bias in the database is surprising considering protist comprise most of the diversity within the eukaryotes5. Beyond this bias in genomics sequencing there is a lack of thorough information documenting the morphological diversity within the microbial eukaryotes. Eukaryotes tend to large genomes with many chromosomes, smaller populations, and more variance within gene families6. This makes them more difficult to study than bacteria and archaea. Within the microbial eukaryotes members of the SAR supergroup (Stramenopiles, Aveolates, Rhizaria) are particularly underrepresented in environmentally relevant studies. Vorticella is one interesting species which was first recorded in 1676 by Antonie van Leeuwenhoek7 and although previously brought into culture has few extensive microscopy initiatives to create a digital record of cellular morphology. This group is also interesting evolutionary as they have been discovered in a 200 million year old fossil8. Vorticella is a genera of peritrichs which falls within the Ciliophora (ciliates). Peritrichs fall into two monophyletic clades, the Mobilida and Sessilida9. The Vorticella fall into the Sessilida which are characterized by have a cell body (zooid) and a contractile stalk. Vorticella are filter feeding ciliates that live in 1 two forms throughout their lifes; a free-living telotroch and a stalked trophont attached to a substratum via a holdfast10. The Vorticella zooid consists of a mouth like opening called the peristome where there are bands of cilia (composed of microtubules) which are used to generate a current that draws food particles too the zooid. These currents create micro-eddies which have been show to have a maximum flow velocity of 360 μm/s and can move particles 450 μm away from the peristome11. Structurally the zooid has been described as having a small food vacuole and a J shaped macronucleus and single micronucleus12. This is characteristic of many ciliates, whereby the micronucleus acts as the germ line and does not express the genes13. One of the most unique aspects of the Vorticella genera is its contractile stalk. The stalk is unique within the protists due to its adherence to a variety of surfaces, including but not limited to crustaceans11, algae10, diatoms14, and detritus11. The stalk houses a spasmoneme (the contractile organ), a sheath, and a fibrillar matrix. The spasmoneme can coil up and retract in less than a millisecond in response to calcium signaling15. While most contractile stalks rely on ATP the Vorticella system utilizes calcium ions11. Other members of the Peritrichs such as Zoothamnium16 and Carcheslium17. The proteins in the stalk of Vorticella is believed to be a member of the centrin family of calcium binding EF hand proteins. This includes both centrin and caltractin. Electron micrography has determined that the stalk is able to attach to the substrate by an adhesive disc12. When separating the stalk from the zooid it has been suggested that G-protein signal transduction plays a role18. These interesting factors make Vorticella an ideal organisms to study as a pure culture to facilitate a better understanding about lifestyle and regulation in the Peritrichs. This project looked at Cedar Swamp near the Marine Biological Laboratory. Cedar Swamp has a highly diverse community of microbial eukaryotes as seen in Figure 1. Vorticella was enriched for from these samples when conducting experiments for enriching aerobic thiosulfate oxidizing bacteria. This is interesting because the related species Zoothamnium has been shown to have a symbiosis with a sulfur oxidizing chemolithoautotrophic bacteria which play important roles in nutrition for this organism19. 2 Figure 1. Various eukaryotes seen under wet mouns from samples collected in Cedar Swamp in July 2018. Methods Enrichment Initial enrichments for Vorticella were conducted by inoculating a 125mL flask containing 50mL of Freshwater aerobic thiosulfate autotroph medium (DI water to 1000 ml, 100X FW Base 10 ml, 1M Sodium Nitrate 5 ml, 100mM Potassium Phosphate 0.1 ml, 1M MES 5 ml, Vitamins 1 ml, Trace elements 1 ml, 1M Na Bicarbonate 5 ml, 1M Na Thiosulfate 25ml) with detrital leaf matter from the sediment surface of Cedar swamp (quantity was ~5 -10grams). Wet mounts of the sample were conducted prior to inoculation to record community. The sample was incubated at 30°C for 2 weeks. Isolation Densities of enrichment were monitored and imaged using a Zeiss Axioskop fluorescence microscope. When densities were high (e.g at least 2 individuals per 20 µL wet mount) six well plates were set up containing 5mL of Freshwater aerobic thiosulfate autotroph medium (FATAM) 1µL of yeast extract, and a glass coverslip (22x22) placed at the bottom of the well. From the enrichment 500µL was used to inoculate each of the six wells and left at room temperature for 24-48 hours. Each well was monitored daily under a Zeiss inverted microscope 3 looking for Vorticella with stalks affixed to the slide covers. When Density was at least 10 individuals the slide was carefully removed and rinsed 3 times with clean media before transferring to a new six well plate. Some individuals were also transferred in a 50µm-100µm glass pipette with a Sutter XenoWorks Micromanipulator System into individual wells. These were similarly monitored using a Zeiss inverted microscope daily. Immunostaining When slides had a dense enough population, some were selected and carefully removed from the six well plates and placed into 2% Paraformaldehyde (diluted in PBS) for 2.5 – 5 minutes. Slides were washed 3X in 1X PBS. Following this some slides were placed directly in 0.1% Triton-X for 10 minutes to permeabilize cells. Some slides were covered in 50µL of 0.8% low melting point agarose to embed cells. Following drying this slide was also permeabilized in 0.1% Triton-X for 10 minutes. Slides were then washed 3X with 1X pbs and transferred to a clean well. To this slide 20µL of a 1:200 concentration of mouse α-TAT1 anti-tubulin conjugated to Alexa Fluor 488 was added and left to incubate for 4 hours at room temperature. Slides were then washed 3X with PEMBALG buffer (500ml PEM, 500ml BALG: 5g - 1% BSA , 0.5g - 0.1% NaN3, 9.1g - 100mM lysine, 2.5ml - 0.5% gelatin cold water fish). PEM was made with 100mM PIPES- pH 6.9, 1mM EGTA, 0.1mM MgSO4. Once slides were washed in PEMBALG 20 µL of 1µg/uL of 2-(4-amidinophenyl)-1H -indole-6-carboxamidine (DAPI) was pipetted and left in the dark for 10 minutes. Slides were then washed in 1X PBS 3 times and mounted on a slide with Thermofischer pro-long antifade. Slides were analyzed on a Zeiss Axioskop fluorescence microscope and a Nikon eclipse Ti2 inverted microscope. Images were analyzed in ImageJ for those images takon on the Zeiss scope and NIS-Elements for those taken on the Nikon. Results Initial visual inspection of Vorticella is seen in Figures 2A-H. The Vorticella were highly enriched at 30°C, but when returned to room temperature while in co-culture a large amoeba species overcame the enrichment. This is seen in Figure 3A-D. Interestingly these amoeba in figure 3A and figure 3D were consuming a Vorticella zooid. It is hypothesized that maintaining the culture at 30°C helped to initially select and enrich for Vorticella as other ciliates and amoeba tend to be temperature sensitive. 4 Figure 2. Various images of Vorticella throughout the enrichment. Photos 2E-G are shown in DIC, while Photos A-D and H are shown in Bright Field. All images were taken on a Zeiss Axio camera and edited in imageJ. Figure 3. Images of unknown ameoba that began growing in the enrichment when placed at room temperature. All images were taken on a Zeiss Axio camera and edited in Image J. 5 Visual analysis of the Vorticella enrichments under an inverted scope showed that although individuals picked using a micromanipulator were isolated and alive after four days they had not replicated. Due to this focus was placed on maintaining the dilution to extinction culturing method and enriching the Vorticella by transferring slides into clean media regularly. To conduct immunostaining the method of embedding the cells in 0.8% agarose was attempted first. Results indicated that embedding the cells in agarose did not improve the quality of imaging.