The capacity for regeneration is a common feature of our most distant metazoan relatives. The comb rows of some cnetophores grow anew if ablated, via onsite cell reorganization (Tamm 2014). Cnidarians,such as Hydra can reform a polyp from fragments of individuals or even entirely dissociated cells, thanks to the stem character of their cells (Bosch 2007). Sponges possess similar capacities: some can regenerate by the formation of gemmules, cellular bodies which transform into a functioning individual at fast rates (Thoms et al. 2008). Even more impressive is the ability of these animals to reform a fully functioning adult individual from dissociated cells. It has been observed in multiple sponge species (Zhang et al. 2003, Wilson 1907). The cells even discriminate between cells coming from other species, although not always, other individuals, and either aggregate only with themselves, or as in the case of some larvae, other individuals (Jarchow and Burger 2008, McGhee 2006, Leith 1979). It seems like sponges have organizational capabilities that enable for this flexible regeneration.

            The poriferan body plan is organized around an aquiferous transfer system, which enables its filter feeding. The structures and cells are highly mobile and different cell types are found loosely in the tissues, as are spicules, a form of mineral endoskeleton. Sponges are sometimes even capable of moving on the substrate. Growth is indeterminate and the organization of the aquiferous system subject to change (Ruppert et al. 2004). With such a dynamic organisation, the regeneration capacities of the sponge are perhaps unsurprising. But what are some underlying mechanisms?

            The formation of sponge clones from dissociated cells was first observed by Wilson, at the beginning of the 20^th century on degenerating animals in culture, with spurred him to come up with a method of squeezing chunks of tissue through cloth to separate the cells in water artificially (Wilson, 1907). Dissociated sponge cells first clump into so-called primmorphs, in which they regain the high telomerase activity that characterizes them and that was lost with dissociation. There is therefore a first stage with no cell division but simple reorganization, and reformation of the outside layer, the pinacoderm (Custodio et al. 1998). This pattern is known for multiple species of sponges (Custodio et al. 2008, Sipkema et al. 2003). Initial formation of these aggregates is known to occur through “aggregation factors”, molecules that bind to receptors on the external surface of other sponge cells. Their concentration is known to have an influence in aggregate formation success (Lavrov and Kosevitch 2014) therefore initial cell concentration should matter in the formation of primmorphs.

            Indeed, this is what Sipkema et al found: primmorphs formed were larger when cell concentrations were higher in the culture (2003). But does this trend vary within related species? Amphimedon queenslandica seems like an ideal model sponge to comparatively assess the primmorph formation. Indeed, its genome is sequenced (Srivastana et al. 2010), and lends itself well to developmental biology studies (Degnan et al. 2008). We therefore chose it and another Nipahtidae sponge to observe initial primmorph formation at different cell densities in this study.

We aimed to address the questions: What is the morphology of the primmorphs formed? How does sponge cell reaggregation vary with decreasing cell concentration? How does this vary across the species of sponge?

Our prediction was that the cells would reagreggate, with the most concentrated treatments having either more aggregates, or larger ones. Accordingly, the lesser-concentrated treatments would have smaller aggregates or none at all. We expect to see a threshold in concentration at which the cells are so sparse that they are unlikely to come into contact to reaggregate. The aggregates observed could have gone beyond the primmorph stage and reformed some choanocyte chambers, possibly have produced new spicules. We expect the size and shape of aggregates, and capacity to aggregate to be different across species in general.

Study sponges: Niphatidae

            The experiment was performed on live animals harvested from the class aquarium originally collected at Heron Island, at University of Queensland, QLD. After preliminary assays of experimental procedure, two species of sponges were selected to enable for comparison of the results: Amphimedon queenslandica and another species. The second sponge was identified with the help of a classmate. A small sample of tissue was bleached for a week and observed in the compound microscope in order to see the sponge’s spicules.

            Both of the study organisms were individual haplosclerid sponges from the family Niphatidae, with the second sponge possibly being of the genus Amphimedon but not confirmed. Therefore, they are perhaps close relatives.The niphatid family contains 13 genera (van Soest 2015), and is defined by characteristics in spicules (Garcia Santos et al. 2014). Some aggregates from other sponges of the order Haplosclerida have been successfully obtained in lab conditions (Holmes and Blanch 2008), so aggregation was likely possible in our species.

Cell squeeze and dilutions

            We obtained cell tissue for each of the sponges from one individual only to avoid confounds. For Amphimedon queenslandica, a piece of roughly 5 cm³ was cut off the sponge, whereas the other niphatid individual was completely scraped off its coral rubble support. The sponge tissue was always kept is seawater to avoid stress.

            The cells were mechanically dissociated using a cell squeeze apparatus. It consisted of a syringe with a mesh bottom, through which the cells were pushed into filtered seawater. The solutions obtained in this way were estimated for cell density using a hemacytometer. We equalized the cell densities between the solutions of the two individuals. We then proceeded to dilute the solutions into different treatments and added equal volumes for each treatment (Table 1). The sponge solutions were kept for a week at room temperature and with a normal light cycle.


Table 1: Cell concentration of the dissociated solutions.

Observations and image acquisition

            After a week, the solutions were assessed for aggregate structure and presence, and images were acquired. All of the solutions were first observed and photographed under a dissecting microscope. The aggregates of most concentrated solutions only, because of time constraints, were then observed under the dissecting microscope in order to have a look at their structure. The tissues had to be crushed to get trough the thick layers of cell. The most concentrated solutions were also stained with DAPI and trypan blue using standard protocols. This was done in order to see which of the cells were living (DAPI) and which were debris (trypan). Only these treatments were selected because we predicted they were the most plausible to have succeeded in terms of having structured aggregates. 

The cells of both of the sponge species formed aggregates after a couple of hours, even though there were no living cells detected with the imaging techniques and bacteria were abundant in the cultures. Since sponges feed on them (Ruppert et al. 2004), this could be an indicator of bad health. The death of the primmorphs could be attributed to many factors, without meaning that these two species do not have the capacity for regeneration. The bacterial and fungal contamination of the cultures has been a problem in other studies (Sipkema et al. 2003). Additionally, the mechanical disaggregation of tissue and agitation causes damage to the cells. Ideal temperatures for the maintenance of primmorphs are much lower than room temperature, at which our cultures were kept (Custodio et al. 2005). With this in mind, it is hard to imagine how cell reaggregation could occur in natural conditions, even though it seems it should be adaptive in stressful times. 

In most respects the aggregates from both species seemed similar in structure. They both contained spicules, which most likely were debris from the squeeze, although their production anew cannot be ruled out. Additionally, Sipkema et al. found that their aggregates had a cuticle covering the pinacorderm that rendered the cells impossible to see, which leaves the possibility open that some living cells were present. However the integration of debris in the primmorphs could also be a cause of the cell deaths (Sipkema et al. 2003). This could be remediated to in the future by taking the debris out of the culture, as Sipkema et al. (2003) did. This seemed to be a problem in both cultures.

An interrogation that arises from the fact that the primmorph cells were dead is whether dead cells aggregated or whether they were living when the process took place. Indeed, it would be of interest to know whether the aggregation factors could perform their tasks without living cells for biotechnological applications for example.

The aggregates showed interesting patterns in terms of shape and abundance. The second niphatid species seemed to have aggregates of a more branched nature, which could mean it aggregates faster than Amphimedon q. This was in spite of the concentrations for Amphimedon solutions being higher (Table 1). This configuration could be the result of fusion of multiple spherical aggregates, such as Sipkema et al. (2003) observed. However, in their observations, the primmorphs resulting from the fusion returned to their original rounded shape. A possible interpretation is that the cells died before returning to that shape, or that they needed more time to do so.

Visually, the initial concentration of cells was a good predictor of the size of the tissue aggregates for both species. This is consistent with Sipkema et al.’s findings (2003). Additionally, our concentrations were in the same range as those in their study. For Amphimedon, it seems that the threshold concentration for the formation of these aggregates is between 1.53 and 0.76 x 10⁶ cells per mL. It was not detected for the other species, so it must be below 0.11 x 10⁶ cells per mL, which would be over a tenfold difference. However, it must be taken into account that the estimate of concentrations in this study was coarse, as the sponge cells were difficult to quantify.

Our aggregates certainly did not go past the primmorph stage, but other studies have been successful in obtaining functioning aggregates and functional adults. After this initial stage, the cells go through multiple morphological stages as they increase in complexity (Eerkes-Medrano et al. 2014). The ability to do this has not really been linked with evolutionary relationships (Holmes and Blanch 2008). It would be of interest to pursue aggregation experiments on our two species, and more niphatids and determine how closely related they are to see if the concentration thresholds vary within the group.

In conclusion, formation of primmorphs occurred and took on a similar form in both species, even though the second niphatid seemed to have gotten more advanced on the process of becoming functional aggregates. As we predicted, aggregates decreased in size with concentration, but also in shape complexity. A threshold concentration was only found in Amphimedon, and the other species is able to form aggregates at lower concentrations in general. Future studies should take into account the external environment and stress factors identified here to obtain aggregates that can be used for developmental biology studies.

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