Convergent and parallel evolution in a voltage-gated sodium channel underlies TTX-resistance in the Greater Blue-ringed Octopus: Hapalochlaena lunulata.

The natural history and pharmacology of tetrodotoxin (TTX) has long intrigued biologists. This toxin has a remarkable distribution that spans two domains of life (Bacteria and Eukarya). Within Eukaryotes, TTX has only been identified in animals but is known to be present in over five-dozen species of phylogenetically distant Metazoans. Despite decades of work, the origin and biosynthetic pathways of TTX remain unresolved. Investigations in puffer fishes and salamanders have provided insights into the acquisition of auto-resistance to TTX through the evolution of voltage-gated sodium ion channels (VGSCs) that have reduced binding affinity for TTX. To date there have been no studies of these proteins in tetrodotoxic Blue-Ringed Octopuses. Here we report data demonstrating that the Greater Blue-ringed Octopus (Hapalochlaena lunulata) expresses a VGSC (HlNaV1) gene with mutations that reduce the channel's TTX-binding affinity and likely render the organism TTX resistant. We identified three amino-acid substitutions in the TTX-binding site of HlNaV1 that likely confer TTX-resistance to both the channel and the organism. These substitutions are associated with organismal TTX-resistance in other TTX-bearing taxa and are convergent with substitutions that have evolved in fish, salamanders, and some TTX-resistant invertebrates.

A PUFFERFISH (TETRADON NIGROVIRIDIS) AVAILABLE IN THE COMMON PET TRADE HARBORS LETHAL CONCENTRATIONS OF TETRODOTOXIN: A CASE STUDY OF POISONING IN A CUVIER'S DWARF CAIMAN (PALEOSUCHUS PALPEBROSUS).

Many pufferfish possess tetrodotoxin (TTX). Unaware of this fact, the owner of a 3-mo-old pet Cuvier's dwarf caiman ( Paleosuchus palpebrosus ) fed the caiman a green spotted pufferfish ( Tetraodon nigroviridis ), acquired from a local discount department store. The caiman was nonresponsive within an hour of consumption of the fish. The caiman was presented for veterinary evaluation but died despite intensive medical care. High-performance liquid chromatography and a competitive inhibition enzyme immunoassay were used to determine whether the pufferfish was tetrodotoxic and whether the deceased caiman had TTX in its system. Skin and liver of the pufferfish harbored high concentrations of TTX, and the caiman had TTX in the blood, liver, and kidney. The clinical signs and presence of TTX in the caiman suggest that the caiman succumbed to tetrodotoxicosis. The implication is that lethally poisonous species are available commercially and pose a danger to other pets and possibly small children.

Microdistribution of tetrodotoxin in two species of blue-ringed octopuses (Hapalochlaena lunulata and Hapalochlaena fasciata) detected by fluorescent immunolabeling.

Blue-ringed octopuses (genus Hapalochlaena) possess the potent neurotoxin tetrodotoxin (TTX). We examined the microdistribution of TTX in ten tissues of Hapalochlaena lunulata and Hapalochlaena fasciata by immunolabeling for fluorescent light microscopy (FLM). We visualized TTX throughout the posterior salivary gland, but the toxin was concentrated in cells lining the secretory tubules within the gland. Tetrodotoxin was present just beneath the epidermis of the integument (mantle and arms) and also concentrated in channels running through the dermis. This was suggestive of a TTX transport mechanism in the blood of the octopus, which would also explain the presence of the toxin in the blood-rich brachial hearts, gills, nephridia, and highly vascularized Needham's sac (testes contents). We also present the first report of TTX in any cephalopod outside of the genus Hapalochlaena. A specimen of Octopus bocki from French Polynesia contained a small amount of TTX in the digestive gland.

An improved competitive inhibition enzymatic immunoassay method for tetrodotoxin quantification.

Quantifying tetrodotoxin (TTX) has been a challenge in both ecological and medical research due to the cost, time and training required of most quantification techniques. Here we present a modified Competitive Inhibition Enzymatic Immunoassay for the quantification of TTX, and to aid researchers in the optimization of this technique for widespread use with a high degree of accuracy and repeatability.

Ontogeny of tetrodotoxin levels in blue-ringed octopuses: maternal investment and apparent independent production in offspring of Hapalochlaena lunulata.

Many organisms provision offspring with antipredator chemicals. Adult blue-ringed octopuses (Hapalochlaena spp.) harbor tetrodotoxin (TTX), which may be produced by symbiotic bacteria. Regardless of the ultimate source, we find that females invest TTX into offspring and offspring TTX levels are significantly correlated with female TTX levels. Because diversion of TTX to offspring begins during the earliest stages of egg formation, when females are still actively foraging and looking for mates, females may face an evolutionary tradeoff between provisioning larger stores of TTX in eggs and retaining that TTX for their own defense and offense (venom). Given that total TTX levels appear to increase during development and that female TTX levels correlate with those of offspring, investment may be an active adaptive process. Even after eggs have been laid, TTX levels continue to increase, suggesting that offspring or their symbionts begin producing TTX independently. The maternal investment of TTX in offspring of Hapalochlaena spp. represents a rare examination of chemical defenses, excepting ink, in cephalopods.

Behavioral and chemical ecology of marine organisms with respect to tetrodotoxin.

The behavioral and chemical ecology of marine organisms that possess tetrodotoxin (TTX) has not been comprehensively reviewed in one work to date. The evidence for TTX as an antipredator defense, as venom, as a sex pheromone, and as an attractant for TTX-sequestering organisms is discussed. Little is known about the adaptive value of TTX in microbial producers; thus, I focus on what is known about metazoans that are purported to accumulate TTX through diet or symbioses. Much of what has been proposed is inferred based on the anatomical distribution of TTX. Direct empirical tests of these hypotheses are absent in most cases.

Intra-organismal distribution of tetrodotoxin in two species of blue-ringed octopuses (Hapalochlaena fasciata and H. lunulata).

In-depth studies on the intra-organismal distribution of toxin may yield valuable clues about potential ecological functions. The distribution of tetrodotoxin (TTX) in previously unexamined tissues of two species of blue-ringed octopuses, wild-caught Hapalochlaena fasciata and Hapalochlaena lunulata from the aquarium industry, was surveyed. Tissues from each individual were examined separately. Tetrodotoxin was detected in posterior salivary gland (PSG), arm, mantle, anterior salivary glands, digestive gland, testes contents, brachial heart, nephridia, gill, and oviducal gland of H. fasciata. By contrast TTX was found only in the PSG, mantle tissue, and ink of H. lunulata. The highest concentrations of TTX resided in the PSG of both species; however, the arms and mantle contained the greatest absolute amounts of TTX. Minimum total amounts of TTX per octopus ranged from 60 to 405 microg in H. fasciata and from 0 to 174 microg in H. lunulata and correlated well with the amounts in the PSG. Transport of TTX in the blood is loosely suggested by the presence of the toxin in blood-rich organs such as the gill and brachial hearts. The distributional data also suggest both offensive and defensive functions of TTX.

Parallel arms races between garter snakes and newts involving tetrodotoxin as the phenotypic interface of coevolution.

Parallel "arms races" involving the same or similar phenotypic interfaces allow inference about selective forces driving coevolution, as well as the importance of phylogenetic and phenotypic constraints in coevolution. Here, we report the existence of apparent parallel arms races between species pairs of garter snakes and their toxic newt prey that indicate independent evolutionary origins of a key phenotype in the interface. In at least one area of sympatry, the aquatic garter snake, Thamnophis couchii, has evolved elevated resistance to the neurotoxin tetrodotoxin (TTX), present in the newt Taricha torosa. Previous studies have shown that a distantly related garter snake, Thamnophis sirtalis, has coevolved with another newt species that possesses TTX, Taricha granulosa. Patterns of within population variation and phenotypic tradeoffs between TTX resistance and sprint speed suggest that the mechanism of resistance is similar in both species of snake, yet phylogenetic evidence indicates the independent origins of elevated resistance to TTX.

A resistant predator and its toxic prey: persistence of newt toxin leads to poisonous (not venomous) snakes.

The common garter snake (Thamnophis sirtalis) preys upon the rough-skinned newt (Taricha granulosa), which contains the neurotoxin tetrodotoxin (TTX) in the skin. TTX is toxic, large quantities are present in a newt, and highly resistant snakes have the ability to ingest multiple newts; subsequently snakes harbor significant amounts of active toxin in their own tissues after consuming a newt. Snakes harbor TTX in the liver for 1 mo or more after consuming just one newt, and at least 7 wk after consuming a diet of newts. Three weeks after eating one newt, snakes contained an average of 42 microg of TTX in the liver. This amount could severely incapacitate or kill avian predators, and mammalian predators may be negatively affected as well.