Squid symbiotic bacteria: friend or foe?

Some squid team up with symbiotic bacteria, which supply light and help the squid to disguise itself. Speaking to Chris Smith, Cheryl Whistler from the University of New Hampshire explains how quickly the animals establish the right relationship with the right bacteria, including cherry-picking the mutants that make the most light...

Cheryl - We work with a squid species called Euprymna scolopes, more popularly called the Hawaiian Bobtail squid and it lives on near-shore areas at the Hawaiian Islands and it associates with a single species of bacterium called Vibrio fischeri. And Vibrio fischeri is a bioluminescent bacterium. So, it produces bioluminescence or light and when it associates with the squid, it lives in a particular special organ that the squid has on the bottom of its body. And when it’s swimming at night, and it’s out looking for mates or food, predators will actually sit on the bottom of the reef and look up for shadows. And when a squid passes over the top of a predator, if it is casting light from the bottom of its body, it just looks like a beam of moonlight and it’s completely camouflaged from the predators.

Chris - So, how does the squid team up with the bacterium to tell it, “Right. I want the light now”?

Cheryl - Good question. So when the squid first hatch, they actually don’t have any of the bioluminescent symbionts. They have to recruit them from the sea water. And they do this by providing attractins that the bacteria can sense and then they swim into the light organ down through ducts and enter the light organ and then start growing there. The problem is if the squid were not selective about who it let into its light organ, it could very easily let in a pathogen or a squatter that doesn’t belong there and that doesn’t produce bioluminescence. So the squid has actually developed really exquisite specific mechanisms to recognize who its symbiont is and to only let its symbiont into its light organ. Once the bacterium is there, the squid feeds it and when the bacterium reaches a nice robust population, it automatically glows and it does so by a method called quorum sensing where the bacteria actually communicate with each other and tell each other to turn on light together.

Chris - So getting the right population of bacteria and getting the right numbers of bacteria that want to cooperate is absolutely critical for the survival of the squid and for the survival of that bacterial strain as a whole.

Cheryl - Absolutely. So the bacteria can live outside the squid but they don’t live a good life. And the squid certainly, the theory is without the camouflage, they would be much more susceptible to predation.

Chris - So, what was the question you were seeking to ask here?

Cheryl - We were interested in understanding how the squid recognized their specific symbiont and how does their symbiont communicate to them that they are the beneficial partner that should be allowed to live in that light organ? And we did it by looking at natural diversity that exists in non-symbiotic populations. So can bacteria that have never seen the squid before through very subtle changes and their DNA, basically evolving through mutation, can they find solutions to the problem that the squid provides in terms of barriers to prevent the wrong symbiont from getting in?

Chris - So how did you do it? Take some squid, take some bacteria, mix them up, and start a family growing and then take some of those bacteria and keep repeating that to slowly select for bacteria and see how they change.

Cheryl - Well, we thought we might have to do it slowly but in fact, it happened very rapidly. So, what we did as we took bacterial strains that were naive to squid. We took strains from sea water that never encounter squid. We took the strains from other hosts like fish and we grew them up in culture and then dropped in a cohort of juvenile squid who had never seen a symbiont yet. And then we let the squid basically select from the natural genetic diversity that arose in culture and then we passage them from that squid to the next squid by allowing the squid to vent out the bacteria. This is one of the things that does is it actually grows up a population and then every morning, it vents out or dumps out 95 per cent of the culture so that they can grow a nice fresh culture and be glowing very brightly at night. So when they go back into the water, they're potentially now genetically adapted to that light organ environment.

Chris - You say potentially but is that actually what happens? Do you see a refinement of the population driven by this selection from a squid?

Cheryl - Absolutely. And this is one of the remarkable things about the experiment is that the mutations likely arose in culture when the populations where very large. But the first squid didn’t have only the beneficial mutations in their light organ population. It was a mixed population. By the second squid, we were able to detect the beneficial mutation that was literally a needle in a haystack in the initial inoculum.

Chris - And what did change?

Cheryl - It was really kind of interesting because it wasn’t a different mutation in every line of squid. So, we did squid in parallel and we used several different strains. And what we found was that five lineages acquired beneficial mutations and the mutations mapped to the same gene. And they were not remarkably dramatic mutations. They were literally tiny little point mutations – so nucleotide changes. And just that change was enough to transform a non-symbiont into a really “kick ass” symbiont.

Chris - Love the phrase! So, there must be something specific about that gene. What does it do that gene, do you know?

Cheryl - Yes. It’s a protein whose job it is to turn off behaviours. And these strains that were not natural symbionts have this repressor blocking the expression of traits that were necessary to be a good symbiont. And what the mutations arose did is that they didn’t completely obliterate the activity of this repressor. They just modified it ever so slightly. So by modifying how this repressor interacted with its targets, the bacteria were able to modify not just one behaviour but a suite of behaviours that were linked to each other and that were necessary to be a symbiont. And these included their metabolism and included their ability to produce light at the right level and a really key trait was that it changed the way they produced their sugary coats that they coat their selves with. They are called polysaccharides. And these polysaccharides allow the bacteria to form aggregates or biofilms that protect them from things like oxidative stress, and chemicals and it also protected them from the squid’s immune cells. So when immune cells monitor who is coming into the light organ, they literally will glob on and kill any bacterium that isn’t the right bacterium. But this change in the sugary coat of these cells allowed them to slip pass the immune cells and get in to the light organ and do their job as symbionts.