The molecular evolution of cephlopod venoms

A project undertaken at the Department of Biochemistry & Molecular Biology, The University of Melbourne, and supervised by A/Prof Bryan Fry

New insights into the evolution of venom systems and the importance of the associated toxins cannot be advanced without recognition of the true biochemical, ecological, morphological and pharmacological diversity of venoms and associated venom systems. A major limitation of the use of venom proteins has been the very narrow taxonomical range studied. Entire groups of venomous animals remain virtually completely unstudied. One such group are the coleoids (cuttlefish, octopuses, and squid). In particular, our pilot study on Antarctic octopus venoms is the only one published on polar octopus venoms (Undheim et al 2010a).

Venoms play a range of adaptive roles in the animal kingdom; killing, paralyzing, immobilizing or pre-digesting prey, as well as defence against predators and deterring competitors (Fry et al 2009a). In animals relying on toxins for prey capture and handling, the evolution of the molecular components of venom is often tightly linked to diet and trophic ecology through an evolutionary arms race between predator and prey. Due to the strong selection pressure that results from this arms race, and because toxin genes are members of multigene families, toxins often show very rapid evolutionary rates. The accelerated evolution may be in a diversifying manner, such as conotoxins , or convergent, like the fish-specialised venoms of the unrelated sea kraits and sea snakes.

One group that uses venom in the capture of otherwise tricky or potentially dangerous prey are octopuses, which are known to prey on a range of taxa, including molluscs, crustaceans, fish, and even birds. The octopus venom apparatus terminates in the toothed salivary papilla contained within the buccal mass, which is connected to the paired posterior salivary glands through ducts running adjacent to the oesophagus. The inverse relationship between the size of venom gland and the size of the buccal mass and beak, is at its most extreme in the species Adelieledone polymorpha from the Australian East Antarctic waters.

As well as functioning as a reservoir, the salivary papilla is used to inject saliva into prey, often with great force, through either puncture wounds caused by the beak or holes drilled mainly by the physical actions of the radula . This results in immobilisation of prey, such as by hypotension and paralysis followed by death in crustaceans , but seemingly only temporary paralysis or hypotension in molluscs. Although small prey is often handled without the use of venom, many octopods switch to the use of venom once the use of physical force becomes inefficient. In large, shelled prey, for example, small holes are often drilled through which the octopus injects its venom, which then acts by paralyzing and/or killing the prey as well as aiding in the detachment of tissue from the exoskeleton.

The production of toxic saliva in octopod posterior salivary glands was first recognized at the end of the 1800's. Despite findings as early as 1906 that the toxin was a protein particularly potent against crustaceans, it was long thought that the paralysis and death observed in envenomated prey was due to the actions of various amines isolated from octopod PSG, such as tyramine, histamine, tyramine, acetylcholine, octopamine and serotonin. However, the findings that these amines at realistic concentrations only caused the initial symptoms of "overexcitability" in envenomated crabs, and were unable to reproduce the irreversible paralysation achieved by injection of crude saliva, suggested a more complex venom composition. This resulted in the description of Cephalotoxin (Ctx), a protein mixture consisting of four proteins, including at least one glycoprotein, originally extracted from the PSG of the cuttlefish Sepia officinalis, and later Octopus vulgaris. Ctx was reported to have a number of activities, including inhibition of respiration in crabs, inhibition of blood coagulation in both crabs and humans, and paralysation of crabs and cockroaches.

Since then, several proteinaceous toxins have been isolated and their activities described from the salivary glands from a range of octopodid species, for example Eledone cirrhosa, Hapalochlaena maculosa, Octopus dofleini and O. vulgaris. Being responsible for the serious symptoms (even human fatalities) associated with bites from members of Hapalochlaena, and proving useful in areas like clinical research, much of cephalopod toxicological research has been directed towards tetrodotoxin (TTX) and TTX-like compounds (Fry et al., 2009b). However, studies have shown that like in many other marine organisms, Hapalochlaena TTX is produced by endosymbiotic bacteria and distributed not only in the salivary glands, but also all other parts of the body.

Relatively little is known about the presumably endogenous salivary toxins found in octopods. It seems, however, that various proteases and neurotoxins are important venom constituents. In one of our HSF funded studies, we found evidence for a basal radiation of the toxin type that is a mutated form of the peptidase S1 molecular scaffold (Fry et al. 2009b). Moreover, we showed that the molecular diversity in the functional residues of all the protein types, coheres to the pattern seen in multigene toxin families that have undergone adaptive radiation through positive selection (Fry et al., 2009b). This basal extensive duplication and functional diversification is consistent with observations on protease activities in octopodid species belonging to phylogenetically relatively distant subfamilies, such as the eledonine E. cirrhosa  and octopodine O. vulgaris, where ten and eight, respectively, caseinolytic proteases were identified.

Like proteases, neuro/myotoxins seem to be a common feature of most octopodid venoms. Small neuropeptides with potent hypotensive properties have been isolated from PSG extracts from species belonging to both Eledoninae and Octopodinae; eledoisin from Eledone aldrovandi and E. moschata , Octopus tachykinins (OctTK) from Octopus vulgaris, and a peptide-transcript in O. kaurna homologous to that coding for OctTK (Fry et al., 2009b). Similarly, non-TTX-like neuro/myotoxic proteins have been found in PSG extracts from O. dofleini, O. vulgaris (Ctx), and E. cirrhosa. We constructed H. maculosa and O. kaurna cDNA libraries that revealed six novel frameworks that displayed little homology to any previously sequenced peptides, venom or non-venom (Fry et al 2009b); this finding underscores the biodiscovery potential of octopoid venoms. Antarctica is rich with octopus biodiversity, with 12 genera currently recognized as present in Antarctic waters, of which 5 genera are endemic (Adelieledone, Bathypurpurata, Megaleledone, Pareledone, and Praealtus. The main radiation is thought to have occurred after the separation of the Antarctic continent and formation of the Antarctic circumpolar current approximately 34 million years ago.

The members of the unique Antarctic octopod clade that are not restricted to the deep sea can be found throughout the Antarctic shelf and continental slope where they have undergone extensive radiation, particularly in the case of Pareledone, with each species usually having a fairly limited geographic, and often bathyal, range. While the species radiation has been attributed to the effect of the shelf of isolated island groups combined with glaciation cycles, there are large overlaps in species distributions, with often several species appearing sympatrically in an area. As sympatry is often associated with trophic niche partitioning, this may again lead to a range of adaptations, including venom diversification.

Of particular interest is the convergent adaptation to cold water predation, once in the Arctic and separately in the Antarctic. Both lineages have radiated into the deep sea, facilitated by the fundamental basic deep sea predation occupying a similar temperature range to the polar. Thus, polar adaptations and deep-sea radiation are linked. The Antarctic octopuses spread outwards to the deep sea in all directions. However, they did not penetrate the Arctic ice. In contrast, the Arctic octopuses spread outwards and one genus was successful enough to pentetrate the Antarctic, in the form of the derived Benthoctopus genus, which occupies 1000-1500 meter zones in the Australian East Antarctic region. Our pilot study (Undheim et al 2010a) indicates that the venoms of the Antarctic octopuses have undergone temperature-specific adaptations. The specific compositions of the venoms or the mechanisms by which they have biochemically differentiated their venoms for sub-zero activity remains to be elucidated. This is the sole publication on the venoms of Antarctic octopuses. Our genetic study of the evolutionary relationships (Undheim et al 2010b) allowed a proper phylogenetic context for the interpretation of the variations in venom.

The work funded by the HSF will continue through the subsequent award to A/Prof Fry by the ARC of a Future Fellowship, with a specific focus on the convergence for cold water adaptation between Arctic and Antarctic octopuses.

Papers available for download

Undheim EAB, Norman JA, Thoen HH, Fry BG (2010) Genetic identification of Southern Ocean octopod samples using mtCOI. CR Biologies 333: 395-404

Undheim EAB., Georgieva DN., Thoen HH., Norman JA., Mork J., Betzel C, Fry BG. (2010) Venom on ice: Adaptive evolution of Antarctic octopus venoms Toxicon 56(6):897-913

Fry BG, Roelants K, Norman J, King G, Tyndal J, Lewis R, Norton R, Renjifo C, Rodriguez de la Vega RC. (2009) Toxicogenomic multiverse: convergent recruitment of proteins into animal venoms Annual Reviews: Genomics and Human Genetics 10:483-511.

Fry BG, Roelants K, Norman J (2009) Tentacles of venom: Toxic protein convergence in the Kingdom Animalia. Journal of Molecular Evolution 68(4)311.

Figure 1. With an Antarctic Giant Octopus in the East Antarctic

Figure 2. With a Nautilus at Osprey Reef, Coral Sea

Figure 3. With a Reef Cuttlefish on the Great Barrier Reef

Figure 4. An assortment of octopuses captured in nets set at 1000m in Antarctica

Figure 5. A unique, soft-bodied type of octopus captured in Antarctica

Figure 6. A particularly beautiful octopus capture in Antarctica at a depth of 2000m