Anole Annals

Communication is a prerequisite of human society. But when you look, smell and hear around, you rapidly realize that we are not the only ones to communicate. Indeed, plants, fungus, animals and other living things such as bacteria show extraordinary ways to “talk” to each other.

The diversity of animal signals  (e.g. visual, acoustic, chemical) and their evolution has particularly fascinated biologists for decades. Etymologically, the term “communication” implies to share information between a signaler and a receiver, but the interplay between the two remains extremely challenging to study, especially in animals in their natural environment.

The use of robots in ecology is in its infancy, but promises great advances in the study of animal communication. Lizards are not outdone with this robotic revolution (see previous AA posts and several others should be forthcoming!). The use of lizard robots allows researchers to control the signal stimulus directly in the field and to address questions related to species recognition, mate choice and evolution of aggressiveness, for instance.

In a recent paper, Dave Clark and collaborators went further by designing an interactive lizard robot! Yes, the study is about Galapagos lava lizards, but it deserves great attention in the Anole Annals community as understanding the processes underlying lizard communication is of pressing interest in anole research and the interactive robot may help to understand its evolution. The authors presented a bio-mimetic conspecific robot and tested whether an immediate or a postponed 30s response of the robot to the subject’s display influenced the display latency and duration of the focal individual. They found that immediate display response by the robot to a subject’s bobbing display stimulated the focal lizard to display more rapidly and more often than when the robot’s response was delayed. These results suggest that subjects perceived a rapid response from their robotic contestant as being more aggressive than a delayed response.

This study is the first interactive robot playback experiment with lizards in natural conditions and it demonstrates that the response ‘timing of the receiver is a crucial factor of lizard communication.

 

Reference:

Clark, D.L., Macedonia, J.M., Rowe, J.W., Austin, M.R, Centurione, I.M, Valle, C.A. (2019). Galápagos lava lizards (Microlophus bivittatus) respond dynamically to displays from interactive conspecific robots.  Behav Ecol Sociobiol 73: 136. https://doi.org/10.1007/s00265-019-2732-6

Read all about it in the latest post from Daffodil’s Photo Blog.

After a bit of a hiatus, Daffodil’s Photo Blog is back with its splendid anole photos and natural history tidbits. Check out the recent offering.

From the pages of Nova.

BY KATHERINE J. WU TUESDAY, AUGUST 27, 2019 

Compared to mammals, reptiles have a weird way of reproducing—and in the spring of 2017, that put Ashley Rasys in something of a pickle.

For months, the University of Georgia biologist was struggling to come up with a way to tinker with the genes of the brown anole (Anolis sagrei), a petite, pointy-faced lizard native to Cuba and the Bahamas.

The reptile had initially caught Rasys’ eye because of, well, its eyes. People with albinism often have poor vision due to problems with their foveae, the dense pits of cells at the back of the eyes that confer visual acuity. While foveae are lacking in most mammals, they’re present in lizards—making them intriguing candidates for studying the genes that impact foveal function.

There was just one problem: Reptiles aren’t easy to genetically manipulate. In other common laboratory animals, like mice and zebrafish, a tool called CRISPR has made DNA editing a breeze. The procedure typically involves injecting freshly fertilized eggs with gene-editing machinery, creating a change that would propagate when the cell divided.

But a few quirks ruled out that particular strategy in these lizards. Female anoles can store sperm for many months before fertilizing their eggs internally, making it difficult to time the introduction of the CRISPR cocktail. Anole fertilization also cues the formation of a soft, delicate eggshell that’s hard to penetrate without damaging the embryo.

That meant Rasys and her advisor, Doug Menke, had to get creative. So they decided to shift the injection back a developmental step, targeting eggs still maturing in the females’ ovaries. “At this point, they’re just hanging out in the lizard, waiting to be fertilized,” Rasys says.

The procedure took more than a year to perfect. But in the fall of 2018, Rasys, Menke, and the rest of their team hatched the world’s first gene-edited non-avian reptile: a red-eyed albino anole with near-transparent skin. According to the team’s study, published today in the journal Cell Reports, its birth marks a breakthrough for the field of developmental genetics, and hints that similar experiments may be possible in some of the other 10,000-plus species of non-avian reptiles that scuttle the Earth.

“This technology is really important and exciting,” says Martha Muñoz, an evolutionary biologist and anole researcher at Yale University who was not involved in the study. “This really opens up the door for other groups to think outside of traditional model organisms [like mice and zebrafish]…the sky’s the limit.”

With albinism in mind, Rasys and her colleagues set out to mutate the anoles’ tyrosinase gene, which governs pigmentation and has been linked to foveal function in humans. Manipulating this gene, Rasys explains, also made for an easy marker of success: If the procedure ended up generating albino anoles down the line, they’d be pretty tough to miss.

After rounding up 21 female brown anoles from the wilds of Orlando, Florida, the researchers gently anesthetized the lizards and opened them up. In anoles, the ovaries are transparent, making it easy to eyeball their contents “like a train of developing eggs,” Menke says.

The team selected 146 of these growing eggs and injected them with the classic CRISPR recipe: a pair of molecular scissors and a series of DNA-binding “guides” that would show them where to cut—in this case, the tyrosinase gene.

The researchers then had to wait another three months or so for the females to fertilize and lay the eggs. And even when this generation hatched, they thought there’d likely be more work to do, Rasys says. Since the CRISPR concoction had been delivered to eggs that were later fertilized by unaltered sperm, the offspring were expected to be hybrids—half edited, half unedited. These lizards then would need to be bred further to yield albinos, which must inherit the mutation from both parents for the trait to manifest.

But as Rasys watched her first clutch of gene-edited eggs grow, she noticed something strange. About a week before they were due to hatch, most of the embryos had darkened from pink to gray—an indication that they’d started producing pigment. A handful, however, retained their initial pallor, even as they continued to swell in size.

A few days later, Rasys arrived at the lab to find a newly-hatched, inch-long albino, stretching its ghostly pink legs. “It was so exciting to see it,” she recalls. “I thought, ‘It’s so cute.’”

In total, four out of the team’s 146 CRISPR-injected embryos were obvious albinos, surprising the entire team. There’s no way to know exactly what happened, but Menke’s leading theory is that the CRISPR components remained active in some of the eggs long enough to work their magic on both the maternal and paternal copies of the tyrosinasegene.

Genetic screening revealed another five embryos to be the half-edited hybrids the team had initially expected. And when the researchers partnered one of these CRISPR mutts with an unmanipulated mate, the mutation was passed on to some of the pair’s offspring, suggesting the edited gene was heritable.

There’s still plenty of tinkering to do, Menke says. As they report in the study, the team’s gene-editing success rate was around 6 percent—a figure that pales in comparison to the near-perfect efficiency rates that have been reported in zebrafish and mice.

But just showing gene-editing is possible in this system is a big deal, says Ambika Kamath, a behavioral ecologist at the University of California, Berkeley who was not involved in the study. Albinism implications aside, anoles have long been studied by evolutionary biologists and ecologists. In their native Caribbean, the lizards have split into many lineages, but understanding this diversification “has primarily been a historical science…involving stitching together patterns that happened a long time ago,” Muñoz says. “By extending CRISPR to Anolis, we can now mechanistically test some [evolutionary] hypotheses.”

As more applications surface, however, “we don’t want to be releasing CRISPRed lizards into the wild willy-nilly,” Kamath says, without a better understanding of how these sorts of introductions would affect the population at large.

And it might be more than lizard lives at stake. Menke thinks the team’s technique is likely to work in a variety of reptiles, many of which share the anole’s mode of reproduction. There’s even the possibility, he says, that the method could be adapted for birds, which are cut from the same evolutionary cloth. Scientists have hatched CRISPant chicks in the past, but as in lizards, bird embryos are hard to pinpoint at the single-cell stage, making current editing procedures complex and laborious.

Carolyn Neuhaus, a bioethicist at the Hastings Center who was not involved in the study, cautions that as CRISPR continues to be debuted in more and more organisms, the how, when, and in whom of gene editing will need to remain transparent. Though many experiments—including the ones in this study—have the potential to advance science and human health, she says, technology like this shouldn’t be used in a new species “just because it’s there.”

“We rely on scientists to create accurate and reliable knowledge, and that’s a huge responsibility,” she says. “With the CRISPR craze…I just hope it happens as mindfully and carefully as possible.”