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Q&A: U Vermont Researchers Using Proteomics to Investigate Evolution of Digestive Functions in Wasp Colonies

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pickett_1.JPGName: Kurt Pickett
Position: Assistant professor in biology, University of Vermont, 2007 to present; curator of invertebrates, Zadock Thomson Natural History Collection, University of Vermont, 2007 to present
Background: Postdoctoral fellow, American Museum of Natural History, 2004 to 2006; PhD in entomology, Ohio State University, 2003

ballif.JPGName: Bryan Ballif
Postion: Assistant professor, biology, University of Vermont, 2006 to present; co-director Vermont Genetics Network Proteomics Facility, 2008 to present
Background: Postdoctoral associate, Harvard Medical School, 2003 to 2006; postdoctoral associate, Fred Hutchinson Cancer Research Center, 2001 to 2003




Researchers at the University of Vermont were recently awarded a National Science Foundation grant for $385,432 to study the social structure of a group of wasps including yellow jackets and hornets. As part of their work, set to start in May, they will perform de novo sequencing of proteins, and integrating proteomics data with genomic and morphologic data.

Specifically, the researchers are interested in the digestive function of the wasp colony. The colony functions as a kind of organism in which different members of the colony are "assigned" different tasks and it appears that only larvae in the colony are able to digest the food that workers bring back. The larvae eat the food and produce a saliva rich in sugar and amino acids, which is then eaten by the workers.

The proteomics portion of the research will be directed at deciphering the protein aspects of this division of labor in the colony, and then to trace the evolution of this division.

ProteoMonitor last week spoke with Kurt Pickett and Bryan Baliff, two of three principal investigators on the project, about their research. Below is an edited version of the conversation.

Describe for me the social structures of these wasps and yellow jackets.

Kurt Pickett: The grant is … to do a variety of things with yellow jackets and hornets. The grant scope is broad and [for a] field called taxonomy, which is my field, which includes things such as phylogenetics, nomenclature, genetics, [and] behavior. It's a very broad field. It's the sort of field that all biologists were engaged in at the time of Darwin.

The project is to look at all these details of a single group of social wasps, the yellow jackets and hornets — there are 69 species in this group — and we're going to decipher the evolutionary tree for the group, using a variety of genetic loci, using anatomical and behavioral characters, and also protein characters.

We're going to analyze aspects of their social behavior. There are a variety of interesting things about this group. Like all social wasps … they partition certain aspects of their labor, so certain members of the group do some things, and other members of the group do other things.

The most basic of that division is reproduction where there's a single or a few individuals that reproduce and the other individuals do not. And this is the same sort of phenomenon that's going on in your body right now. There's a group of cells that are reproductive cells and the rest of your cells are sterile.

This is the most fundamental division, and within the yellow jackets and hornets this division of labor occurs in an extreme form where the queen is extremely different morphologically than the workers. She tends to be much larger than them. The workers have a variety of morphological traits that the queen lacks.

So they have weaponry and specialized mandibles. Obviously, most of the workers are engaged in colony construction and defense, whereas the queen stays in the colony and lays eggs and initiates cells into which the eggs are laid.

Her role is basically reproduction, everybody else's is everything else.

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The interesting thing about this group is that our preliminary data suggests that there are some other kinds of partitioning going in the group. And the one that we're particularly interested in has to do with digestion.

It appears that the larvae of the colony may be in some species the only individuals in the colony that can digest food. And so we know that, like in lots of social systems, the workers will forage for food and they come back to the colony — and the larvae are sessile: they can't really move or do anything to themselves — and the workers will feed the larvae.

In this group an interesting thing happens: the larvae produce saliva, which is known to be rich in sugars and amino acids, and the larvae will upon sort of request by the workers, produce saliva, which the workers then eat.

This caught the attention of some people in the early 1960s and there were some suggestions that there may be some partitioning of digestive labor.

For preliminary data for this grant, we did some investigations that tried to look at what's really going on here … and our preliminary data do suggests that in some species, the larvae are perhaps the only members of the colony that can digest food.

Just like in your body, you have cells that are perfectly totipotent, they have the same genes, they could all do everything, but we don’t do it that way — some cells do the digestion for the rest of the cells.

And the same sort of multi-cellular-like division of physiological labor appears to be happening in these social wasps such that the larvae are, in a sense, the stomach of the colony.

The proteomic piece of the grant is to try to decipher the details of that protein division of labor, and then to trace the transformation from a primitive state where everyone can digest food into the derived evolutionary state where perhaps only larvae can digest food.

We believe our preliminary data are suggestive that this transformation took place around the base of the yellow jackets and hornets. Since we will have a fully resolved evolutionary tree, we'll be able to trace the evolution of this interesting hyper-colonial-level phenomenon.

When you say that when you're trying to decipher the protein part of this division of labor, are you trying to create some sort of proteomic map of these creatures?

Bryan Ballif: To a degree. What we're looking at is the digestive components, in particular, for the larvae. We're looking at the saliva that they give up at the request [of the workers] and we want to examine this saliva for 1) protein activity, and 2) protein content.

First, we would like to see if the saliva is capable of digestion, so we will challenge the saliva in biochemical assays to see how well it can digest proteins relative to the same type of salivary liquids that we get out of the workers.

And we will try to see if it is capable of digestion of proteins.

Second, we would like to know [the] components … in these salivary liquids so we can identify which ones are participating in this, ultimately using this information to understand how this social behavior has evolved.

Will you be creating a protein interaction map?

BB: Not so much for looking at protein-protein interactions in this regard. Cataloging the proteins that are present will be an important and somewhat of a daunting task for us, but that will be the first priority.

We don't get lots of material out of these larvae, so being able to conduct a standard kind of protein-protein interaction map would be challenging at this point, so we're not looking for that initially.

KP: I think it's important to realize that …when people do this kind of proteomic [experiment] they typically work with very well characterized genomes and in some instances, well characterized proteomes, or at least they have a genetic scaffolding to work with.

We're really doing something here that's not been done before. There are some other members of the order, hymenoptera [whose] genomes have been sequenced. They are extremely distant relatives of the wasps that we're studying.

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That's part of the challenge. The other part of the challenge, as Dr. Baillif alluded to, is collecting the saliva itself.

We have to try to find these colonies, which are high in trees and underground, and then we have to rip into these colonies while hundreds if not thousands, sometimes tens of thousands, of workers try to kill us. And then we have to try to get their saliva.

These are all things that we're figuring out how to do. Now, we did pieces of all of these for the preliminary data, and we're very optimistic, but we are still in the preliminary stages and we do believe we're going to be able to complete the work, but it's a little different from typical proteomic-type analyses.

Can you describe how you're approaching this de novo protein sequencing work?

BB: There are a couple of things that need to happen. The first is we need to have methods to preserve these samples in the field where they will be collected. We're working those collection methods out, and we believe now that we have a way to desiccate them out in the field and keep them from degrading.

Then we need to be able to use some kind of a de novo approach in order to identify what these proteins are.

So we're taking a multiple prong approach … First of all, we're using the program PepNovo [developed by the computational mass spectrometry group at the University of California, San Diego] as a way to get sequence information that would allow us to get small fragments of proteins to which we can then hope to map back to particular sets of proteins by the number of peptides that we generate.

At the same time, we are taking the data and we are searching against known proteomes, those genomes [where] we can look for these proteins including the other sequenced genomes such as Apis genome in the same genera hymenoptera.

Even though they're distantly related, we hope that we will also get some matches based on that information.

Ultimately, we believe that we will also incorporate some measure of DNA sequencing into our approach, so that we can hurry it along if we can have more genomic and/or cDNA information.

Contrast the proteomics work with the genomics work and how they'll complement each other.

BB: When we want to look at the phylogenetics of organisms, we want to look at multiple traits. The genomics will be one aspect of it, but given that the proteins that will be functional here in this social behavior are in the saliva of these organisms, we believe that understanding which proteins are present and are responsible for these sorts of behavior will allow us to focus on the genes that have been most influential in the evolution of this behavior.

KP: I think it's important to recognize that when a lot of people do proteomics or even genomics they're typically looking at a single species … and they characterize that.

What we're doing here is looking at 69 species and then some close relatives of that to try to understand how things have changed within that group over time, how we've moved from a situation where everybody can digest food to a situation where only some members of a colony can digest food.

We're trying to characterize these proteins that are responsible for this digestion. We're trying to characterize in which species there is differentiation in the ability to digest food, and then we're trying to look on the evolutionary tree where and when and how this transformation took place.

In a very broad sense, I believe we are looking at the kinds of changes that were the same kinds of changes that gave rise to multicellularity.

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Primitive multicellularity surely derived from colonies of single cells that partitioned their labor over time and became intimately organized into multicellular creatures, which then the colony itself became its own individual.

And I think the same kind of thing is happening at this level in social wasps … and the term we use often to describe that is often called the 'superorganism,' where the individual cells are not cells but are multicellular individuals themselves, which are partitioned into these tasks.

If it's true, for example, that only larvae can digest food, then we can imagine that the larvae represent a digestive tissue of the superorganismic level, which is the colony itself.

We're trying to fuse, if you will, or bridge typical proteomics, which is often focused on one or a few key lab taxa and take that and use what's known there … to bridge these things together so that we can understand something about the evolution of protein systems in these wasps and how they've evolved through time into very interesting divisions of physiological labor.

Does looking at these wasps and yellow jackets specifically allow you to answer certain kinds of biological or evolutionary questions that you wouldn't if you were looking at other creatures, even those with similar social structures?

KP: Absolutely, the family of social wasps that we're studying is really special in that there are a lot of these transformations that I'm talking about, so the very base of the family has completely solitary wasps that do what solitary creatures do: they live alone, they hunt for themselves, they don't like being around other animals.

Even mating is a very tenuous procedure. They don't like being with other animals.

And there is this transformation, and there's a variety of grades, many wasps representing grades of transformation, from that to this advanced sociality where they live in these giant groups and they partition their labor as we see in the yellow jacket.

The interesting thing is that even though we're dealing with these highly social wasps … there is apparently this transformation from one primitive condition, digestive totipotency, into this diversification where some caste of animal in the colony can digest food and other castes cannot.

So there's division of labor nested with division of labor nested with other divisions of labor, and in this group of wasps, you can trace those transformations on the evolutionary tree.

So I do think this group of wasps is special in that it represents all of these transitions.

Aside from de novo sequencing, what other proteomics work will be included in this research?

BB: I guess you're asking how else we will be using the information that we get from the mass spectrometer to identify the proteins.

This is a really exciting problem, one that many biologists in the world are trying to address, and it's not a simple one. Ideally, we would have genetic information that would allow us to create a proteome database against which we can search when we're trying to identify the proteins, and ideally that's what we would want.

And we should continue encouraging … not only the sequencing of genomes but also the generation of cDNA libraries that could give us expressed sequence tags for particular types of tissues.

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So, that's going to be one of the approaches that we're going to be using, as well. We're going to be generating ESTs from the particular organs that are creating the salivary liquids and we'll be using that to help identify what the proteins are.

Are you investigating only the saliva or are you going to look at other parts of the digestive system?

KP: In the larvae, it's saliva for sure. In the adult, we are also going to be looking at mid-gut regurgitation. The adults will regurgitate food to each other and to the larvae.

It's the larvae in particular we know [that] process this food, and then they create this saliva that has this nutritional value, whereas the adults digest food in their mid-guts and regurgitate that.

Are you doing anything with the queen?

KP: We're collecting these fluids from all of the different life stages — workers, and queens, and larvae, and even the male, although we don't believe the males are involved in any way, but as long as you're ripping into a colony, you may as well collect all the data that you can get.

How would the application of this have any bearing for other types of proteomics research?

BB: As you know, there are so many different types of organisms that are under investigation by lots of different types of investigators, and being able to use all of the tools that modern biotechnology provides is a goal for all of us.

Just attempting and solving some of these problems in the collection of the data and the analysis of the data and learning what the pitfalls are and what the solutions will be, we hope, will break ground to allow a lot of other investigators to begin to ask questions that they would never have been able to ask before.

We hope and anticipate the types of studies and the types of ways that we find solutions to identifying these proteins will enable and help a lot of other investigators.

I think it's important to remember as well that even though we've collected a lot of tandem mass spectra already, and we still have that data, it won't go away, so as we get more and more information, this data will reveal more and more.

And what I mean by more and more information is that as we get more and more genetic information on the different organisms, we will be able to use our mass spec data again and again until we can draw from it all of the fruit possible.

What do you expect to find?

KP: I think we've already shown that some of these wasps are engaging in this division of labor and we expect to characterize the mechanisms to some extent of that division of labor — how is it that they've partitioned their digestive labor?

And we also believe that this will have an impact on shedding light on the same sort of mechanisms that took place during the origins of multicellularity, so there's this very broad, general theoretical approach or sort of outcome, that we think this model system will shed light on that.

And obviously that sort of thing has already happened. You don't see a lot of transitionary forms of that today, so this I think will have some impact there.

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