Cephalove

To recap the last post on the Euprymna/Vibrio symbiosis: Euprymna scopoles (also known as the Bobtail squid) is a tiny species of squid that has two light organs in the underside of its mantle. Vibrio fischeri is a species of bacteria, of which some varieties can live inside of the bobtail squid’s light organs. These bacteria produce light, which the squid uses for camoflauge.

The story of how the two evolved together to make the working symbiosis is long, complex, and as of now incomplete – scientists are still piecing together all of the many adaptations that allow these two species to live together. I’ll try to bring both of us, dear reader, at least one step closer to making sense of it in this post.

A juvenile bobtail squid. Photo by Loh Kok Sheng (click through to see his blog.)

The important thing about this symbiosis is that it is selective. The squid has these little pouches which are just perfect for bacterial growth, but only one species of bacteria is found there. This involves a number of processes of selection – for example, the squid’s immune system sends cells into the crypts of the light organs to eat up invading bacteria, and the lining of the light organs secrete antimicrobial chemicals (like nitric oxide.) Vibrio fischeri has evolved to allow it to prosper under these conditions. The first step of the symbiosis, though, involves the bacteria and the squid finding each other. The ocean is big, and there are lots of bacteria and squid in it; how do these two get together so reliably?

Nyholm and McFall-Ngai address this in a 2003 paper that examines what they call “the first site of symbiont specificity”: the mucus that coats the opening of the light organ crypts in juvenile squid. Let’s start from the beginning:

A baby bobtail squid hatches from its egg. (Awwwww! So cute!) While it developed, its bacterial partner was nowhere to be found. After it hatches, though, a colony of V. fisheri will become established in its light organs within mere hours. Nyholm and McFall-Ngai looked at the surface of the mantle where the crypts open to the seawater and found that specialized cells in this area secrete mucus in response to the seawater that it would normally encounter right after hatching. This mucus helps trap bacteria, which can then colonize the light organ. V. fisheri normally make up about 0.1% of the bacteria found in seawater, though, so in order to beat out the competition, they must have some ability to interact with the light organ in a special way. Nyholm and McFall-Ngai hypothesized that the mucus layer on the outside of the light organ was key to this specificity, and conducted a number of experiments to test this idea.

First of all, they found that if they exposed hatchling bobtail squids to seawater without V. fisheri in it, all sorts of bacteria could be found in the light organ mucus. However, when they used seawater with small amounts of V. fisheri in it (again, on the order of one-tenth of one percent of the total bacteria in the water,) the colonies that formed in the mucus were almost exclusively V. fisheri. This indicates that this mucus excretion has some role in establishing the specificity of this symbiotic relationship, in that it somehow “screened out” all of the other species of bacteria that might have taken hold in the mucus and started to multiply.

Image of a V. fisheri colony in a bobtail squid light organ, labeled with green fluorescent protein. Aa, anterior appendage; pa, posterior appendage. (from Nyholm and McFall-Ngai, 2003)

They also determined that most of the V. fisheri present when they took their measurements had been collected from the water; this is in contrast to a scenario where a few cells were captured and then multiplied. To do this, they used a chemical called nalidixic that prevents cell replication while not affecting cell growth – when exposed to this chemical, bacteria won’t divide, they will simply elongate. By looking at how long V. fisheri cells grew in the light organ mucus, the experimenters determined that the cells were growing at a low rate in the mucus – in fact, they were growing much more slowly than they do in a plain culture! Thus, it’s unlikely that a few cells were captured by the mucus and then dividing into the large colonies they found; rather, there may exist some way for V. fisheri to selectively adhere to the mucus and be efficiently collected from the water (the authors say that this is unlikely, but not completely ruled out – it seems to me a likely explanation, especially taking into account the results of a series of studies that I’ll write on soon.)

The authors than tried using killed V. fisheri, to see if there is something specific to the presence of the bacteria (for example, some component of their outer membrane) that inhibits the growth of other bacteria. They found that, although killed V. fisheri could still adhere to the light organ mucus, they did not prevent the growth of other species of non-symbiotic bacteria. This implies that the bacteria perform some active process that prevents the growth of other bacteria in the light organ and allows V. fisheri to establish its dominance there, even though the mere presence of V. fisheri bacteria doesn’t kill other kinds of bacteria.

This symbiosis, then, which occurs very quickly and very specifically, depends (as most great things do) on mucus. Somehow, V. fisheri interacts with the squid’s secretions to beat out it many competitors. Interestingly, though (and I won’t cover the methods here, for time’s sake) the authors also found that the V. fisheri that colonize the crypts initially are not necessarily able to produce luminescence. It seems that the species of bacteria is selected during the initial stages of colonization, but that later on, specific strains that are better able to produce light are selected for while those that do not produce light are expelled or die off – each stage of selection no doubt involving a complex set of signals between the squid and the bacteria.

Thanks for reading!

Nyholm, S., & McFall-Ngai, M. (2003). Dominance of Vibrio fischeri in Secreted Mucus outside the Light Organ of Euprymna scolopes: the First Site of Symbiont Specificity Applied and Environmental Microbiology, 69 (7), 3932-3937 DOI: 10.1128/AEM.69.7.3932-3937.2003

I’ve recently gotten into microbiology (I got a book on protozoans, and I’m hooked,) so I decided to try to find something microbiological to write about. Lo and behold, after a few Pubmed searches, I came upon some papers about an bioluminescent bacteria called Vibrio fischeri. Of course, not just any bacteria would do for a blog post – this one is special. It lives inside the Hawaiian bobtail squid, Euprymna scolopes, in two special light organs. There, it finds a nice comfy home, and the squid can use the bacteria’s light-producing ability for countershading, so that it is harder to catch and eat. It is easy to see how this is good for both the squid and the bacteria.

(As a brief aside, the genus Euprymna is among the cutest cephalopod taxa. Go ahead and do a Google image search for the string “euprymna”, if you don’t believe me.)

The squid houses its tiny symbionts in a set of paired organs called the light organs (there’s a nice, straightforward anatomical term for ya!) These organs contain many convoluted cavities lined with epithelial cells in which the bacteria live. They connect to the inside of the mantle cavity (which is actually the outside of the body, as the mantle is open to the seawater through the funnel,) through ducts, so that the inside of the light organ is actually continuous with the squid’s mantle epithelia. Vibrio fischeri infect young squids by making their way through these connecting ducts and colonizing the cavities in their light organs. This process has been the subject of a number of very interesting studies; I’ll come back to this later, though.

At the present moment, let’s forget about the bacteria and consider the light organ from the squid’s perspective. The light organ’s structure was described in 1990 by McFall-Ngai and Montgomery. They found that the organ had a structure that was specialized for the projection of light downwards from the squids body. Specifically, the organ is set up so as to maximize the amount of light that leaves the squids ventral surface (its belly, or underside.) In addition to the cavities for the light producing bacteria to live in, the light organ contains a specialized “reflector” that helps to prevent light from escaping from the top of the organ, and a clear lens beneath the light organs to allow light to exit the squid’s body from the bottom. These structures are closely associated with the animal’s ink sac, which it has muscular control of, and so the shape of the reflector and lens can be changed by the action of the squids muscles. This allows the squid to adjust the characteristics of the light that leaves its light organ – specifically, it allows the squid to control how much light leaves the light organ. This is very important, as it’s what makes the light organ useful.

This image shows the light organs of the Hawaian bobtail squid. They are the pair of white and black organs below the eyes. From McFall-Ngai and Montgomery, 1990.

This is a cross section of the light organ. For orientation, imagine that the squid in the above photograph was lying on a table, and we cut it in half horizontally through the light organ, so that one half had the head and tentacles and the other half had the tip of the mantle. From McFall-Ngai and Montgomery, 1990.

How do we know what the light organs are used for, though? They look a lot like they would be used for countershading – that is, the lightening of an animals downward-facing side that prevents predators below them from seeing their silhouette against the light from the surface. Suggestive anatomy is not enough to make a firm conclusion about their function, though – we have to show that the light organs actually function to provide counter-shading.

Let’s fast-forward to 2004, where after stepping out of our Scienterrific Time-Travel Machine (TM) we will crack open the current issue of the journal “Marine Biology” to find an article by Jones and Nishiguchi that purports to show that the bobtail squid really does use its light organs for counter-shading. Successful bioluminescent countershading requires that the animal be able to regulate the amount of light it gives off in response to the amount of light that falls on it from the surface. In this way, it can match its appearance to that of the surface light, and blend in.

To demonstrate that the bobtail squid can do this, Jones and Nishiguchi used the following procedure: A squid was placed in a small container, slightly larger than its body, to prevent movement (the small size of the apparatus may have interfered with the results by stressing the animals, but after removing animals who appeared to be distressed by the experimental procedures, they got consistent results.) Then, a light was turned on at the top of the container. A fiber optic probe placed at the bottom of the container allowed the authors to measure the amount of bioluminescence the squid generated.

They found that the amount of light the squid’s light organ released was proportional to the amount of light they hit its dorsal surface (the upward-facing surface of the squid.) This is pretty good evidence for active counter-shading (also called counter-illumination.) This is the benefit that the squid derives from the symbiosis – it can more effective hide from predators below it. This at first seems a bit puzzling, because the bobtail squid spends much of its time buried in the sand. On those occasions when it leaves its sandy hiding place, however, it is very vulnerable, and these are the times when light organ really, *ahem*, shines.

Now, let’s speculate a bit about the evolutionary history of the light organ (in very general terms, of course.) We have a mechanism for selection: both the squid and the bacteria benefit from the symbiosis. The squid gets a counter-shading mechanism that allows it to escape being eaten, and the bacteria gets a reliable and fertile place to grow and reproduce. But what is the cost of evolving this symbiosis? For one, when they evolve specialized mechanisms for coexisting with their host’s immune system, the bacteria might give up the flexibility to live in other environments (this is not the case in this symbiosis, as there are both free-living and symbiotic forms of V. fischeri, but it is a general problem in the evolution of symbiosis.) The squid’s immune system, following the same tack, had to evolve special mechanisms to allow the bacteria to colonize it, which may have fitness-reducing side effects. Furthermore, being part of a symbiosis makes the animals dependent on each other (in this case, perhaps not entirely dependent upon each other, but at least dependent upon each other in terms of achieving optimal fitness.) As a symbiosis evolves and becomes more completely co-dependent, the organisms involved are increasingly restricted to only those habitats and conditions that their symbionts can also live in. Nevertheless, this symbiosis has evolved, indicating that such problems are not actually detrimental enough in this case to preempt the beneficial effects of the symbiosis.

The ways that the bacteria and the squid have evolved live together on a molecular level are manifold and complex. One particular problem sticks out like a sore thumb: how is colonization of the squid’s light organ so selective? The squid has these nice little crypts that are apparently well-suited for bacteria to live in, and yet they are only colonized by a single species of bacteria. In fact, this problem has been extensively studied. It turns out that, during colonization, the V. fischeri and E. scolopes are engaged in a sort of biochemical dance (if you will excuse a student’s romanticism,) mutually sensing and reacting to each other, and putting out chemical and physical signals in a coordinated fashion to successfully live together. This actually presents many problems: the bacteria must be plentiful, but stay contained; they must survive exposure to the squid’s immune system, but not be allowed to infect the squid as a whole. Other bacteria must be excluded from the crypts. The initial colonization must take place, with the bacteria finding the right place on the squid to enter and moving through the ducts of the light organ to its crypts. This process is of interest to molecular biology because it is a case where molecular signals between an endosymbiont and its host have been identified, leading to its use as a model system for the development of such symbioses in general.

I know, I know, you want to hear about the molecular biology, but it’s already the end of the article! Don’t worry – I’ll get to that in a dedicated post soon enough. It’s a complicated subject, and it’s far enough out of my area of knowledge that I’ll need a little time to learn about it before I can write about it. For now, thanks for reading, and stay curious!

For more info on the Bobtail squid-Vibrio symbiosis, check out this great page by J. Graf. Also, as always, I encourage you to find and read the studies I cited for yourself.

McFall-Ngai, M., & Montgomery, M. (1990). The Anatomy and Morphology of the Adult Bacterial Light Organ of Euprymna scolopes Berry (Cephalopoda:Sepiolidae) Biological Bulletin, 179 (3) DOI: 10.2307/1542325

Jones, B., & Nishiguchi, M. (2004). Counterillumination in the Hawaiian bobtail squid, Euprymna scolopes Berry (Mollusca: Cephalopoda) Marine Biology, 144 (6), 1151-1155 DOI: 10.1007/s00227-003-1285-3