Lorenzo Lagostina Microbial Diversity 2015

Morphological and behavioral characterization of Corallomyxa tenera.

Corallomyxa tenera is a poorly known reticulose amoeba belonging to a clade that comprises Gromia, Haplosporidia and Foraminifera (Tekle et al 2007). Foraminifera are considered an old lineage inside the eukaryotic domain (Pawlowsky 2002) thus suggesting that Corallomyxa spp. is also likely an old representative of this domain. Considered the lack of phylogenetic resolution present for unicellular eukaryotes more efforts in genomic and morphological characterization are needed to reconstruct the early evolution of this lineage.

Corallomyxa is a large multinucleate naked amoeba that forms an intricate network that can reach areas of square centimeters. The network is substituted in older cultures (in vitro) by an extensive plasmodium rich in vacuoles and nuclei. Under certain conditions, as starvation, anoxia, and other undefined circumstances the amoeba produce fruiting bodies, spherical structures attached to the network that are packed with nuclei and vacuoles.

The original goal of this work was to characterize the morphology and the analyze the transcriptome of Corallomyxa tenera in its two distinctive life stages, the plasmodium and fruiting bodies. Due to delays the transcriptomic analysis had to be postponed and it will be not discussed in the present report. Here I present the morphological and behavioral characterization of an highly intriguing eukaryote.

Materials and methods

Corallomyxa tenera was isolated from Sippewissett marsh sediments in summer 2014 and grown in presence of Maribacter. The media (SaltyScott) used was seawater based, MOPS 1µM, 0.1 g tryptone per liter and 0.1 yeast extract per liter. Tissue flasks and MatTek glass bottom dishes of different volumes were used as growing support to be ready for imaging on inverted microscope.

One vibrio isolated by group 4 during MD2015 was used as alternative food source.

Fluorescent E. coli were used as alternative food source to track phagocytosis process.

Media for axenic cultures were prepared as follows:

1liter SW base, 1 g of yeast extracts, 1ml MOPS 10 mM.

Antibiotics used:

Gentamicin final concentration 10 µg/ml

Ampicillin final concentration 100 µg/ml

Tetracycline final concentration 50 µg/ml

Chloramphenicol final concentration 35 µg/ml

Erythromycin final concentration 20 µg/ml

PCR amplification

Amplification of 18S rRNA gene was performed with the primer set 360E (f) and 1200E (5’ CCC GTG TTG AGT CAA ATT AAG C 3’) (r). Cycling conditions: an initial denaturation at 95°C for 2 min, followed by 30 cycles at 95°C for 20 s, 53°C for 45 s and 72°C for 1 min. Final extension at 72°C for 3 minutes. For analysis of 16S rRNA the primer pair 8f 1391r (bacteria) and 333f (5'- TCC AGG CCC TAC GGG - 3’) 1391r (5'- GAC GGG CGG TGW GTR CA -3') (archaea) were used. Cycling conditions: an initial denaturation at 95°C for 2 min, followed by 30 cycles at 95°C for 20 s, annealing temperature of 55°C (bacteria) and 52°C (archaea)for 45 sec, 72°C for 1 min and 3 min as final extension step.

Microscopy

Video and imaging were taken on inverted microscope Zeiss Axio Observer.Z1. Image editing and analysis were performed using (Fiji Is Just)Image J. The plugin TrakMate was used to calculate mean and maximal velocities of particles.

Fluorescent dyes were added to the media SaltyScott to reach the following working concentrations:

Calcofluor-white 40 µg/ml

Hoechst 33258 1 µg/ml

FM4-64 8 µg/ml

Fluorescent bacteria 0.2 µl per ml

Oregon Green 1 µM

Mitotracker orange 250 nM

Lysotracker 100 nM

cAMP was diluted in different concentrations in SaltyScott media. Final concentrations tested were 50 pM, 1 nM and 100nM. monoFISH

MatTek dish was used as support for hybridization. Corallomyxa (attached to the glass) was dehydrated with ethanol washes directly in the dish with 50, 80 and 100% ethanol. The rest of the protocol used (hybridization, incubation and washes) are from Manz et al 1992.

Probes used: Eub I-III

Arch915

Observations and discussion

Different growing conditions were tested, including anaerobiosis, dark, light, shaking, steady and with media flow. Corallomyxa grew at the same rate (no quantification performed) colonizing a 50 mm MatTek petri dish in a about 48 hours under all conditions but in the anaerobic chamber. After 10 days in the anaerobic chamber completely obscured the media, while buds were present but totally unaltered, indicating that anaerobic environments are not suitable for its development. After 10 days of anaerobic conditions the culture was left exposed to normal atmosphere, and in 4 days its characteristic network was observable (fig 1).

Fig 1. Corallomyxa tenera in a young developmental stage. The plasmodium network is developed only nearby the fruiting bodies and is still relatively slim.

Considered that this species was isolated from surface of brackish sediments seems likely that this organisms need to face periods of anoxia or micro- oxygenation, and the production of fruiting bodies could be a dispersal strategy as well as a way to escape anoxia.

a

Morphology and behavior a

Fruiting bodies were massively produced in case of starvation (absence of bacteria in the media). Fruiting bodies formation could be stimulated by irradiation with any light in between 600nm to UV light. Fruiting bodies start to form also in organisms living in fresh media with bacteria available when the network reached high density. b Fruiting bodies start to form attached to the surface, the network start to collect nuclei and vacuoles inside an anastomosis. The growing structure gaining volume increase signal in tubulin, presumably indicating an increase in rigidity of the structure (fig 2a,b and c). The buds during maturation become spherical and are rising from the bottom of the surface, eventually being released as “flying balloons”.

Calcofluor-white dye stained the surface of the c fruiting bodies, indicating presence of cellulose or chitin as structural reinforcement (fig 3). These polymers have been reported to be present in some amoeba’s cysts in the literature (Fouque et al). Calcofluor stained also nuclei inside the fruiting bodies, indicating that, in case of real and not unspecific staining, the nuclei are also embedded in cellulose/chitin layer.

Under temptative axenic growth, buds have been observed to be phagocytized in case of nutrient limitation (scarcity of bacteria due to presence of different Fig 2. Bud formation. a) DIC image b) antibiotics in the media). Link to Tubulin stained c) DNA stained. All scale bars are 20 µm. video “buds are good for you” in the appendix.

Buds have been recorded to explode and release a huge amount of vacuoles (link to the video “bud explosion” in the appendix).

Nuclei inside fruiting bodies are in constant motion.

Strikingly also in the plasmodium network nuclei are in constant Fig 3. Fruiting bodies stained with calcofluor-white dye. Pseudocolored. Note the stained nuclei inside the buds. Scale bar 25 µm. motion. Nuclei have been tracked and calculated to move inside the network with a mean velocity up to 2 µm/s, but they have been recorded to reach 14 µm/s (fig 4).

Nuclei can change dramatically their a shape when accelerated inside the network (see link “Nuclei movement” in the appendix).

These speed are similar to vacuoles’ speed recorded for the freshwater network forming amoeba Reticulomyxa (Koonce et al).

Cytoplasmic stream and mitochondrial movement have also been recorded for Corallomyxa (link in the appendix). b

Plasmodium waves of 0.2 µm/s have been recorded (link in the appendix). Their function is unclear, but they clearly help in reshaping the network after their passage. Should be interesting relate their direction of movement to known protist’s chemoattractants.

Experiments with addition of block of agarose containing different Fig 4. Nuclei movement tracks. a) Mean velocities b) Maximal velocity concentrations of cAMP or amminoacids did not produce any notable effect.

monoFISH

Different attempt to obtain pure eukaryotic DNA for genomic sequencing were made, unsuccessfully, as PCR amplification of archaeal and bacterial 16S rRNA always yielded amplicons.

In order to asses presence of endosymbionts or associated bacterial communities with the amoeba, monoFISH was used. Probe for general bacteria (EubI-III) and archaea (Arch915) were used.

Bacterial probe did not gave any signal. This is probably due to a failure in the hybridization process, since many bacteria were present as biofilm attached to the bottom of the dish, so at Fig 5. Fruiting body collapsed after monoFISH. In yellow fluorescence of archaeal probe Arch915. least some fluorescence should have been detected. Archaeal probe on the other hand attached to the fruiting bodies. This result is also debatable, since the fluorescent signal seems to arise uniformly from the buds, without providing any hint on cellular morphologies of putative archaea. It is possible that the archaeal probe bounded unspecifically to the buds. PCR amplification of archaeal 16S anyway provided positive amplification from filtered fruiting bodies, but to have a definitive confirmation on the presence of archaea sequencing of the amplicons should be done.

Fig 6. Late developmental plasmodium stage. Nuclei in cyan stained with Hoechst 33258.

Outlook

Further efforts in characterizing putative bacteria/archaea with Corallomyxa tenera should be done, as well as trying to cultivate axenically the organism in order to be able to obtain genetic material valuable for high quality sequencing. Transcriptomic analysis coupled with behavioral and morphological observation of different life stages of the organisms could provide valuable information on the biology and life style of this elusive and fascinating eukaryote.

References

Tekle Y.I., Grant J., Cole J.C., et al. 2007. Multigene Analysis of Corallomyxa tenera sp. nov. Suggests its Membership in a Clade that Includes Gromia, Haplosporidia and Foraminifera. Protist, Vol. 158, 457—472

Manz, W., R. Amann, R., Ludwig, W., et al.(1992).Phylogenetic oligodeoxynucleotide probes for the major subclasses of Proteobacteria: Problems and solutions. System. Appl. Microbiol. 15:593-600.

Pawlowsky J and Holzmann M. Molecular phylogeny of Foraminifera – a review. 2002. Europ. J. Protistol. 38, 1–10

Fouque E., Trouilhé M.C., Thomas V. et al. 2012. Cellular, Biochemical, and Molecular Changes during Encystment of Free-Living Amoebae. Eukaryot Cell. 11(4): 382–387.

Koonce M. P., Euteneuer U., Schliwa M. 1986. Reticulomyxa: a new model of intracellular transport. J Cell Biol. 145-59.

APPENDIX

Link to videos:

Nuclei movement: https://www.youtube.com/watch?v=atvoRZ-WuBY

Fruiting body explosion: http://youtu.be/PuKvP6H5tBI

Cytoplasmic streaming: https://www.youtube.com/watch?v=RmB-3oih-VY

Buds are good for you: https://www.youtube.com/watch?v=sBXkFW7rct0

Mitochondrial flow: https://www.youtube.com/watch?v=JI6Z2cTrZzY

Plasmodium reshaping itself: https://www.youtube.com/watch?v=OXdS51F9NzY