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.”

We’ve had previous posts on parasites of anoles (for example, here), but now a new paper in Herpetology Notes adds to the literature on this topic, reporting blowfly parasitism of Anolis parvauritus from northwest Ecuador.

Congrats to Jon Suh for this fabulous photo of Anolis grahami, native to Jamaica but introduced to Bermuda. It highlights the paper on ecological interactions among introduced anoles of Bermuda by Stroud et al.

Dominica Geographic filed this report on Claire Dufour’s research on Anolis oculatus, which we have reported on previously.

Is the Zandoli Getting Stronger?

July 25, 2019 / No Comments

Dr. Claire Dufour is an ecologist and evolutionary biologist; a postdoctoral fellow of Harvard University, and currently a postdoctoral fellow and teacher at the University of Montpellier, in France. Claire is interested in the dynamic interactions between sister species and their environment. Her focus is on environmental pressures – such as competing or invasive species – on the evolution of ecological, morphological, and behavioural traits of animals such as insects, mammals and reptiles. This work has led her to take a scientific interest in the two competing species of anole (tree lizards) here in Dominica; one of which is the endemic zandoli (Anolis oculatus), the other, the invasive Puerto Rican anole (Anolis cristatellus).

For a number of years, Claire has been studying the co-existence of these two species in Dominica, how they react and behave towards each other, and how co-existing and dealing with extreme weather events such as hurricane Maria have affected behaviour and physical morphology. Her research has suggested a number of interesting outcomes that will require further study.

When the invasive species of anole arrived in the port area around Woodbridge Bay several years ago, its spread around the island was rapid, but it followed a pattern; namely the coastal road system. Claire and other scientists, such as Jacqui Eales who first recorded and studied the invasive species (see Dominica Traveller, second edition), speculate that this movement was probably caused by transportation in vehicles. The invasive anole hitched rides and occupied predominantly a coastal territory (and still does), whereas the endemic zandoli was also present in the interior where it had developed and spread over many, many years. At first, the relationship was feared to be antagonistic in nature, with the native zandoli coming under threat. Claire believes this has now changed and that the two species have settled into a form of coexistence. Moreover, she believes that the native zandoli is now doing better and is, in fact, probably the dominant species.

In order to study anoles, many environmental and morphological attributes are noted and recorded, most of which are too detailed and complex to get into here, but examples include: the location of the tree (or other structure) that the anole is inhabiting; where specifically on the tree the anole likes to hang out (trunk, branches, higher canopy, etc.); any behavioural characteristics (for example towards other anoles); and the physical attributes of the anole itself (size, weight, colour, etc.). A further experiment has been carried out to record behavioural characteristics that involves the innovative use of mimetic robots that are designed to represent both anole species. Claire presents the robots to the lizards and records their behaviour towards specific actions and signals that she is able to control remotely and unobtrusively.

Claire’s research has resulted in many interesting findings, one of which recently made headlines in the scientific community. Claire discovered that, with regard to the anoles she measured and recorded after hurricane Maria, their ability to grip with their toepads had increased by a factor of ten compared to her pre-hurricane tests. In concluding her work this year, Claire says that this attribute appears to have not only continued but has actually increased.

The likely, though unproven, explanation is that the anoles that survived the hurricane by clinging onto trees perhaps already had a strong grip compared to those that perished, and that they may have passed on this genetic trait as part of their morphological evolution. Further studies are needed and Dominica Geographic will follow Claire’s work.

Dominica is a draw for scientific researchers such as Claire because of its relatively unspoilt natural environment, its isolation as an island (it could be viewed as a very large laboratory), and its regionally and locally endemic species. One of the first things that Claire does when she comes here for her field work is engage with local people to get their views of what they have noticed about the two species of anole. Anecdotal evidence provided by local people is often an important foundation for further field research.

If you are interested in recording anoles or any other aspect of natural science in Dominica, here is one fun way to get started. Download the iNaturalist app to your smartphone and start photographing and recording the plants, flowers, birds and other critters you see when you are out walking (yes, you have to get out and explore !). Think about becoming a Dominica ‘citizen scientist’ and helping to record information about the island’s natural environment, and sharing your findings with the world.

Dominica ought to be producing more of its own scientists; encouraging, educating, and financially supporting young people with the interest and the potential. Many visiting researchers are very open to engaging and sharing their work with local people but often find it difficult to do so. More could be done to provide them with the facilities they need (classroom, lab, accommodation and so on); facilities that could also be used by a home-grown scientific community.

Over on Biology of Mexican Herps, Levi Gray has started a new series where he examines some currently recognized Mexican Anolis to discuss whether, in fact, they are valid species. In the first installment, he discusses the case of Anolis utowanae. Check it out!

Photo by Peter May (https://media.eurekalert.org/multimedia_prod/pub/web/191913_web.jpg?w=650)

A year ago, we mentioned reports of brown anoles (A. sagrei) in Germany, green anoles (A. carolinensis) in southern Spain and the Canary Islands (reviewed here), as well as a report of Cuban knight anoles (A. equestris) also on the Canary Islands, and asked if there were other sightings. Now we have one!

Reader Rick Wallach writes in:

Yet another newspaper article on brown anoles supplanting green anoles. This one from The Times of Apalachicola and  Carabelle in Florida. The text of the article is reprinted below, but it contains maps and other figures that you’ll have to go to the original to see. Also, the article included the video above.

Cuban anoles at war with native Carolinians

By James Hargrove Special to the Times

The brown (a.k.a. “festive”) anole, A. sagrei, is slowly making its way around the world. Latest report: Panama City, Panama!