Introduction

The seawater on coral reefs tends to have low concentrations of many nutrients that sponges and corals, which are the main reef builders, need to survive. Because of these low nutrient concentrations, corals and sponges tend to use nutrients very efficiently and can thrive in these nutrient poor conditions. In fact, corals in particular, are sensitive to changes in nutrient types and concentrations, with too many nutrients negatively affecting coral growth and survival. Therefore, the types and concentrations of nutrients, as well as the sources and sinks -that is, where do they come from and where do they go? - for these nutrients are of interest to coral reef researchers to better understand changes to the coral reef ecosystem and how these changes might affect the organisms that live on coral reefs.

Because sponges are constantly pumping water through their bodies, we were interested in testing the hypothesis that sponges alter the amount and types of organic nutrients in the seawater on coral reefs. Sponges act as a filter on coral reefs removing bacterial cells and dissolved nutrients from the water column. The nutrients we examined include metabolites like amino acids and vitamins that are dissolved in the water and collectively known as dissolved organic matter (DOM).

These nutrients are important to many coral reef organisms from microbes to the reef building corals. However, scientists still don’t understand the factors that influence the types and concentrations of nutrients on coral reefs. In our project, we examined the type and concentration of DOM ( = DOM profile) in four main types of seawater:

1) “ambient seawater” collected on the reef but not immediately near a sponge

2) “exhalent seawater” that was released from a sponge that was host to a dense community of microbes (scientific name: Ircinia campana, shown below as the red sponges)

3)  exhalent seawater from a sponge with few microbes (scientific name: Spheciospongia vesparium, shown below as the gray sponge)

4)  seawater collected away from the reef (“off reef”).

 

We expected to see that the exhalent seawater would have a very different DOM profile from the off reef seawater, while the ambient seawater would have a profile that looks like a mix of the exhalent and off reef profiles.

Methods

The collected seawater samples (~ 1 L each) were brought back to the laboratory where they were filtered to remove particles including bacteria. This filtered seawater was then used for three different types of analyses:

1) Some of the water in each sample was used to measure total organic carbon (TOC), which is a broad measurement of how much DOM is present in the water. 

The DOM was extracted from the rest of the seawater using solid phase extraction, where we pass the water through a cartridge and the nutrients stick to the cartridge material. Then we can elute the DOM in a small volume so we can detect it with our instruments.

The eluted DOM was then used in:

2) Untargeted metabolomics analysis. For this analysis, samples were run on a mass spectrometer that yields a list of mass values and a relative concentration. This means we get a detailed profile (types and concentrations) of compounds present in each sample. With this we can compare the types and concentration of compounds between sample types, giving us an idea of how similar or different the profiles are between sample types.

3) Targeted metabolomics analysis. For this analysis, samples were run on a different mass spectrometer that looks for specific compounds for which we know the identification. This gives us a list of known metabolites such as vitamins and amino acids that are present in each sample and the concentration of these compounds. For example, we observed that the amino acid tryptophan was higher in concentration, on average, in the exhalent seawater compared to the inhalant seawater.

Results

We saw three main trends in our data:

The first is that the profile of DOM in the exhalent seawater is different from that of the inhalant and off reef seawater. 

One way that we can see this is by comparing the types of compounds within each seawater type, as shown in the figure below. When we do this, we see that the exhalent seawater has the highest number of \"unique\" compounds - in other words, those found only in the exhalent samples.

 

This figure shows the number of unique and shared compounds across all three sample types indicated by the three circles. There were 1639 compounds in all three sample types. The numbers in the circle represent the number of compounds found in each sample(s). As an example, the exhalent seawater contained a total of 2362 compounds; of these, 334 were found only in the exhalent samples while 347 were found in both the exhalent and off reef samples.

Additionally, there were 400 compounds that were significantly different (either higher or lower in concentration) between the exhalent and inhalant seawater. The majority of these significant compounds were elevated in the exhalent seawater compared to the inhalant, indicating that the sponges are contributing a suite of compounds to the surrounding seawater. Clearly, the sponges (including the microbes that live within the sponge) are changing the DOM profile of seawater as they pump it through their bodies!

The results described above are at least part of the reason that the exhalent seawater samples were so different in terms of the type and concentration of DOM. Lastly, when all of the compounds in each sample are compared based on their presence and concentration in each sample, the exhalent samples stand out as different from the other samples. In the figure below, the inhalant and off reef samples group together while the exhalent samples form a separate group. 

The analysis shown in the figure below uses the DOM profile of each sample to plot samples close together if they are similar or further apart if they are different.

 

The analysis shown in this figure is called non-metric multdimensional scaling (nMDS for short) and uses the DOM profiles to compare samples. The closer together the dots are, the more similar the DOM profiles are between those samples. The exhalent samples all group together on the right, while the inhalant and off reef samples group together on the left. In this analysis, the axes do not represent a specific trait or factor, rather they represent a scale that shows the similarity of the DOM profiles (type and concentration of compounds in each sample). This is why they are labeled "Axis 1" and "Axis 2". In the legend, "In" = inhalant and "Ex" = exhalent.

Second, the sponge-derived DOM (compounds in the exhalent seawater) did not appear to change the profile of DOM in the surrounding seawater near the sponges (inhalant).

We can tell this in part because the inhalant samples group more closely with the off reef samples in the analysis shown above. If the inhalant seawater samples had a DOM profile that was more strongly influenced by the exhalent seawater then we would expect the the inhalant samples to be in between the exhalent and off reef samples in the figure above.

However, we did see something else that was really interesting. Previous studies have shown that sponges remove DOM from seawater. Our results from the metabolomics analysis showed that it is more complex than this. Sponge may remove a large portion of DOM, but more specifically, sponges are adding some compounds to seawater and remove other compounds, effectively changing the DOM profile of seawater as it passes through the sponge. You can see this in the figure below which shows the maximum amount of each metabolite that we estimate would be released from each sponge each day. The amount is in nanomoles of the metabolite per day and as a comparison, the concentration of amino acids in shallow near-shore seawater is ~15 to 50 nanomoles per liter of seawater. It is easy to imagine that a whole reef full of sponges could add a substantial portion of amino acids and other metabolites to the surrounding seawater. You'll see that some metabolites have negative values, this means the sponge is removing this compound from the seawater.

Third, the DOM profile in the exhalent seawater of two sponge species (I. campana and S. vesparium) were highly similar

The overwhelming majority of compounds (85%) in the DOM profiles were found in the exhalent seawater samples of both species. 

We also used a statistical test, a t-test, which is used to determine if the averages of two samples are significantly different, to compare the concentrations of compounds in the exhalent seawater between the two sponge species. There were no significant differences in concentrations of compounds between the the exhalent seawater of two sponge species. There were however, a few compounds that were only found in seawater exiting from I. campana and others that were only found in the exhalent seawater of S. vesparium.

These results indicate that the DOM profile of the exhalent seawater of the two sponge species are highly similar. This was surprising because the microbial communities of the two sponges are very different and we thought this would influence which nutrients and how much of the nutrients would be removed or added to the seawater. It is possible that the metabolism of the sponge is a greater factor than that of the microbial community in altering the DOM profile of seawater. If this is true, then all sponges may alter the DOM profile in a similar way (a \"sponge signal\" in DOM) and we might be able to predict how a community of sponges would alter the nutrients in seawater on a coral reef. 

However, we have only sampled from two sponge species; thus, further work is needed to determine if the DOM profiles from other sponge species are similar to those observed in this study. Additionally, there were some compounds that were detected in the exhalent seawater of I. campana only or S. vesparium only, yet we do not know if these compounds are significant to the biology of the sponges or to other organisms on the coral reef. Lastly, we do not yet know if the DOM profile in the exhalent seawater changes over time or in response to environmental factors such as temperature, nutrients, or light levels. All of these questions represent opportunities for future research that can now be addressed because of the significant foundational work that this study provided. 

Conclusion

This study showed us several exciting and interesting results. We saw for the first time, that sponges are doing more than just removing organic nutrients from the seawater, rather, they are adding some compounds and removing other compounds! This means there is some influence that sponges have on the availability of organic nutrients in seawater. However, we also saw, at least, in the region where we sampled, that this influence from the sponges does not change the overall profile of nutrients in the seawater on the reef.  As such, this work raises several intriguing questions:

1) Is there more of an impact from the seawater released by sponges on the overall reef DOM profile in areas where there are more sponges? 

The area we sampled was nearshore and had relatively few sponges compared to an offshore reef. It is possible that we could see more of the \"sponge signal\" in the DOM profile of reef seawater in areas with more sponges. However, it may be that given the sheer volume of seawater flowing in and out over a coral reef that the changes that sponges make to DOM composition of seawater is still small. To address this question, we would need to perform a larger version of the current study, including different locations and more sponge species. We have used the results generated in this study in a proposal to the National Science Foundation that would allow us to do a similar experiment on a larger scale. We did not receive funding initially, but we did receive positive and constructive feedback on the proposal and we will try again! This is typical for large proposals and it often takes a couple of iterations to be successful in obtaining funding.

2) Does the exhalent seawater from sponges, which we now know contains a different composition of nutrients from the rest of the reef seawater, benefit the growth of other organisms, particularly corals?

In other words, are any of the compounds that the sponges release (or remove) beneficial for coral growth or resilience? The aquaculture of corals provides a perfect avenue to address this question. In aquaculture, small corals are typically grown on an artificial substrate in the ocean or in tanks in a coral nursery, then transplanted to a coral reef when they are big enough (https://en.wikipedia.org/wiki/...). During a recent coral reef conference, where we presented our work to other researchers and managers, we spoke to a scientist who works on coral aquaculture about collaborating to address this question. We are currently working with the researcher on a proposal that would allow us to see how the addition of sponges to coral nurseries affect coral growth and other health indicators for the corals, and determine how much of the \"sponge-derived DOM\" is utilized by the corals.

Other questions and future goals include: 1) better understanding the ecological implications of specific compounds that are released by sponges (are certain organisms using these compounds? do they have an impact on which organisms can grow in the area? how much is used right away and how much, if any, makes it off the reef?), 2) understanding the role that sponge metabolism vs. microbial symbiont metabolism have in structuring the DOM profile in the exhalent seawater, and 3) determining other factors that may influence how sponges alter the DOM profile of seawater.

Acknowledgements:

The banner image was taken by Dr. Jessica K Jarett. The artwork used in the first figure was created by Krin Grande.

Thank you to all of our backers, this work would not have been possible without you!