Human cells help researchers understand squid camouflage

Squids and octopuses are masters of camouflage, blending into their environment to evade predators or surprise prey. Some aspects of how these cephalopods become reversibly transparent are still "unclear," largely because researchers can't culture cephalopod skin cells in the lab. Today, however, researchers report that they have replicated the tunable transparency of some squid skin cells in mammalian cells, which can be cultured. The work could not only shed light on basic squid biology, but also lead to better ways to image many cell types.

The researchers will present their results at the spring meeting of the American Chemical Society (ACS).

For many years, Alon Gorodetsky, Ph.D., and his research group have been working on materials inspired by squid. In past work, they developed "invisibility stickers," which consisted of bacterially produced squid reflectin proteins that were adhered onto sticky tape. "So then, we had this crazy idea to see whether we could capture some aspect of the ability of squid skin tissues to change transparency within human cell cultures," says Gorodetsky, who is the principal investigator on the project.

The team at the University of California, Irvine focused their efforts on cephalopod cells called leucophores, which have particulate-like nanostructures composed of reflectin proteins that scatter light. Typically, reflectins clump together and form the nanoparticles, so light isn't absorbed or directly transmitted; instead, the light scatters or bounces off of them, making the leucophores appear bright white.

"We wanted to engineer mammalian cells to stably, instead of temporarily, form reflectin nanostructures for which we could better control the scattering of light," says Gorodetsky. That's because if cells allow light through with little scattering, they'll seem more transparent. Alternatively, by scattering a lot more light, cells will become opaque and more apparent. "Then, at a cellular level, or even the culture level, we thought that we could predictably alter the cells' transparency relative to the surroundings or background," he says.

To change how light interacts with cultured cells, Georgii Bogdanov, a graduate student in Gorodetsky's lab who is presenting the results, introduced squid-derived genes that encoded for reflectin into human cells, which then used the DNA to produce the protein. "A key advance in our experiments was getting the cells to stably produce reflectin and form light-scattering nanostructures with relatively high refractive indices, which also allowed us to better image the cells in three dimensions," says Bogdanov.

In experiments, the team added salt to the cells' culture media and observed the reflectin proteins clumping together into nanostructures. By systematically increasing the salt concentration, Bogdanov got detailed, time-lapse 3D images of the nanostructures' properties. As the nanoparticles became larger, the amount of light that bounced off the cells increased, consequently tuning their opacity.

Then, the COVID-19 pandemic hit, leaving the researchers to wonder what they could do to advance their investigation without being physically in the lab. So, Bogdanov spent his time at home developing computational models that could predict a cell's expected light scattering and transparency before an experiment was even run. "It's a beautiful loop between theory and experiments, where you feed in design parameters for the reflectin nanostructures, get out specific predicted optical properties and then engineer the cells more efficiently -- for whatever light-scattering properties you might be interested in," explains Gorodetsky.

On a basic level, Gorodetsky suggests that these results will help scientists better understand squid skin cells, which haven't been successfully cultured in a laboratory setting. For example, previous researchers postulated that reflectin nanoparticles disassemble and reassemble to change the transparency of tunable squid leucophores. And now Gorodetsky's team has shown that similar rearrangements occurred in their stable engineered mammalian cells with simple changes in salt concentration, a mechanism that appears analogous to what has been observed in the tunable squid cells.

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Do octopuses, squid and crabs have emotions?

Octopuses can solve complex puzzles and show a preference for different individuals, but whether they, and other animals and invertebrates, have emotions is being hotly debated and could shake up humans' moral decision-making, says a York University expert in animal minds.

Most countries don't recognize invertebrates, such as octopuses, crabs, lobsters and crayfish, as sentient beings that can feel pain, but the United Kingdom is considering amendments to its animal welfare legislation that would recognize this.

"A London School of Economics (LSE) report commissioned by the U.K. government found there is strong enough evidence to conclude that decapod crustaceans and cephalopod molluscs are sentient," says York University Professor and philosopher Kristin Andrews, the York Research Chair in Animal Minds, who is working with the LSE team.

Andrews co-wrote an article published today in the journal Science, "The question of animal emotions," with Professor Frans de Waal, director of the Living Links Center at Emory University, which discusses the ethical and policy issues around animals being considered sentient.

Andrews points out it has long been thought in Western culture that other animals don't feel pain or have emotions. "It's been a real struggle even to get fish and mammals recognized under welfare law as sentient. So, it's pretty cutting-edge what seems to be happening in the U.K. with invertebrates."

Pre-verbal human babies were considered not to feel pain up until at least the 1980s. It is still thought by many that animals, including invertebrates, don't feel pain and only have unconscious reactions to negative stimuli. However, research on mammals, fish, octopuses, and to a lesser extent crabs, has shown they avoid pain and dangerous locations, and there are signs of empathy in some animals, such as cows -- they become distressed when they see their calf is in pain.

Recognizing the sentience of invertebrates opens a moral and ethical dilemma. Humans can say what they feel, but animals don't have the same tools for describing their emotions. "However, the research so far strongly suggests their existence," says Andrews, is working on a research project called Animals and Moral Practice.

"When we're going about our normal lives, we try not to do harm to other beings. So, it's really about retraining the way we see the world. How exactly to treat other animals remains an open research question," says Andrews. "We don't have sufficient science right now to know exactly what the proper treatment of certain species should be. To determine that, we need greater co-operation between scientists and ethicists."

There may be a point when humans can no longer assume that crayfish, shrimp, and other invertebrates don't feel pain and other emotions.

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New species of extinct vampire-squid-like cephalopod is the first of its kind with 10 functional arms

New research led by scientists at the American Museum of Natural History and Yale shows that the oldest ancestors of the group of animals that includes octopuses and vampire squids had not eight but 10 arms. The study, which describes a new species of vampyropod based on a 328-million-year-old fossil that had not been previously described, pushes back the age of the group by nearly 82 million years. The details are published today in the journal Nature Communications.

"This is the first and only known vampyropod to possess 10 functional appendages," said lead author Christopher Whalen, a postdoctoral researcher in the Museum's Division of Paleontology and a National Science Foundation postdoctoral fellow in Yale's Department of Earth & Planetary Sciences.

Vampyropods are soft-bodied cephalopods typically characterized by eight arms and an internalized chitinous shell or fin supports. Because they lack hard structures, Vampyropoda are not well represented in the fossil record. The new study is based on an exceptionally well-preserved vampyropod fossil from the collections of the Royal Ontario Museum (ROM). Originally discovered in what is now Montana and donated to ROM in 1988.

Whalen and coauthor Neil Landman, a curator emeritus in the Museum's Division of Paleontology, identified the fossil specimen as a completely new genus and species that dates to about 328 million years old, making it the oldest known vampyropod and extending the fossil record of the group by about 82 million years. In the new study, they also describe its 10 arms -- all with preserved suckers -- corroborating previous scientific arguments that the common ancestor of vampyropods had 10 arms as well.

"The arm count is one of the defining characteristics separating the 10-armed squid and cuttlefish line (Decabrachia) from the eight armed octopus and vampire squid line (Vampyropoda). We have long understood that octopuses achieve the eight arm count through elimination of the two filaments of vampire squid, and that these filaments are vestigial arms," said Whalen. "However, all previously reported fossil vampyropods preserving the appendages only have 8 arms, so this fossil is arguably the first confirmation of the idea that all cephalopods ancestrally possessed ten arms."

Two of the cephalopod's arms appear to have been elongated relative to the other eight arms, and its torpedo-shaped body is reminiscent of today's squids. The fossil was given the name Syllipsimopodi bideni. The genus name is derived from the Greek word "syllípsimos" for "prehensile" and "pódi" for "foot" -- because this is the oldest known cephalopod to develop suckers, allowing the arms, which are modifications of the molluscan foot, to better grasp prey and other objects. The species name is to honor the recently inaugurated (at the time of paper submission) 46th President of the United States, Joseph R. Biden.

"Syllipsimopodi may have filled a niche more similar to extant squids, a midlevel aquatic predator," said Landman. "It is not inconceivable that it might have used its sucker-laden arms to pry small ammonoids out of their shells or ventured more inshore to prey on brachiopods, bivalves, or other shelled marine animals."

Based on the age, characters, and phylogenetic position, the fossil challenges the predominant arguments for vampyropod origins, and the authors propose a new model for coleoid (internally shelled cephalopod) evolution.

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