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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

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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.

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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

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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.

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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.

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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. This can be seen in Figure 4A-H. In Figure 4A-D no discernable structure can be determined. Particularly in Figure 4A and 4E (the brightfield images) a lot of interference is seen with bacteria and yeast growing in culture which have also been embedded in the agarose. In Figure 4F some structure can be seen near the peristome where the feeding cilia are, but it is not consistently seen in other Vorticella zooid structures on the same slide. The bright green spot is on figure 4F appears to be a cyst of another unknown eukaryote and swamps out the fluorescent signal of the Alexa Flour 488.

Figure 4. Immunostaining results of a Vorticella enrichment conducted on a slide in a six well plate. A-D show the bright field, A488 FITC(475/530nm), DAPI (495 nm/ 517 nm) , and combined A488 and DAPI images respectively for one cell on a slide embedded in 0.8% agarose. E-H show the bright field, A488 FITC, DAPI, and combined A488 and DAPI images respectively of another cell on this slide. Scale bars and editing done in imageJ.

Staining done without embedding in agarose appeared to show a more distinct structure as seen in Figure 5A-D. In 5A the cilia at nearest the peristome can be seen. Additionally, in figure 5A a vestigial line of concentrated tubulin can be seen. This is believed to mark the posterior tuft of cilia maintained during the telotroch life stage can be seen. In Image 5A the macronucleus is seen to form a J shape, no micronucleus is seen.

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Figure 5. This shows immunostaining results on slides that was staining directly. 5A is the FITC (475/530nm). 5B is DAPI (495 nm/ 517 nm). 5C is the bright field image. 5D is the combined FITC and DAPI image. Scale bars and editing done in imageJ.

In figure 6A you can see one frame of a Z-stack taken on a Nikon eclipse Ti2 inverted microscope showing stains for Alexa Flour 488 (mouse α-TAT1 anti-tubulin) and DAPI (Nuclei). Figures 6B and C are 3-D renderings of this Z-stack and show Alexa Flour 488 (mouse α-TAT1 anti-tubulin) and DAPI (Nuclei) respectively. This image interestingly captures the two life stages, telotroch and trophont. In the upper left corner of figure 6A and 6B a Vorticella zooid in the telotroch phase can be seen and is identified based on the posterior tuft of cilia seen. Two of the remaining (top middle, bottom) three zooids appear to be in the trophont phase and can be seen to have cilia protruding from the peristome. A third (top right) appears to be dying as the membrane around the peristome has bursted and appears to be blebbing out. In figure 6C, which shows the nuclei stained with DAPI, the J or semi-circular shape indicated in the literature can be seen. This was tested further and shown more clearly in Figure 7B where the micronucleus is also seen.

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Figure 7. (A) 3D rendering of the Z-stack of a Vorticella stained with the α-TAT1 anti-tubulin Alexa Flour 488 shown. (C) 3D rendering of the z the Z-stack of a Vorticella stained with DAPI fluorescence shown. All images were analyzed in NIS-Elements.

Figure 6. (A) This depicts an image still of the Z-stack taken on Vorticella stained with DAPI and α-TAT1 anti-tubulin. (B) 3D rendering of the Z-stack in A with the α-TAT1 anti-tubulin Alexa Flour 488 shown. (C) 3D rendering of the z-stack in A with DAPI fluorescence shown. All images were analyzed in NIS-Elements.

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Figure 8. 3D rendering of the Z-stack of a Vorticella stained with the α-TAT1 anti-tubulin Alexa Flour 488 shown. All images were analyzed in NIS-Elements.

Interestingly the contractile tail of the zooid on the bottom of Figure 6A and the zooid in Figure 7A appears to be taking up some amount of stain throughout the coil. This was tested further through replication and in Figure 8 a clear image is seen of microtubule centers throughout the length of the contractile tail. It appears that these microtubulin centers are evenly distributed throughout the length of the spasmoneme.

Discussion & Conclusion

Although this project has not yet achieved a pure culture the Vorticella sp. of interest were enriched and growing quickly. Based on the lack of replication in cells sorted individual using a micromanipulator it may be that an unknown symbiosis is occurring that is preventing the Vorticella sp. from replicating. This should be further tested to determine if it is a bacterial

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symbiont that was simply not enriched enough in the transfer vessel or if there was another stress related factor that led to this.

Immunostaining of the cells for microtubulin indicated a conserved structure amongst this group of Vorticella sp. The telotroch phase has a clear tuft of cilia along the base and some of this structure is maintained in the adult zooid. It is also inferred from the images that when the peristome is closed completely some of the bands of cilia still protrude from the mouth. Microtubulin stain has also indicated that there is an organized pattern of microtubulin throughout the length of the contractile stalk. It is unclear what the role of this is and further research needs to be done. It has been suggested by previous research that these may be either mitochondria and/or calcium storage site20,21.

Staining of the nuclei with DAPI yielded photographic support for the nuclei structure described in the literature. This macronucleus is semi-circular, while the micro-nucleus is a small round circle that sits within the semi-circular macronucleus. Previous research has suggested that the micronucleus is a germ line cell so individuals that did not appear to have a micronucleus may have recently divided and may be reforming this micronucleus.

This study provides a foundation for future research on this subgroup. Continued efforts will be put forth to get this Vorticella sp. into isolation and eventually towards a unique genome. If isolated and sequenced this organism would serve as a useful model for understanding physiology and regulation.

Acknowledgements

Thank you to Sarah Guest who helped teach me microscopy, helped me troubleshoot throughout the process, and provided invaluable insight. Scott Dawson for support and encouragement throughout the way. Rachel Whitaker and George O’ Toole for all their work in putting together the Microbial diversity course. All the MBL support staff, professors, teaching assistants, and course assistants who helped make the microbial diversity course possible.

Data Accessibility

Images and videos used in this report will be deposited to the microbial diversity 2018 course servers. If other data is needed and not readily available, please contact the me.

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