Wednesday, October 26, 2016

A new toxin from the Blue Coral Snake

(a) Specimen of Calliophis bivirgatus, the blue coral
snake (Photo by Tom Charlton). (b) Dissected preserved 
112 cm Calliophis bivirgatus specimen with 29 cm elongated 
venom glands (arrows).
The Asian Coral snakes in the genus Calliophis feed upon other snakes, including other snake-eating venomous species of Elapidae such as kraits (Bungarus) and king cobras (Ophiophagus). A unique evolutionary scenario ensues, a chemical arms race between predator and prey in which the risk of role reversal becomes a key selection pressure driving the evolution of toxins that rapidly render prey incapable of retaliation or escape. Snakes that hunt animals capable of inflicting serious retaliatory wounds often release their intended prey after envenomation. In this situation, selection may favour the evolution of toxins that rapidly disable prey, either to prevent it moving too far to be recovered or to prevent the possibility of it attacking and injuring the snake.

With its combination of electric blue dorsolateral stripes and neon red head, tail, and ventral scales, the blue coral snake, Calliophis bivirgatus, is arguably one of the world’s most striking species of snake. An encounter with one is high on the list for many reptile enthusiasts and nature photographers visiting southern Thailand, Malaysia, Singapore, and western Indonesia. The species is of additional interest to anatomists and toxinologists studying the evolution and diversification of the snake venom system as it (along with its congener C. intestinalis) possesses novel elongated venom glands that extend up to one quarter of the length of its body. It is also of medical significance as, in spite of only a small handful of confirmed bites, it has been responsible for at least one human fatality, is suspected of causing at least one more, and has no known antivenom. In spite of these high levels of interest, the venom has been subject to relatively few studies. Studies that examined the toxin content of the venom concluded that all the three-finger toxins present were exclusively cytotoxic in their effects. However, this limited scope of activity attributed to the venom was reflective of the very narrow scope of assays performed and cytotoxicity was largely assumed based on similarity of partial sequences to other toxin types from other snakes rather than full activity characterisation. One study, which examined the usefulness of Taiwan antivenom, preincubated the venom with antivenom (a clinically unrealistic situation) and even then required very high doses to exert any meaningful level of inhibition.

In a new paper Yang et al. (2016) show that the venom is unique in producing spastic paralysis, in contrast to the flaccid paralysis typically produced by neurotoxic snake venoms. The toxin responsible, is named calliotoxin (δ-elapitoxin-Cb1a), a three-finger toxin (3FTx). The calliotoxin molecule has a form of neurotoxicity, previously known from cone snail and scorpion venoms, and is identified for the first time from the venom of a snake. Calliotoxin shifts the voltage-dependence of NaV1.4 activation to more hyperpolarised potentials, inhibits inactivation, and produces large ramp currents, consistent with its profound effects on contractile force in an isolated skeletal muscle preparation. Voltage-gated sodium channels (NaV) are a particularly attractive pharmacological target as they are involved in almost all physiological processes including action potential generation and conduction. Accordingly, venom peptides that interfere with NaV function provide a key defensive and predatory advantage to a range of invertebrate venomous species including cone snails, scorpions, spiders, and anemones. Enhanced activation or delayed inactivation of sodium channels by toxins is associated with the extremely rapid onset of tetanic/excitatory paralysis in envenomed prey animals. A strong selection pressure exists for the evolution of such toxins where there is a high chance of prey escape. However, despite their prevalence in other venomous species, toxins causing delay of sodium channel inhibition have never previously been described in vertebrate venoms. Here we show that NaV modulators, convergent with those of invertebrates, have evolved in the venom of the long-glanded coral snake. Calliotoxin represents a functionally novel class of 3FTx and a structurally novel class of NaV toxins that will provide significant insights into the pharmacology and physiology of NaV. The toxin represents a remarkable case of functional convergence between invertebrate and vertebrate venom systems in response to similar selection pressures. These results underscore the dynamic evolution of the Toxicofera reptile system and reinforces the value of using evolution as a roadmap for biodiscovery.

Yang DC, Deuis JR, Dashevsky D, Dobson J, Jackson TN, Brust A, Xie B, Koludarov I, Debono J, Hendrikx I, Hodgson WC. 2016. The Snake with the Scorpion’s Sting: Novel Three-Finger Toxin Sodium Channel Activators from the Venom of the Long-Glanded Blue Coral Snake (Calliophis bivirgatus). Toxins. 8(10):303.

Monday, October 24, 2016

Loss and Re-emergence of Legs in Snakes

This image depicts mouse embryos with the ZRS from cobra or 
python inserted into their genomes, replacing the normal gene regulator. 
Their truncated limb development is visible in the comparative 
bone scans. Credit: Kvon et al. Cell 2016
Snakes lost their limbs over 100 million years ago, but scientists have struggled to identify the genetic changes involved. A Cell paper publishing October 20 sheds some light on the process, describing a stretch of DNA involved in limb formation that is mutated in snakes. When researchers inserted the snake DNA into mice, the animals developed truncated limbs, suggesting that a critical stretch of DNA lost its ability to support limb growth during snake evolution.

"This is one of many components of the DNA instructions needed for making limbs in humans and, essentially, all other legged vertebrates. In snakes, it's broken," says Axel Visel, a geneticist at the Lawrence Berkeley National Laboratory and senior author on the paper. "It's probably one of several evolutionary steps that occurred in snakes, which, unlike most mammals and reptiles, can no longer form limbs."

Today's serpents have undergone one of the most dramatic body plan changes in the evolution of vertebrates. To study the molecular roots of this adaptation, Visel and his colleagues started looking at published snake genomes, including the genomes from basal snakes such as boa and python, which have vestigial legs -- tiny leg bones buried in their muscles -- and advanced snakes, such as viper and cobra, which that have lost all limb structures. Within these genomes, they focused specifically on a gene called Sonic hedgehog, or Shh, involved in many developmental processes -- including limb formation. The researchers delved further into one of the Shh gene regulators, a stretch of DNA called ZRS (the Zone of Polarizing Activity Regulatory Sequence) that was present but had diverged in snakes.

To determine the consequences of these mutations, the researchers used CRISPR, a genome-editing method, to insert the ZRS from various other vertebrates into mice, replacing the mouse regulator. With the ZRS of other mammals, such as humans, the mice developed normal limbs. Even when they inserted the ZRS from fish, whose fins are structurally very different from limbs, the mice developed normal limbs. However, when the researchers replaced the mouse ZRS with the python or cobra version, the mice went on to develop severely truncated forelimbs and hindlimbs.

"Using these new genomic tools, we can begin to explore how different evolutionary versions of the same enhancer affect limb development and actually see what happens," says Visel. "We used to be mostly staring at sequences and speculating about molecular evolution, but now, we can really take these studies to the next level."

To identify the mutations in the snakes' ZRS that were responsible for its inactivation during snake evolution, the researchers took a closer look at the evolutionary history of individual sequence changes. By comparing the genomes of snakes and other vertebrates, they identified one particularly suspicious 17 base-pair deletion that only occurred in snakes; this deletion removed a stretch of the ZRS that has a key role in regulating the Shh gene in legged animals.

The research team turned back the evolutionary clock, restoring the missing 17 base pairs in an artificially created hybrid version of the python ZRS, and tested the edited DNA in mice. Those that carried this evolutionarily "resurrected" ZRS in their genome, replacing their normal regulator, developed normal legs. However, Visel cautions that the evolutionary events were probably more complex than just the one deletion: "There's likely some redundancy built into in the mouse ZRS. A few of the other mutations in the snake ZRS probably also played a role in its loss of function during evolution."

Of course, snakes aren't the only vertebrate animals that lack arms and legs -- some lizards, eels and other fish, and marine mammals, for example, have also adapted limb reduction to varying degrees and likely underwent a slightly different evolutionary process. "Loss of limbs has occurred multiple times independently during animal evolution, and it's safe to assume that mutations affecting other genes were involved," says Visel. "It's a complex problem, but with the introduction of genome-editing tools, we can finally start tying specific DNA changes to alterations in body shape more systematically."

Evgeny Z. Kvon, Olga K. Kamneva, Uirá S. Melo, Iros Barozzi, Marco Osterwalder, Brandon J. Mannion, Virginie Tissières, Catherine S. Pickle, Ingrid Plajzer-Frick, Elizabeth A. Lee, Momoe Kato, Tyler H. Garvin, Jennifer A. Akiyama, Veena Afzal, Javier Lopez-Rios, Edward M. Rubin, Diane E. Dickel, Len A. Pennacchio, Axel Visel. Progressive Loss of Function in a Limb Enhancer during Snake Evolution. Cell, 2016; 167 (3): 633 DOI: 10.1016/j.cell.2016.09.028

Francisca Leal, Martin J. Cohn. Loss and Re-emergence of Legs in Snakes by Modular Evolution of Sonic hedgehog and HOXD Enhancers. Current Biology, 2016; DOI: 10.1016/j.cub.2016.09.020

Sunday, October 23, 2016

Aegean wall lizards switch foraging modes in a human-built environments

Male of Erhard's Wall Lizard (Podarcis erhardii) in the ruins of Ag. Achilleos
 on the small island in Lake Mikri Prespa. Author: Jeroen Speybroeck
The Aegean Wall Lizard, Podarcis erhardii inhabits the Balkan peninsula and the Aegean islands. On the mainland it ranges from Albania, the Republic of Macedonia and southern Bulgaria to the northeastern part of the Peloponnese peninsula in Greece. Donihue (2016) tested for foraging mode switching between populations of the Aegean wall lizard, Podarcis erhardii, inhabiting undisturbed habitat and human-built rock walls on the Greek island of Naxos. He observed foraging behavior among 10 populations and tested lizard morphological and performance predictions at each site. He also investigated the diet of lizards at each site relative to the available invertebrate community.He  found that lizards living on rock walls were significantly more sedentary—sit and wait—than lizards at nonwall sites. He also found that head width increased in females and the ratio of hind limbs to forelimbs in both sexes increased as predicted. Diet also changed, with non-wall lizards consuming a higher proportion of sedentary prey. This study demonstrates microgeographic variability in lizard foraging mode as a result of human land use. In addition, these results demonstrate that foraging mode syndromes can shift intraspecifically with potential cascading effects on local ecological communities. Lacertids are considered a clade of active foraging species and the populations on Naxos from habitats that reflect the pre-human landscape in Greece  were active foragers.

Donihue CM. 2016. Aegean wall lizards switch foraging modes, diet, and morphology in a human‐built environment. Ecology and Evolution. DOI: 10.1002/ece3.2501

Saturday, October 22, 2016

The snake that ate a lizard, that ate an insect

An interpretive drawing of SMF ME 11332a overlaid on a„ photograph. The lizard, 
Geiseltaliellus maarius (orange), is preserved in the stomach of the snake (white). 
The lizard was swallowed headfirst, and the tail does not appear to have been shed 
during the encounter with the snake. The position of the insect in the abdominal cavity
 of the lizard is indicated in outline (blue).Juliane Eberhart, Anika Vogel. 
A recent paper in  Palaeobiodiversity and Palaeoenvironments Smith and Scanferla (2016) report a fossil snake from the middle Eocene (48 million years ago) Messel Pit, in whose stomach is a lizard, in whose stomach is an insect. This is the second report of a vertebrate fossil containing direct evidence of three trophic levels. The snake is identified as a juvenile of Palaeopython fischeri on the basis of new characters of the skull; the lizard is identified as Geiseltaliellus maarius, a stem-basilisk; and the insect, despite preserved structural colouration, could not be identified. The lizard, G. maariusis is thought to have been an arboreal species, but like its extant relatives may have foraged occasionally on the ground. Another, larger specimen of G. maarius preserves plant remains in the digestive tract, suggesting that omnivory in this species may have been common in larger individuals, as in extant Basiliscus and Polychrus. A general picture of the trophic ecology of P. fischeri is not yet possible, although the presence of a lizard in the stomach of a juvenile individual suggests that this snake could have undergone a dietary shift, as in many extant boines.


Smith KT, Scanferla A. Fossil snake preserving three trophic levels and evidence for an ontogenetic dietary shift. Palaeobiodiversity and Palaeoenvironments. 2016:1-1.

Thursday, October 20, 2016

A new Andean Shadow Snake and the Diaphorolepidini tribe

Nicéforo María's Shadow Snake, Synophis niceforomariae
The genus Synophis contains a number of enigmatic species, distributed primarily in the Andean highlands of northern South America. Their extreme crypsis and rarity has precluded detailed study of most species. A recent flurry of collection activity resulted in the accession of many new specimens, and the description of 4 new species in 2015, doubling the number of described taxa. However, lingering questions remain regarding the assignment of many new and historical specimens, the morphological limits and geographical ranges of the species, and their phylogenetic relationships. In a new paper Pyron et al. (2016) analyze new and existing morphological and molecular data to produce a new molecular phylogeny and revised morphological descriptions. They also validate the previously unavailable tribe name Diaphorolepidini Jenner, Pyron, Arteaga, Echevarría, & Torres-Carvajal, describe a 9th species, Synophis niceforomariae and offer the new Standard Names in English and Spanish for the group: the Andean Shadow Snakes and Culebras Andinasde la Sombra, respectively. The authors suggest  cryptic and undiscovered diversity undoubtedly remains within the genus. The tribe Diaphorolepidini is based upon the most recent common ancestor of Diaphorolepis wagneri Jan, 1863, Emmochliophis (Synophis) miops (Boulenger, 1898), and Synophis bicolor Peracca, 1896. The new species, Synophis niceforomariae occurs in the Andean highlands of north-central Colombia, Antioquia department, near Medellín, ~1300–1700m, with possible populations south of Medellín, ~900m.

Pyron RA, Artega A, Echevarria LY, Torres-Carvajal OM. 2016. A revision and key for the tribe Diaphorolepidini (Serpentes: Dipsadidae) and checklist for the genus Synophis. Zootaxa. 4171(2):293-320.