The intricate world of cone snail venoms

A great paper authored by Sébastien Dutertre and colleagues was recently published by Nature Communications:

ResearchBlogging.orgDutertre, S., Jin, A., Vetter, I., Hamilton, B., Sunagar, K., Lavergne, V., Dutertre, V., Fry, B., Antunes, A., Venter, D., Alewood, P., & Lewis, R. (2014). Evolution of separate predation- and defence-evoked venoms in carnivorous cone snails Nature Communications, 5 DOI: 10.1038/ncomms4521

In this post, I will explain what this research is about and why it’s cool, but first, let’s go through all the background knowledge needed.

Conus species. Source: Olivera (1997).
Conus species. Source: Olivera (1997).

Over 600 species of Conus snail (Duda & Kohn, 2005) sounds like a lot of animals, but it’s a mere 7% of all the diversity of the Conoidea superfamily (refer to Bouchet et al. (2011) for taxonomic details). Despite this, Conus is one of the most well-known of all marine snails – they’re super-easy to collect, the shells are large and beautiful with their varied patterns, and it’s easy to get a morbid fascination with their ability to kill a human with their venoms.

That’s right, venoms with a plural. The genomic regions coding for these toxins exhibit some of the highest molecular evolution rates known (Chang & Duda, 2012). Consider that each of these species can produce 50-200 different toxins, all of which are species-specific(Terlau & Olivera, 2004). This means that, on average, there are at least 600000 different toxins to discover – an unparalleled hyperdiversity that has warranted the erection of a database dedicated to them, the ConoServer.

They’re all classified as conopeptides or as conotoxins. They fall within 15 superfamilies with stereotypical structures (Terlau & Olivera, 2004). They are small, 15-40 amino acids long, and consist of a conserved core pattern with a highly-variable region that is responsible for the multitude of effects conotoxins have. Toxins are grouped together to form a venom. The physiological action of the venoms is classified into cabals (Olivera, 1997). For example, fish-hunting cones have a lightning-strike cabal and a motor cabal: the first paralyses the victim, the other blocks neuromuscular function.

This hyperdiversity stems from a few conserved genetic sequences: the toxins of one species have exact homologs in other species, and the homologisation can be so clear that conotoxins are very useful for species-level phylogenetics. Homologous peptides are not only similar at a structural level, but also at a pharmacological level. This is how we classify conotoxins.

Evolution of Conus. Source:
Evolution of Conus. Source: Duda & Palumbi (2004)

Conus are predatory snails that have neither speed nor weapons, so they depend on their venom for survival and capturing food.They are roughly classified according to their main prey: molluscs, worms, hemichordates, or fish, although they are not limited to these. The analysis by Duda & Palumbi (2004) revealed that cone snails most likely first evolved as worm-hunters, with several independent switches to fish-feeding and one to mollusc-feeding.

One of the main uses of the venoms is thus in acquiring food. There is as much diversity in venom effects as there are venoms. Most are the usual paralysing neurotoxins, but there are some with weirder effects, such as sleep inducers (White et al., 2000).

Venom duct. Source:
Conus venom duct. Source: Norton & Olivera (2006)

This predatory mode is a huge departure from the ancestral herbivory of snails, so it’s no surprise that there are unique anatomical differences that evolved in cones (and, more broadly, in all the other toxoglossate gastropods, which are ancestrally venomous). The conotoxins are synthesised and secreted in a venom gland that runs along the foregut. The evolution of this gland and what organ it arose from is currently enigmatic and controversy still abounds even after more than a century of study (Amaudrut, 1898). The most accepted explanation is that it’s a derived form of the mid-oesophagal gland of other gastropods as originally postulated by Amaudrut (Ponder & Lindberg, 1997), although one can also justify a view that it is a novel organ (Smith, 1967).

Radular tooth of Conus imperialis. Source:
Radular tooth of Conus imperialis. Source: Kohn et al., 1972

In order to detect prey, the snails extend a long, flexible proboscis much like a tongue (Greene & Kohn, 1989). Once a victim is found, the venom is injected by a single highly-specialised hollow, harpoon-like radular tooth that is shot into the victim. In fish-hunting species, the process from sensation to firing is blindingly quick, taking place in less than 1 ms (Schulz et al., 2004). This tiny amount of time includes the filling of the tooth with the venom (Salisbury et al., 2010). This is very important for understanding the significance of the paper, so keep it in mind.

Conotoxins are incredibly specific in their action – they have been so strongly selected for (Duda & Palumbi, 1999) that individual toxins are specific to only a few receptors (Sharpe et al., 2001). The composition of the venoms is very tightly associated with the prey (Remigio et al., 2008).Besides making them very effective hunters, this has also made them very useful for neurological and pharmacological research, as well as a potential source of new drugs, as exemplified by the conotoxin ω-MVIIA, known on the market as ziconotide or Prialt, approved for treating severe chronic pain (Miljanich, 2004). It is 800 times more potent than morphine (Xia et al., 2006) but without the addictive properties. It works by blocking calcium from entering nerve ends, and by inhibiting the release of neurotransmitters from nociceptive nerves, those nerves that are responsible for sensing pain. Another conotoxin with therapeutic potential is ACV1, which has been found to accelerate the recovery of damaged nerves, as well as to inhibit nicotinic acetylcholine receptors responsible for pain transmission (Livett et al., 2006).

Of course, the venom is not only useful for attacking prey, but also in defence. And here is where the paper comes in.

Collected venom levels. Source: Dudertre
Collected venom levels. Source: Dutertre et al. (2014)

The researchers collected fish-, mollusc-, and worm-hunting cones from Queensland, Australia, and kept them in aquaria. Carefully, they coaxed the cones to emit venoms under different circumstances: predation and defence. This alone produced an interesting result: defensive cones consistently produce and inject twice as much venom as attacking ones.

Composition of collected venoms. Source:
Composition of collected venoms. Source: Dutertre et al. (2014)

The differences don’t stop there. Not only do they produce double the amount of venom when defensive, the defensive and predatory venoms aren’t even the same! In fact, as the mass spectrometer graphs above show, the defensive venoms (green) are much more complex than predatory ones (blue), with very little overlap in the toxins in the case of C. marmoreus.

What is the implication of this? As I tried to emphasise previously, Conus toxins are incredibly specific, so it stands to reason that they have different effects. And it is indeed so. One of the species they were experimenting on, C. geographus, is responsible for several human deaths (and is the reason why you probably shouldn’t be trying to replicate this paper at home). But when the researchers tested the predatory venom, the effect was pretty limited – only the Prialt compound, ω-MVIIA, and its sister compounds had an effect. In contrast, the defensive venom blocked every type of receptor tested for, which in a real life situation would lead to paralysis and a swift yet horrible death. In other words, when you get bitten* by a cone, it’s being defensive, not trying to attack you.

Now, I want you to recall how cones produce their venom. They have the venom duct, and it secretes venom and directs it to the tooth in less than 1 ms. Knowing this, an obvious question will pop into your mind: How does the snail produce such different venoms so quickly, in situations that they can’t possibly predict and prepare for?

To me, this is the most fascinating question in this study. How did the researchers answer it? They used transcriptomics: they analysed how much mRNA was being expressed along the venom duct of one Conus geographus. The results are visualised below.

Transcriptomic analysis of Conus geographus venom duct. Source: Dutertre et al. (2014)
Transcriptomic analysis of Conus geographus venom duct. Source: Dutertre et al. (2014)

Even without knowing what it is you’re looking at, there is an obvious demarcation between the proximal and distal parts of the ducts. If you don’t see it in the main graph, look at the two on the right. The 1417 Da molecule and the 3175 Da molecule do not coexist, and we can see the same pattern of limited overlap in the main graph.

So what do these graphs mean? The y-axis is distance along the venom duct. The x-axis’s unit is Da, which is Dalton, the unit of atomic mass that we use in the molecular biosciences. Higher Da means heavier molecule. As mentioned before, conotoxins are small molecules, and they tend to range between 1000 and 4000 Da, as shown in this graph.

The peaks above the graph represent various molecules of a certain weight, and the height of the peak represents how much of the molecule is present.

So what the graph shows is the distribution of mRNAs along the venom duct, and a look at the graph shows that there is some sort of demarcation between points 6 and 7, with the mRNA contents changing noticeably. This line emerges at the separation between the proximal and distal duct. Because mRNAs get translated directly into the protein sequences, i.e. the conotoxins, what this means is that the conotoxins produced in the proximal duct are different from those produced in the distal duct.

The weights of the individual mRNAs secreted in the proximal duct are consistent with the conotoxins used in defence, and the weights of those in the distal duct are consistent with predatory conotoxins.

Hypothesised control of venom secretion. Source: Dutertre et al. (2014)
Hypothesised control of venom secretion. Source: Dutertre et al. (2014)

Which leads to the conclusion visualised above. The proximal duct produces the defence venoms, the distal duct produces the predatory venoms, and the two venoms are chemically and pharmacologically distinct. The authors hypothesise that upon encountering prey or a threat, the oesophagal ganglion, part of the molluscan brain, triggers both the appropriate part of the duct and the bulb. The duct secretes the venom, and the bulb muscularly pushes it out towards the tooth.

Such a mechanism, with the regionalisation and differentiation of venoms for different purposes, seems to be fairly unique, and it adds a whole new dimension to the already exciting study of cone snails, although there is the important caveat that more research is needed: an investigation needs to be done to see if such neuronal pathways even exist, and let’s not forget that the transcriptomic analysis was done on only one species.

Conussnails are one of the biggest stories of evolutionary success to be found. Since the origin of the Conidae 55 million years ago, Conus has been characterised by extremely fast speciation rates. The majority of the 600 species alive today originated in a Pleistocene adaptive radiation, exhibiting a speciation rate more than 3-4 times that of other snails, a rate that is even higher in some places, like the Cape Verde islands where 10% of cone snail diversity resides endemically due to an explosive local radiation (Monteiro et al., 2004).They owe this success to their venoms and, as this paper shows, to the incredibly precise ways they’ve evolved to administer them.

Footnote:

*: I’m not sure whether bitten or stung is the right term. You get stung by a cone, but the stinging is done by a tooth, so it technically is biting you. Or are jaws and chewing prerequisites for biting? Linguists, help. This is important.

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