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