Spitting cobras have a similar defense strategy that has independent ancestry

Three groups of spitting cobras from different geographical locations possess a pain-inducing venom to hurt and blind their predators. Despite similar function, spitting venom does not appear to be ancestrally shared between these snakes. Instead, the authors of a paper describe the trait as striking example of convergent evolution.

The research, by a large international team of scientists, is the cover story of Science magazine, Jan. 21.

The team's multidisciplinary approach compared gene, protein and functional data of 17 cobras (elapids) from different regions. Cobra venoms are all composed of specific neurotoxins and cytotoxins, but the researchers found that the venoms of the African spitting cobras, the Asian spitting cobras, and the South African rinkhals have a distinctly different composition.

The venom difference is related to function. All cobras use venom in self-defense, but these three types of spitting cobras have the unique ability to eject their venom in a way that "plays no role in prey capture, targets specific sensory tissues, and is the only long-distance, injurious defensive adaptation among almost 4,000 species of snakes," the researchers said. 

These spitting cobras are unique in that they use their venom "to cause enhanced pain" for their predators. They are very accurate and can spit at their predators 50 to 60 times.

`Unexpected' results on the molecular level

Current Science Daily asked venom biologist Nicholas Casewell, head of the Centre for Snakebite Research and Interventions and chair in tropical disease biology at the Liverpool School of Tropical Medicine, why the researchers characterized their results as "unexpected."

"We already knew that this trait of venom spitting had evolved independently on three occasions in these three closely related groups of snakes," Casewell said. "The question was to understand whether the evolution of spitting had consequences on venom composition. Evolutionary processes are reliant on a number of things that happen over many millions of years. And what was surprising here is that although we showed all three venoms have enhanced pain-causing ability, they have all reached this point by an interaction of very similar toxins. So there's an element of repeatability here." 

Casewell added that this is fascinating from an evolutionary adaptation perspective.

"That's interesting from an evolutionary perspective because there are actually very few examples where convergence at the molecular level results in convergent changes to an adaptive trait, in this case venom spitting," Casewell said. "What we found on a facile level is very logical--we might see similar changes in animals that have evolved similar traits. But at the molecular level, it was more surprising that the mechanisms that have been utilized to reach that endpoint also have parallels."

Defensive venom

How did researchers conclude the particular venoms involved are defensive in nature, and not just a way of subjugating prey, Current Science Daily asked.

"This is really important," Casewell replied. "Although we showed that there were clear differences between spitting cobras and non-spitting cobras in the composition of what those venoms did, this could have just been an artifact of these lineages honing their venom for different prey items."

To check whether this was the case, the researchers devised a counter test. 

"We carried out an experiment where we used mice as an animal model to effectively mimic a prey item," Casewell said. "We found there was no significant difference in the potency of spitting cobra and non-spitting cobra venoms to that prey item. There was variation across the group, but there were no patterns associated with spitting.  

"That gave us a really strong piece of evidence that the changes we see only in the spitting cobras versus the non-spitting cobras are likely to have occurred in response to this parallel evolution of venom spitting as a defensive trait."

Adaptations and convergent evolution

How did this defensive weapon of spitting venom develop over time? 

"What we hypothesized, with good bits of evidence, is a process over many millions of years' time whereby different adaptations have been layered upon each other to end up with this really fantastic defensive weapon that we see today in terms of spitting," Casewell said. "This involved behavioral adaptations and morphological adaptations as well as molecular adaptations in the context of the venom toxins themselves." 

He described the preadaptation of cobras to raise the first third of their bodies as a defensive warning sign, flaring their hood, a good starting position for venom spitting.

"The other thing that the cobras have is that they've already evolved cytotoxins which we do not typically see in the venom of any of their relatives," the biologist said. "And these cytotoxins, as their name suggests, can destroy cells. It certainly stands to reason that if enough of this venom were to get into your eyes it may be painful, just by cell death--dead cells releasing components and causing pain," Casewell said.

"In this paper we're saying that the convergent evolution of phospholipase-based toxins in spitting cobras has enhanced that pain-causing ability. And we showed that the combined action of the phospholipase A-2 toxins in the spitting cobras, which are largely absent in the non-spitting cobras, are able to really enhance the pain caused by cyotoxins."

Casewell also noted other adaptations, such as modified fangs, which progressed from not being able to spit, to flinging venom imprecisely, to "very high-quality jets" that are very precise and reach up to 2.5 meters.

"We see, as with most evolutionary processes of a complex adaptation, you can see this incremental effect over many millions of years' time leads to this honed, exquisite system that exists today," Casewell said.

The role of hominids

A fascinating hypothesis of the study is that hominids are likely to have influenced the development of defensive pain-causing venom in spitting cobras.

"There is a reasonable amount of evidence to support a potential role of ancient hominids in being the stimulating pressure for the evolution of venom spitting," Casewell said. "The pieces of evidence that we provide in the paper are the correlation in terms of divergence times between the evolution of venom spitting in two of the spitting lineages, and key dates of hominid evolution."

"This split in Africa between bonobos and chimpanzees correlates very well with the timing of the origin of venom spitting in African spitting cobras," he added.  "And then in Asia, we find a date much later when Asian spitting cobras evolved this trait, which correlates roughly with the first timing roughly with hominids migrating into that continent.

"We know that primates were certainly major prey items for snakes previously. And we know from behavioral observation that many primates will actually actively seek out and kill snakes, sometimes using weapons. We know that they also have forward facing eyes, which make them a very nice target for spitting, whereas other animals that have been postulated to drive spitting, like ungulates, have more side-facing eyes which would perhaps be less targeted by venom spitting."

Casewell also said, "Other than that, there are no other plausible species that may have produced this selection pressure. There's no natural history observations of carnivore species being spat at by spitting cobras. In contrast, people are spat at by spitting cobras all the time. We've come together with this hypothesis that perhaps hominids were an important selection pressure on snakes, just as snakes have been proposed to be an important selection pressure on primates and our relatives as well."

A collaborative effort 

Casewell highlighted the collaborative nature of the project, involving people from all over the world with different expertise.

"Typically, these people are devoting their time and energy with no formal or financial support, but because they found the question interesting," he said. "That was the really the reason that we were able to form such a comprehensive analysis."

Casewell noted it's difficult getting support for basic questions like the one they studied, but that the basic studies have allowed them to make advances in snake bite treatment. 

"Cobras are a very important  source of snake bites in Africa and Asia," he said. "Now we understand a lot more about the toxins that are in cobra venoms and what we need to do to tackle them."

A 10-minute video, "Secrets of the Spitting Cobra," can be found here.