Deep under the ocean, as the octopus explores around on the ocean floor, it needs to sense its surroundings in order to survive. Thankfully, it has not one or two but eight arms to help it in its sensing. We know that octopuses are extraordinarily intelligent creatures, but we know very little about how they sense the world around them.
“For the octopus it’s very clear that the arms were doing all kinds of interesting things, constantly sampling their environment, and so we just looked at the arms and thought that there must be something to learn there,” said Nicholas Bellono, an associate professor at Harvard University.
We, as humans, use specialised cells in our noses and eyes and other sensory organs to interact with the world. In the octopus, their arms are lined with suckers that help it sense the ocean floor as it probes around looking for prey to eat. When Bellono and his group first started working with the California two-spot octopus in the lab, they began by isolating the cells on the suckers and asking what they sense. What really drew them in was how the cells responded to chemicals.
When they used sequencing technology to look at which genes are specifically turned on in these sucker cup cells, what they found didn’t look like the typical sensory receptors one would expect – the kinds used for taste or smell, as an example.
“We looked for known chemoreceptors such as olfactory (smell) or taste receptors, things that one would guess, but we didn’t see them. The only things we saw were these proteins that looked like neurotransmitter receptors but they lacked the neurotransmitter binding site,” said Bellono. They had stumbled upon a whole new family of sensory receptors that seemed to be unique to octopuses, which they could now use as a tool to ask what the octopuses sense and respond to.
After screening a large number of chemicals on the novel receptors, they found that they did not detect typical odorants or tastants. Instead, they responded to these hydrophobic molecules called terpenes, made by plants and bacteria and sometimes released by marine invertebrates as a form of a defence mechanism. Hydrophobic molecules repel water, and being poorly soluble, they localise to surfaces. If the octopus is crawling about touching surfaces and doing “contact-dependant chemosensation”, these molecules should be on the objects it is probing.
“What makes these receptors odd is that the types of chemicals they sense are not the type of chemicals one finds floating around in water,” said Corey Allard, a postdoctoral fellow in the lab. “They are hydrophobic and insoluble, and in the marine environment we would find them on surfaces. For the organism to sense them, they would have to physically probe that surface because they aren’t in the water. That’s how its chemotactile, different from other forms of chemical sensing, like smell.”
Based on their findings, they called these receptors in the sucker cups “chemotactile” receptors, as they help the octopus pretty much taste what it touches. In a paper in 2020, Bellono and his group reported their discovery of these receptors and characterized their basic functions. Then, they went a bit deeper, they wanted to really understand this receptor in all its glory.
They set out to obtain the cryo-EM structure of this receptor, to delve deep into its structure and understand how this terpene binds to it. They found that it resembles receptors well established to be involved in neurotransmission, and didn’t look like typical sensory receptors. Further, it didn’t even bind neurotransmitter chemicals, like the typical agonist acetylcholine, suggesting a different role for this receptor in the octopus. How does a receptor change from a neurotransmitter receptor to a sensory receptor? Rather serendipitously, the answer seemed to be hiding in plain sight.
“We were trying and trying to find, in the cryo-EM used for the structural analysis, where the terpene binds to the receptor,” said Allard. “We saw that one of the detergent molecules used to solubilise the receptor for cryo-EM was stuck in a place very similar to where the acetylcholine would bind in the ancestral receptor. As the detergent is also partially hydrophobic, we thought maybe that is where the agonist binds.”
They knew that terpenes, the agonist they were interested in, are hydrophobic, but so are the soap molecules present in the detergent used to dissolve the receptor for their technique. Maybe the soap molecules bind where the terpenes bind, and can activate these receptors in the cells? When they tested this out in vitro, it turned out that that was indeed the agonist-binding site, and the detergent happened to be a very potent agonist by itself. It also appeared to be the site where the terpenes bind. It turned out that the amino acids in the binding site of this octopus receptor were hydrophobic themselves, hence attracting other water repelling molecules like terpenes. These amino acids completely change the structure of the binding site, as compared to how it is in the ancestral neurotransmitter receptor that binds acetylcholine.
“There is a cage like structure in the acetylcholine receptor that binds acetylcholine. In these receptors the cage is gone and there is a shallow, hydrophobic groove filled with hydrophobic amino acids that binds these insoluble, greasy molecules like terpenes,” said Allard. The results from this study were published recently in Nature.
Do other cephalopods have these receptors?
The researchers also wanted to understand if other cephalopods like squids and cuttlefish, related to octopuses, even had these kinds of receptors. Squids, along with their eight arms, also had tentacles to capture their prey and reel them in; they explore surroundings and engage with prey very differently than the octopus.
In a companion study in collaboration with Guipeun Kang and Ryan Hibbs, now at UCSD, they found that squids also had these cephalopod-specific chemotactile receptors, but they had a different function. These receptors responded to a bitter tastant called denatonium which was more hydrophilic in nature. When they tested this experimentally, they found that the squids would reject prey that were coated with this chemical. Some of these receptors were also found to be specific to squids and cuttlefish and were not even present in the octopus arms.
It took the researchers a while to realise the evolutionary significance of these receptors, as they were initially looking at it from a physiological and structural point of view. But a grad student, Wendy Valencia Montoya, in the lab helped them tease apart the differences and look at the receptors through the lens of evolution.
“There was just this tangled bolus of receptors, with no logic to them. Wendy compared the sequences across the different organisms we had looked at, and found these specific lineages having evolved separately in the octopus and the squid,” said Allard. “It really united the behaviour and physiology and made sense of what we had learned.”
Once the evolutionary perspective opened up, a new question arose, how does the binding pocket in these chemotactile receptors change across evolution? This pocket appeared to be an evolutionary hotspot, where the receptor could diversify its structure based on its function. “There is an ancestral receptor with a cage-like pocket, trapping acetylcholine. The octopus receptor is totally different, there is a hydrophobic groove. In the squid, there is a cage-like structure but it is different from the ancestral one, maybe a transitioning structure from the ancestral state to a very different state in the octopus,” said Allard. “Maybe you start binding molecules like acetylcholine, and through evolution you start binding different molecules and diversifying.”
Based on the different structures and sequences of the binding pocket in the receptor, the researchers were able to see how this chemotactile receptor may have evolved differently in these different kinds of cephalopods, as compared to the original acetylcholine receptor. “In addition to having an acetylcholine receptor and a long-separated octopus receptor, something came up in between those two, the squid receptor, detecting a very different molecule involved in a different behaviour,” said Bellono. “It gave insight into how the receptor evolved across different ecology and different behaviours.”
A way to bypass the receptors altogether while understanding what the octopus senses is by focusing on what the octopus arms care about. When Allard recorded electrical signals from the nerves in the octopus arms, he found that the octopus doesn’t seem to respond to these bitter molecules that the squids responded to. “The octopus arms don’t care about bitter things, but squid arms do, which could be related to their ecology and behaviour,” said Allard.
Octopuses always responded to the hydrophobic terpenes though. In the lab, when terpenes were infused into the floor of experimental tanks the octopus were placed in, the octopuses responded to them strongly; their probing behaviour changed when they encountered them. A major open question is which of these (or other related) chemicals are present in the natural environment of the octopuses, and what these chemicals signal to the octopus. Answering the question of whether some prey or predator of the octopus secretes these is something the group is focusing on now.
Octopuses and squids engage in very different predatory behaviours. Squids are ambush predators and octopuses probe around the ocean floor and in crevices for prey. But they both may need to sense insoluble molecules that aren’t present in the water but are present on surfaces. It appears that their chemotactile receptors may have evolved different adaptations in their binding pockets based on what they need. The receptors are truly versatile, with structural and functional differences evident across evolution.
“What’s really cool about these papers is that they span from atomic level analysis to physiology, behaviour and to evolution, said Bellono. “That was important, not only for attacking a question with many approaches, but also conceptually, in these studies.”
Rohini Subrahmanyam is a postdoctoral researcher at Harvard University in a neighbouring lab studying human embryo development using organoids.