Anatomy & Physiology

The general Protopalythoa polyp body plan consists of a thick body wall without a calcareous skeleton, that instead has sand and other debris incorporated into it (Erhardt & Knop 2005; Mather& Bennett 1994), a large body cavity, tentacles, actinopharynx and mesenteries (partitions within the polyp). Mesenteries can be either complete (macrocnemes) or incomplete (microcnemes) (Ryland & Lancaster 2003).

Diagram illustrating a longitudinal cross-section of a single Protopalythoa polyp.  Adapted from Ryland & Lancaster (2003).	Diagram illustrating a cross-section of a Protopalythoa polyp. White arrow indicated the ventral side of the organism, black arrow indicates dorsal side.  Adapted from Ryland & Lancaster (2003).

Protopalythoa polyp under white, 'normal' light.	Natural fluorescence of Protopalythoa polyp under GFPA.
Protopalythoa polyps natural fluorescence under RFP1.	Combined GFPA and RFP1 fluorescence images, created using ImageJ.

 

In order to determine the validity of this assumption, and to examine the organism in greater detail, histological sectioning of two individual polyps from the colony was conducted. It was originally proposed that sectioning would reveal the cell types that the Protopalythoa polyps used when naturally fluorescing. Preparation of polyp samples was carried out on Heron Island Research Station. 
In order to ensure that the polyps would be in a relaxed state (i.e. open, with tentacles extended), Magnesium Chloride was added to the container of seawater that the organism occupied a few drops at a time, until the colony no longer responded to mechanical disturbances. Once sufficiently relaxed, two polyps were surgically removed from the colony using a scalpel and fine-point tweezers. Once these were isolated and in a separate container, the remaining polyps of the colony were returned to a tank of fresh seawater to recover, and were later returned to their natural habitat.
The two separated polyps were then prepared for fixing (preservation) by adding drops of 4% saltwater paraformaldehyde (PFA), and were left to sit for approximately 5 minutes. All of the water was then removed and replaced with enough 4% PFA to submerge the polyps. The sample was then allowed to sit for approximately 24 hours to ensure adequate fixing.Samples were then gradually moved into an Ethanol solution for storage until they could be sectioned and stained back at the University of Queensland. 
Samples were sectioned and submerged in DAPI blue and Phalloidin solution for approximately half an hour to stain. Slides were then removed and drained of excess stain, and two drops of glue added to sample surface, to which a coverslip was applied. Samples were then placed in a refrigerator to set glue and help preserve autofluorescence for image capture.
From looking at slides under a standard compound microscope, with photos taken using a DinoLite camera and DinoXcope software, certain internal features were visible, but were very difficult to distinguish until further research had been undertaken. As a result, images are limited, and further images will be provided at a later date.
Evidence of sand being incorporated into the mesoglea was present, and parts of the mesenteries are visible in the photographs below, illustrating the sectioning of the body cavity within the polyp. These mesenteries could potentially contain either reproductive cells or retractor muscles, but the way that the sections were cut and a lack of knowledge regarding these features it was not plausible to assume they were present. Algae is present in much of the body wall visible in the sections, occurring in ‘pockets’ around the body cavity as well as in tentacle cross-sections, suggesting that symbionts occur in the gastrodermis as well the epidermis of the organism.
Fluorescence images of the sectioned organisms were taken using a Nikon Inverted Eclipse Ti microscope, with NIS-Elements software. These images reveal a mosaic of cell types, with the DAPI stain revealing DNA clumps, and Phalloidin binding to actin within muscle cells. As Phalloidin also fluoresces under GFPA, it was difficult to distinguish between the applied stain and the natural fluorescence that was visible in the polyps while on Heron Island. However, the images that were produced indicated that in the tentacle cross-section, there was a high concentration of algal cells, indicated in the image below by the red fluorescence. The images suggest that this particular species of Protopalythoa is host to a symbiotic zooxanthellae, although further research would need to be conducted to determine which species of symbiont it is. Unfortunately the cell types that fluoresced under GFPA could not be determined.

Cross-section of polyp tentacles. (20x magnification)	Mesentery walls inside the gastrovascular cavity of a sectioned polyp. (20x magnification)	Sectioned polyp body wall, showing incorporated sand grains. (20x magnification)

Longitudinal section of what is believed to be a tentacle. DAPI (blue) is showing up DNA clumps, while green illustrates Phalloidin stained muscle fibres. Red fluorescence in this image is due to the presence of algae. (10x magnification)	Fluorescence image illustrating mesentery walls (right) and what appears to be mesoglea and ectoderm (left). DAPI (blue) and Phalloidin (green) (10x magnification)	Fluorescence image of tentacle cross-section, showing DAPI stained DNA (blue), Phalloidin stained actin fibres (green) and algal fluorescence (red).  (40x magnification)

Natural GFPA and RFP1 fluorescence of single polyp. Combined image created using ImageJ.	Protopalythoa colony under white 'natural' light. The mouth opening is a slightly lighter colour, but other than that the colony does not appear very colourful.	The same colony image, taken under the fluorescence microscope, showing GFPA and RFP1 fluorescence in the Protopalythoa colony. Note the substrate fluoresces red also, making the tips of the tentacles and the oral opening more noticeable. Image combined using ImageJ.