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The Evolution of Spitting Cobra Venom to Ward Off Enemies

A new study reports the emergence, on three separate occasions, of venom spitting to inflict pain for defensive purposes.

by Francie Moehring


26 April 2021


PRF News

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A new study reports the emergence, on three separate occasions, of venom spitting to inflict pain for defensive purposes.

When it comes to venomous snakes, usually we think of these creatures as using venom to kill and eat their prey. However, a new study suggests that in one group of snakes, known as spitting cobras, evolution had a new purpose in mind for their venoms.

 

Research led by Nicholas Casewell, Liverpool School of Tropical Medicine, UK, now reveals that some snakes have evolved to spit venom as a defense mechanism to fight off predators, perhaps including our ancient hominin ancestors. This occurred in three different lineages of spitting cobras. The authors also show that the composition of spitting cobra venom changed over time to strongly activate mammalian sensory neurons and cause intense pain.

 

“I think this is a great study demonstrating the occurrence of convergent evolution, where the same trait developed three times in snakes across different continents,” said Sven-Eric Jordt, Duke University, Durham, US.

 

“What is really interesting,” Jordt continued, “is that in addition to the snakes evolving to have a new way to project the venom, the authors also show that PLA2 is an important component that amplifies the effects of the venom, which is responsible for the pain elicited by the venom of spitting cobras,” said Jordt, who was not involved with the new work. Here he was referring to phospholipase A2s, a family of toxins in cobra species.

 

The research was published January 22, 2021, in Science.

 

Evolution comes up with the same answer three separate times

Previous work had largely focused on venom variation in snakes as being driven by dietary differences as these creatures sought to eat their prey. But the researchers wondered whether venom could evolve for a different purpose, as a defense mechanism, and they looked to spitting cobras as a way to test this idea.

 

Venom spitting in snakes evolved independently three times, twice in Africa and once in Asia, in three species of cobras: African spitting cobras (genus Naja: subgenus Afronaja), Asian spitting cobras (Naja: subgenus Naja), and rinkhals (Hemachatus

 

To understand the evolution of venom spitting, Casewell, co-first authors Taline Kazandjian, Liverpool School of Tropical Medicine, UK; Daniel Petras, UC San Diego, US; and Samuel Robinson, University of Queensland, Australia, along with colleagues, used a variety of techniques including transcriptomics, proteomics, and functional and phylogenetic comparisons between different members of a family of venomous snakes known as elapids; this family includes cobras.

 

“To investigate the convergent evolution of venom spitting in cobras, we used a multidisciplinary approach with numerous different techniques and collaborators from all over the world,” said Casewell.

 

“We first compared 17 widely distributed elapids using phylogenetic comparisons,” said Kazandjian, “which allowed us to pinpoint that venom spitting originated in African spitting cobras between 6.7 million and 10.7 million years ago and approximately 4 million years later in the Asian spitting cobras.” Along with the finding of the origin of spitting in Hemachatus, this provided the authors with the confirmatory evidence that the act of spitting venom in cobras evolved three separate times across the world.

 

Slow motion video of Naja nubiae spitting venom. Credit: ©The Trustees of the Natural History Museum, London and Callum Mair.

 

What’s in the venom? One expected finding, one surprising finding

Next the authors used a top-down proteomics approach to analyze what was in the venom of each species. This is a technique that analyzes intact proteins using mass spectrometry.

 

Previous work had shown that cobra venom is slightly different from that of other snake toxins. In particular, all cobra venoms are dominated by cytotoxic three-finger toxins (3FTXs), which disrupt the cell membrane. The second most abundant toxins found in cobra venoms are the PLA2s. Looking at these and some other toxins allowed the group to make one finding that met their expectations.

 

“When analyzing the venom, we found that spitting cobra venom clustered in three separate clusters [the two groups of African spitting cobras and the group of Asian spitting cobras] that were distinct from the homogeneous cluster of non-spitting cobras,” said Kazandjian (with the sole exception being the Asian spitting cobra Naja philippinensis, which appeared similar to nonspitting species). “This suggested to us that the venom composition is collectively different from the non-spitting cobras.”

 

“This finding was not really that surprising to us as it is consistent with the difference in the venom-induced pathology observed after bites to humans. The bite of another snake or cobra is often deadly, but the bite of a spitting cobra causes a lot of tissue necrosis and inflammation but only in rare occasions death due to the bite itself,” said Casewell.

 

But, when the investigators compared the toxin composition of nonspitting cobra venoms to spitting cobra venoms, they did not find a significant difference in the abundance of cytotoxic 3FTXs (CTXs). This was surprising considering the dominance of these toxins in cobra venoms.

 

The authors also wanted to determine whether the spitting cobra venom was more toxic to mammals than the venom of non-spitting snakes. To do this, they used an assay in which irritation in response to a toxin can be measured in non-sentient chick embryos. Topical application of high doses of spitting cobra venom to the embryos revealed no association between how cytotoxic the venom was and spitting.

 

Spitting cobra venom and nociception: working together is better than working alone

However, using venom that slowly destroys cells in order to cause pain may help a cobra for purposes of predation. But this approach wouldn’t be as useful for defensive purposes. In those instances, inflicting direct – and thus rapid – pain is paramount. So, the authors investigated the nociceptive activity of cobra venoms. For this purpose, they applied venom to mammalian trigeminal neurons. These are sensory neurons derived from trigeminal ganglia that innervate the face and eyes.

 

Using calcium imaging to monitor the activity of the trigeminal neurons, the team saw that all the cobra venoms could activate sensory neurons. The researchers say this is probably because all the venoms disrupt the cell membrane thanks to the cytotoxic activity of the CTXs. But the spitting cobra venoms activated the neurons more strongly than the non-spitting cobra venoms did – exactly what such creatures would need to more effectively inflict pain. What was in the spitting cobra venom that accounted for this?

 

Fractionating the venom from three representative spitting cobras into its components, the team found that only those fractions containing CTXs could activate sensory neurons; fractions containing only neurotoxins or PLA2s were unable to do so. But then a new wrinkle to the story emerged.

 

“Interestingly, we noted that none of the cytotoxic fractions completely reproduced the effects seen with whole venom, which suggested that certain venom components might be acting together to produce the whole effect,” said co-first author Robinson. “We hypothesized that venom PLA2s act in synergy with CTXs to potentiate sensory neuron activation.”

 

The hypothesis turned out to be correct. The CTX fractions were better able to activate sensory neurons when combined with PLA2 fractions. Consistent with that finding, use of a PLA2 inhibitor decreased the ability of the CTX fractions to activate the neurons.

 

“It was very interesting to see that spitting cobras evolved to upregulate PLA2, which acted in synergy with the CTXs to make their venom quite a bit more potent. This synergistic action of the two toxins actually is a quite rare phenomenon,” explained Robinson.

 

Spitting cobra venom indeed had more PLA2, compared to the venom of non-spitting cobras. Also, proteomics results showed that there was major variation in PLA2 between spitting and nonspitting cobra lineages, especially when looking at the African species of spitting cobras. Phylogenetic analysis showed that there was a duplication of the PLA2 gene at the time when venom spitting emerged in the ancestor of African spitting cobras.

 

Finally, additional experiments in a mouse model of venom lethality would show no greater lethality of spitting cobra venom vs. the non-spitting variety. This suggested that it was the composition of spitting cobra venom that really made the difference, and that the evolutionary purpose of the enhanced pain by the spitting cobra venom was defense against potential enemies, rather than an offensive maneuver against prey species.

 

Are our hominin ancestors to blame?

So why did cobra venoms evolve in this way – under what threats could these snakes find themselves that would make venom spitting an effective solution?

 

Certain carnivorous mammals and raptorial birds are common predators of snakes around the world, but the authors view this as an unlikely explanation since predation on spitting cobras by these species is also common when looking at natural history reports. Another potential threat to snakes is being trampled by other animals like ungulates (large mammals with hooves), but the authors view this too as a questionable account of the evolution of spitting; Asian spitting cobras primarily live in heavily forested areas, making trampling less of a threat. Also, the eyes of large ungulates are on the sides, which makes spitting less of a threat to them.

 

Instead, the researchers suspect that the real threat to cobras may have been ancient hominins – pre-humans – for a number of reasons. For instance, evidence points to an influence of snakes on primate neurobiology and behavior. And anthropoid primates (a group that includes monkeys and apes) have been known to kill dangerous snakes with clubs and projectile tools. The authors also point out that African spitting cobras first diverged as recently as 6.7 million years ago, not that long after the divergence of hominins from Pan (bonobos and chimpanzees), which occurred 7 million years ago.

 

“The correlation between when spitting cobras evolved and when the divergence of hominins occurs was quite interesting to us as it points towards the possibility that hominins might have caused significant evolutionary pressures on the ancestors of the spitting cobras,” explained Robinson.

 

“The hominin theory," said Jordt, “is quite speculative, since it is difficult to determine if the number of hominins in the specific geographic areas were truly sufficient to cause the evolutionary pressure for snakes to evolve to spit their venom. Nevertheless, I believe that the hominin theory is plausible.”

 

In terms of future research, the Casewell laboratory says their work points the way towards better treatments for snakebites.

 

“Antivenom for the spitting cobras is not very effective for the inflammation and necrosis that it causes,” said Casewell. “Therefore, we hope to find and develop cheaper alternatives or adjunct therapies to antivenoms that could help solve this problem.”

 

“Often, people bitten by snakes do not know the exact snake that bit them,” Casewell continued, “so finding ways to target common pain-causing components of the toxin pathway such as PLA2 might prove to be beneficial in the future, especially if this adjunct therapy could be a small molecule drug given orally.”

 

Francie Moehring is a freelance writer based in Milwaukee, US.

 

Featured Image: Naja nubiae slow motion spitting. Credit: ©The Trustees of the Natural History Museum, London and Callum Mair.

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