Amira

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Initial Questions

I’ve modified questions 2 & 3 to become more specific, but I haven’t really changed question 1. I’ve been doing more research on coral bleaching, and there are apparently two different hypotheses in the literature for the mechanism by which the alga leaves the coral polyp (the coral poly physically expels the alga after being stressed VS. the zooxanthellae actually leaves the polyp after sensing a stressed environment). Both of these hypotheses examine corals under stressed situations. However, it has been shown that the zooxanthellae can re-inhabit the coral polyp if the stress upon the coral is removed thereby allowing it (or perhaps it “chooses”) to return. There is still very little known about this mechanism of reincorporation (I just made that word up, I don’t know what the real word would be)…so it’s definitely something I’d be interested in looking into.

Question 1: What mechanism drives a zooxanthellae alga to re-inhabit a coral polyp after expulsion via coral bleaching? Can we learn by looking at the evolution of this symbiotic relationship? Is the mechanism similar to the one that expels the zooxanthellae in the first place?


It's a thin line between symbiosis and parasitism by one 'partner' on the other -- so might it be interesting to focus on who's doing the expelling and who's initiating relationship? And, of course, how the costs and benefits to each party play out? Kwoods 13:15, 16 September 2011 (UTC) Also -- a practical question -- do you approach it as 'what mechanism'? Or as 'what is the trigger'? Both interesting, but one may offer a more 'doable' starting point? Can you formulate hypotheses from what you know?

  • I’ve also been looking to the nature of the symbiotic relationship…which can also be seen as slightly parasitic (the coral “enslaving” the algae for its own benefit) although it’s debatable. I wonder if this would be a good direction to go in when examining the “reincorporation” method?

This seems like an interesting question. It seems like you would want to narrow the question down to some competing hypotheses, like maybe: does the coral grab a passing alga, or does the alga seek out the coral? (I'd probably realize why that question was dumb if I knew more about corals and algae, but something like that.) Then thinking two things: what could you measure to distinguish your two competing hypotheses? And, would either of those hypotheses say anything about the evolution of the relationship? What would be really nice if you had something like, "following the logic of X theory of partnership, coral and alga should reunite by mechanism A, but following the logic of Y theory, they should reunite by mechanism B. And we'll tell mechanism A from mechanism B by the following experiment..." I'm sure what I am saying here is dumb in particular details, but I do think that looking for hypotheses - based on some theoretical understanding - would be the next place to go with this question. – Andrew

  • It’s actually really interesting because when I was reading about the symbiotic relationship that corals have with algae, I learned that there are actually two ways that a coral polyp can initially incorporate the zooxanthellae. Method one involves the “mother” coral partitioning new “baby” coral polyps with algae of their own before expelling them into the ocean where they will eventually settle down. Method two (which I think provides more to work from for further research) is when these newer polyps somehow acquire algae of their own as they float through the water column. It’s an interesting question…I still have a lot to learn!

I think that I’ve been having so much trouble formulating specific research questions because I’ve been having a hard time deciding what organism to study. Ideally I would love to study corals or sponges, but I’ve realized just how difficult it is to do lab work (as opposed to field based/wet lab based work) with those organisms. I’ve been reading a lot lately about biofilms (mostly stromatolites), and after a lengthy chat with Betsy, I think I’ve narrowed down my organism of interest to a living biofilm that I could create myself in a lab setting. Depending on the process by which I grow these biofilms, I might want to narrow down my question to something related to their actual development (this would be largely design based) but also want to continue looking at the characteristics of the biofilms themselves:

    • Stromatolites are not biofilms themselves, but large structures (some were house-size) built, over extended periods by biofilms in combination with other processes. They're interesting in a lot of ways, though. Biofilms are also important in fresh-water streams where they form on surface of sediments and can be important primary producers as well as affecting sediment stability... Kwoods 03:58, 11 November 2011 (UTC)
    • Okay, but don't give up on a question HERE because you can't do the lab work -- BOTH because a) this is about thinking about how to design research, not a commitment to doing the particular project, and b) why not think of it as field-based research if you can see a way to do it? Kwoods 03:58, 11 November 2011 (UTC)
      • That's a really good point. I think in that case, my third question would revolve around the palatability of various algae, and my test organism would be a fish with a diverse feeding range (perhaps a sharpnose puffer...I know they try nibbles of almost everything, and will spit out food they don't like). Ahankin 16:45, 11 November 2011 (UTC)

Question 2: How do biofilms form? What accelerates their growth, and what stunts it? Why do these large bacterial colonies exist?

I would also like to look at/ compare biofilms that grow in extreme environments (thermophilic biofilms) to biofilms that grow in milder environments.

  • Note that the first questions are not yet very 'hypothesis-generating' -- 'what' and 'how' need to go towards 'is it because of...' or 'which of these factors is more important...' and so on. The 'compare' question begins to be a bit more productive -- but it may be important to recognize that different biofilms may be as different as totally unrelated oranisms of any other group; thermophilic biofilms and 'mesophilic' ones might be made up of entirely different sets of microorganisms. Don't know if this matters to your question... (also -- 'thermophiles' are about heat; that's only one kind of extreme environment. There are also halophiles that tolerate high salt, etc...)Kwoods 03:58, 11 November 2011 (UTC)

I’m having trouble wording my third question, and would love to talk about it more, but basically I want to do a feeding assay (using fish and various algae) to determine the palatability of each plant (to better understand the presence of these chemical defenses.) This is something I’d like to discuss in class.


Question 3: How does marine sponge morphology (i.e. tube shaped, encrusting, or vase-like structures) contribute to the sponge's antifouling or predator deterrent properties?

Project Proposal

Intro

Q: How does the inter-species variation in the morphology of a marine sponge (phylum Porifera) contribute to the organism’s predator deterrent properties?

After transitioning through a motile larval stage, porifera become sessile at maturity, eventually anchoring themselves to a site permanently. As soft bodied, sessile organisms they possess an inherent need to adapt to an environment they are physically vulnerable to, and cannot escape from, namely a need to be able to defend themselves from predators. Sponges therefore contain chemical defenses found within their tissues that deter predators from over grazing or destroying their structures completely through predation (Pawlik et al. 1995, Wilson et al. 1999, Assmann et al. 2000). These chemical defenses, or secondary metabolites, make the sponge tissue unpalatable to predators, thereby marking it as inedible (Wilson 1999). Previous research indicates that the majority of these marine secondary metabolites are structurally and stereochemically complex in nature (Fenical 1997), suggesting that they are metabolically expensive and therefore must be inherently adaptive, playing a role in the organism’s ecological function (Paul 1992, Pawlik 1993). According to the optimal defense theory proposed by Doyle McKey (1974) an organism channels these chemical defenses to body parts that are most vulnerable to predation in a way that will maximize the organism’s fitness. This theory assumes that resources are limited and the production of these defenses is costly, and are therefore unique to the organism’s individual needs. Previous research by Schupp et al. (1999) found, in the Micronesian sponge Oceanapia that the highest metabolite concentrations could indeed be found in the parts most visible to predators, therefore supporting optimal defense theory and the notion that these secondary metabolites are of ecological importance.

Based on the theory that defensive secondary metabolites may be concentrated in the more vulnerable body parts that are first encountered by predators, a sponge with more bodily protrusions (one that typically grows outwardly and is larger) is subsequently more accessible to predation and as a result would have a greater need for defense compared to a sponge that is less accessible (an encrusting sponge that grows close to the surface of its substratum). My hypothesis is that sponges with vase-like or tubular morphologies would be chemically defended to a higher degree than sponges with encrusting morphologies, thereby rendering vase-like and tubular sponges less palatable than encrusting sponges.

Through a series of bioassays using several species of porifera with varying morphologies and the sharpnose pufferfish Canthigaster rostrata as a generalist predator, I would like to test the relative palatability of these three sponge morphologies (encrusting, vase-like, and tubular).

Methods

Proposed Methods referenced from Burns et al. (2003) and Wilson et al. (1999).

An example of three sponges with distinct morphologies that are found in the Caribbean:

  • Vase-like: Verongula gigantea (Netted barrel sponge)
  • Tubular: Aplysina cauliformis (Row Pore Rope Sponge)
  • Encrusting: Chondrilla nucula (Chicken Liver Sponge)


I am going to measure, in a feeding assay using the sharpnose pufferfish, Canthigaster rostrata, the percentage of each sponge sample eaten by the pufferfish relative to the other samples to determine which samples are least palatable and which ones are most palatable. The sharpnose puffer is a small, omnivorous Caribbean fish with a variable diet, thereby making it an optimal test subject for this experiment. For my sponge extracts I would choose multiple sponge species of each morphology to run this experiment, picking each one from the list of 49 out of 71 sponge species that have shown, experimentally, to contain chemical anti-predatory defenses (Pawlik et al. 1995). This would hopefully give me a better idea of a range, if any, of chemical defenses found within sponges of similar morphologies. An example of one species of each morphology can be seen in “Pictures” section of the wiki below.

Sponge Collection

Sponge samples of different species with the three basic morphologies (vase-like, tubular, encrusting) would be collected around the Bahamas islands during the same period of time. Each sample would be collected by cutting the base of the sponge from the anchored point with a sharp instrument. Sponge samples would be stored immediately in separate bags at -20°C for subsequent extractions.

Extraction

The frozen sponge samples would be cut into small cubes and then placed into a graduated cylinder containing 1:1 dichloromethane-methanol (DCM/MeOH). The DCM/MeOH would work to extract the chemical compounds found within the organism from the sponge tissue. This extraction would be done twice, leaving samples in the DCM/MeOH for upwards of 12 hours. After extracting the chemical compounds, the resulting solution would be concentrated using a rotary evaporator. This crude extract is what would be used in the feeding assays.

Food Preparation

The crude extract from each of the sponge species would be added to mixture of 0.3 g alginic acid 0.5 g of powdered squid mantle. The squid mantles serves as an excellent substrate for the test extracts int terms of texture and overall density. This mixture would be stirred until homogeneously distributed and then loaded into a syringe. The paste would then be injected into a 0.25 M solution of calcium chloride creating long, noodle-like strands of sample. After leaving to harden, each strand would be rinsed in seawater and then cut down into 3 mm long pellets. Control pellets would be created in the same way, without the addition of crude extract. If necessary, food coloring would be added to the control pellets in order to match the color of extract-treated pellets.

Feeding Assays

Feeding assays would be performed twice a day (morning and evening) with 10 sharpnose pufferfish collected in the same areas (around the Bahamas) as the sponge samples. Each individual assay would begin by presenting the pufferfish with a control pellet to insure that they were hungry. If the fish did not eat the pellet, they would be excluded from that particular feeding assay. If the control pellet was eaten, they would be presented with an extract-treated food pellet. If the extract-treated pellet was not eaten (or visibly rejected) the fish would be given a control pellet to determine if the pufferfish was full. A pellet would be considered rejected if the sample was approached by the puffer and then not eaten, or if taken into the mouth and then spit back out. If the control pellet was eaten, the rejected, extract-treated pellet was determined unpalatable for that fish and marked as so. If the control pellet was not eaten, the fish would be considered satiated and the results from that particular assay would be disregarded.

The feeding assay would continue, alternating between treated and control pellets to determine hunger level of each fish before the experimental sample was given. The result of each feeding would be noted, marking samples eaten versus samples not eaten (marked “YES” or “NO”).

Proposed Results

I would expect extracts from the encrusting sponge to be eaten more often and rejected less than those of the tubular or vase-like sponges. Generally, I would expect samples from the smaller growing, less accessible (i.e. fewer protrusions) sponges to be more palatable if they aren’t as heavily chemically defended.

To analyze my results I would count up the “YES” responses for each species of sponge from a week’s worth of feeding assays (twice daily, 7 days a week) and divide each number into the total number of extract samples given. I would then compare results for each species.

If there is a correlation between a sponge’s morphology and the extent to which it is chemically defended I would expect to see Chondrilla nucula with the highest percent eaten out of the total amount of samples given in the feeding assays. I would also expect to see a lower percent eaten for Verongula gigantea and Aplysina cauliformis, with both of these larger structured sponges chemically defended to a similar (higher) extent.


Conclusion

The results of an experiment such as this would be crucial in better understanding the connection between porifera morphology and chemical defense strategy, if there is one. Understanding this connection could in turn provide me with a stronger basis for future studies regarding the ecological importance of secondary metabolites and chemical defenses found within marine organisms.

If there is a connection, a future experiment could shed light on the varying types of secondary metabolites found in different species, or simply identifying and better understanding the compounds that exist within the sponge tissues. Could sponge species that are closer to each other evolutionarily contain similar compounds, or similar concentrations of compounds? Another follow-up question I have, regardless of whether or not there is an inter-species difference in chemical defense production, is if there is a difference in concentration of these secondary metabolites dependant on the location of the tissue of the sponge. Would the tips of a tubular sponge contain higher concentrations of a secondary metabolite than its base due to the fact that it is more exposed and therefore more vulnerable to predation?

Another direction to go in could be to examine the costs versus the benefits of allocating resources towards the production of thee secondary compounds. Studies in plants have shown that there’s a trade-off between allocating resources to defense and allocating those same resources to growth. The same study indicates that the overall allocation of resources to defense is negatively correlated with the plant’s growth rate (Bazzaz et al. 1987) . Could the same be true of marine organisms, namely sponges? Exactly how expensive is it to be producing these secondary metabolites?

Lastly, as of recently, secondary metabolites have demonstrated potentially important activity in pharmacological studies (Kitagawa & Kobayashi 1993), and previous research claims that sponges are the most diverse source of marine natural products due to the plethora of secondary metabolites they contain (Faulkner 2000). The chemical compounds found within sponge tissues have also shown great medical potential as anti-tumor agents, antibiotics, anti-inflammatory agents, and more (Kerr & Kerr 1999). How can knowing more about the ecological context from which these sessile marine organisms originated provide insight into the field of medicine?

Pictures


Citations

Assmann M., Lichte E., Pawlik J.R., Köck M. (2000) Chemical defenses of the Caribbean sponges Agelas wiedenmayeri and A. conifera. Mar Ecol Prog Ser 207:255–262

Bazzaz F.A., Chiariello N. R., Coley P.D.Pitelka P.F. (1987) Allocating resources to reproduction and defense. BioScience, Vol. 37 No. 1

Burns E., Ifrach I., Carmeli S., Pawlik J.R., Ilan M. (2003) Comparison of anti-predatory defenses of Red Sea and Caribbean sponges. Mar Ecol Prog Ser 252:105–114

Faulkner D.J. (2000) Marine natural products. Nat Prod Rep 17:7–55

Fenical W. (1997) New pharmaceuticals from marine organisms. Mar Biotechnol 15:339–341

Kerr R.G., Kerr S.S. (1999) Marine natural products as therapeutic agents. Exp Opin Ther Patents 9:1207–1222

KItagawa I., and Kobayashi M. (1993). Pharmacochemical investigation of marine sponge products. Gazz. Chim. Ital. 123:321-327.

McKey D. (1974) Adaptive patterns in alkaloid physiology. Am Nat 108:305–320

Pawlik J.R., Chanas B., Toonen R.J., Fenical W. (1995) Defenses of Caribbean sponges against predatory reef fish. I. Chemical deterrency. Mar Ecol Prog Ser 127:183–194

Schupp P., Eder C., Paul V., Proksch P. (1999) Distribution of secondary metabolites in the sponge Oceanapia sp. and its ecological implications. Mar Biol 135:573–580

Wilson D.M., Puyana M., Fenical W., Pawlik J.R. (1999) Chemical defense of the Caribbean reef sponge Axinella corrugata against predatory fishes. J Chem Ecol 25(12): 2811–2823