Hapalochlaena Maculosa Research Articles

SNP data reveals the complex and diverse evolutionary history of the blue-ringed octopus genus (Octopodidae: Hapalochlaena) in the Asia-Pacific

The blue-ringed octopus species complex (Hapalochlaena spp.), known to occur from Southern Australia to Japan, currently contains four formally described species (Hapalochlaena maculosa, Hapalochlaena fasciata, Hapalochlaena lunulata and Hapalochlaena nierstraszi). These species are distinguished based on morphological characters (iridescent blue rings and/or lines) along with reproductive strategies. However, the observation of greater morphological diversity than previously captured by the current taxonomic framework indicates that a revision is required. To examine species boundaries within the genus we used mitochondrial (12S rRNA, 16S rRNA, cytochrome c oxidase subunit 1 [COI], cytochrome c oxidase subunit 3 [COIII] and cytochrome b [Cytb]) and genome-wide SNP data (DaRT seq) from specimens collected across its geographic range including variations in depth from 3 m to >100 m. This investigation indicates substantially greater species diversity present within the genus Hapalochlaena than is currently described. We identified 10,346 SNPs across all locations, which when analysed support a minimum of 11 distinct clades. Bayesian phylogenetic analysis of the mitochondrial COI gene on a more limited sample set dates the diversification of the genus to ∼30 mya and corroborates eight of the lineages indicated by the SNP analyses. Furthermore, we demonstrate that the diagnostic lined patterning of H. fasciata found in North Pacific waters and NSW, Australia is polyphyletic and therefore likely the result of convergent evolution. Several “deep water” (>100 m) lineages were also identified in this study with genetic convergence likely to be driven by external selective pressures. Examination of morphological traits, currently being undertaken in a parallel morphological study, is required to describe additional species within the complex.

High-Density Genetic Linkage Map of the Southern Blue-ringed Octopus (Octopodidae: Hapalochlaena maculosa)

Genetic linkage maps provide a useful resource for non-model genomes and can aid in genome reassembly to form more contiguous pseudo-chromosomes. We present the first linkage map of any cephalopod, H. maculosa, composed of 47 linkage groups (LG). A total of 2166 single nucleotide polymorphisms and 2455 presence–absence variant loci were utilised by Lep-Map3 in linkage map construction. The map length spans 2016.62 cM with an average marker distance of 0.85 cM. Integration of the recent H. maculosa genome allowed 1151 scaffolds comprising 34% of the total genomic sequence to be orientated and/or placed using 1278 markers across all 47 LG. The linkage map generated provides a new perspective on HOX gene distribution in octopods. In the H. maculosa linkage map three (SCR, LOX4 and POST1) of six identified HOX genes (HOX1/LAB, SCR, LOX2, LOX4, LOX5, POST1) were located within the same LG (LG 9). The generation of a linkage map for H. maculosa has provided a valuable resource for understanding the evolution of cephalopod genomes and will provide a base for future work.

Adaptive venom evolution and toxicity in octopods is driven by extensive novel gene formation, expansion, and loss.

BackgroundCephalopods represent a rich system for investigating the genetic basis underlying organismal novelties. This diverse group of specialized predators has evolved many adaptations including proteinaceous venom. Of particular interest is the blue-ringed octopus genus (Hapalochlaena), which are the only octopods known to store large quantities of the potent neurotoxin, tetrodotoxin, within their tissues and venom gland.FindingsTo reveal genomic correlates of organismal novelties, we conducted a comparative study of 3 octopod genomes, including the Southern blue-ringed octopus (Hapalochlaena maculosa). We present the genome of this species and reveal highly dynamic evolutionary patterns at both non-coding and coding organizational levels. Gene family expansions previously reported in Octopus bimaculoides (e.g., zinc finger and cadherins, both associated with neural functions), as well as formation of novel gene families, dominate the genomic landscape in all octopods. Examination of tissue-specific genes in the posterior salivary gland revealed that expression was dominated by serine proteases in non–tetrodotoxin-bearing octopods, while this family was a minor component in H. maculosa. Moreover, voltage-gated sodium channels in H. maculosa contain a resistance mutation found in pufferfish and garter snakes, which is exclusive to the genus. Analysis of the posterior salivary gland microbiome revealed a diverse array of bacterial species, including genera that can produce tetrodotoxin, suggestive of a possible production source.ConclusionsWe present the first tetrodotoxin-bearing octopod genome H. maculosa, which displays lineage-specific adaptations to tetrodotoxin acquisition. This genome, along with other recently published cephalopod genomes, represents a valuable resource from which future work could advance our understanding of the evolution of genomic novelty in this family.

Preliminary report of specific behaviours of juvenile greater blue-ringed octopus Hapalochlaena lunulata (Quoy and Gaimard, 1832)

ABSTRACTThe greater blue-ringed octopus (Hapalochlaena lunulata) is one of three venomous octopuses well-known for the iridescent blue rings on the skin and dangerous toxin. However, the understanding of this octopus at the juvenile phase is limited compared to the adult phase. The other two venomous octopuses (Hapalochlaena fasciata and Hapalochlaena maculosa) have also not been thoroughly examined at the juvenile stage. We collected a juvenile greater blue-ringed octopus from the coastal waters of the Ryukyu Archipelago, Japan, and maintained it in captivity for observation. Five days after beginning rearing, iridescent blue rings, similar to those in the adult, appeared on the entire skin of the octopus and continued to be expressed with varying intensity. When the octopus was behaving aggressively it flashed the blue rings. Episodic antagonistic behaviour against the experimenters was also observed.

Genome-wide comparisons reveal a clinal species pattern within a holobenthic octopod-the Australian Southern blue-ringed octopus, Hapalochlaena maculosa (Cephalopoda: Octopodidae).

The southern blue‐ringed octopus, Hapalochlaena maculosa (Hoyle, 1883) lacks a planktonic dispersal phase, yet ranges across Australia's southern coastline. This species’ brief and holobenthic life history suggests gene flow might be limited, leaving distant populations prone to strong genetic divergence. This study used 17,523 genome‐wide SNP loci to investigate genetic structuring and local adaptation patterns of H. maculosa among eight sampling sites along its reported range. Within sites, interrelatedness was very high, consistent with the limited dispersal of this taxon. However, inbreeding coefficients were proportionally lower among sites where substructuring was not detected, suggesting H. maculosa might possess a mechanism for inbreeding avoidance. Genetic divergence was extremely high among all sites, with the greatest divergence observed between both ends of the distribution, Fremantle, WA, and Stanley, TAS. Genetic distances closely followed an isolation by geographic distance pattern. Outlier analyses revealed distinct selection signatures at all sites, with the strongest divergence reported between Fremantle and the other Western Australian sites. Phylogenetic reconstructions using the described sister taxon H. fasciata (Hoyle, 1886) further supported that the genetic divergence between distal H. maculosa sites in this study was equivalent to that of between established heterospecifics within this genus. However, it is advocated that taxonomic delineations within this species should be made with caution. These data indicate that H. maculosa forms a clinal species pattern across its geographic range, with gene flow present through allele sharing between adjacent populations. Morphological investigations are recommended for a robust resolution of the taxonomic identity and ecotype boundaries of this species.

Mating behaviour and postcopulatory fertilization patterns in the southern blue-ringed octopus, Hapalochlaena maculosa

Female octopuses are known to store sperm from multiple males they encounter throughout a breeding season, before laying a single clutch with mixed paternity. Although octopuses display a broad range of precopulatory behaviours, and both sperm competition and cryptic female choice have been hypothesized to occur, the current understanding of how these processes influence resulting paternity remains limited. This study aimed to identify behavioural factors associated with paternity patterns and the capacity of females to bias paternity postcopulation to specific males in the southern blue-ringed octopus, Hapalochlaena maculosa. Genetic markers and controlled, sequential, laboratory pairings of genotyped individuals were used to examine paternity patterns and compare them to relative signatures of male sperm remaining in female oviducal glands after egg laying. Multiple paternity was discovered in all 12 laboratory-reared clutches. There was no indication that the relative time spent in copulation affected the resulting paternity. Males that waited for females to terminate the copulation had greater paternity when they were the first candidate male, but this was not the case among second candidate males. The relative quantities of candidate male alleles detected in female oviducal glands after egg laying were consistent with relative paternity of the candidate males in all but three cases. In one of these, sibship analysis revealed that the male that obtained less paternity than expected was in fact the female's full-sibling brother. Although this study found no evidence for female postcopulatory selection of male sperm, anecdotal evidence suggests that female H. maculosa might benefit from polyandry if chemical processes can favour clutch fertilization by unrelated males. Future studies, investigating paternity bias among genotyped males of varying, but known relatedness to the female, might help to validate this pattern.

Chemical cues correlate with agonistic behaviour and female mate choice in the southern blue-ringed octopus,Hapalochlaena maculosa(Hoyle, 1883) (Cephalopoda: Octopodidae)

Chemoreception cues potentially influence intraspecific interactions of cephalopods, including mate choice. However, at present there is limited empirical evidence demonstrating whether cephalopods can use olfaction to identify the sex or identity of conspecifics. This study examined the responses of the southern blueringed octopus, Hapalochlaena maculosa (Hoyle, 1883), to conspecific odours during controlled laboratory trials. The ventilation rates in aquaria of 25 wild-sourced animals were measured during four treatments: baseline, sea water, sea water containing male conspecific odour and sea water containing female conspecific odour. When used as 'receivers' in trials, female H. maculosa significantly increased their ventilation rates in response to male odours, but not to female odours. However, female response decreased significantly with the receiver's size during female-odour treatments. The ventilation rates of male H. maculosa were statistically similar in all treatments. However, their ventilation rates showed a significant progressive increase over the observation period during male and female-odour treatments. Eighteen of these animals (nine females and nine males) were used in focal-animal trials 1 week after odour-cue experiments. Of these individuals, females were significantly more receptive to copulation attempts, and spent significantly more time per day in copulation, with males whose odours had elicited a weaker ventilation response in prior trials. These results suggest that female H. maculosa can use chemosensory cues to discriminate the sex, and possibly identity, of conspecifics and that this information might influence their mate choice. However, the mechanisms underlying these responses and subsequent copulatory access to females by males remain unknown.

Combined Transcriptomic and Proteomic Analysis of the Posterior Salivary Gland from the Southern Blue-Ringed Octopus and the Southern Sand Octopus.

This study provides comprehensive proteomic profiles from the venom producing posterior salivary glands of octopus (superorder Octopodiformes) species. A combined transcriptomic and proteomic approach was used to identify 1703 proteins from the posterior salivary gland of the southern blue-ringed octopus, Hapalochlaena maculosa and 1300 proteins from the posterior salivary gland of the southern sand octopus, Octopus kaurna. The two proteomes were broadly similar; clustering of proteins into orthogroups revealed 937 that were shared between species. Serine proteases were particularly diverse and abundant in both species. Other abundant proteins included a large number of secreted proteins, many of which had no known conserved domains, or homology to proteins with known function. On the basis of homology to known venom proteins, 23 putative toxins were identified in H.maculosa and 24 in O.kaurna. These toxins span nine protein families: CAP (cysteine rich secretory proteins, antigen 5, parthenogenesis related), chitinase, carboxylesterase, DNase, hyaluronidase, metalloprotease, phospholipase, serine protease and tachykinin. Serine proteases were responsible for 70.9% and 86.3% of putative toxin expression in H.maculosa and O.kaurna, respectively, as determined using intensity based absolute quantification (iBAQ) measurements. Phylogenetic analysis of the putative toxin serine proteases revealed a similar suite of diverse proteins present in both species. Posterior salivary gland composition of H.maculosa and O.kaurna differ in several key aspects. While O.kaurna expressed the proteinaceous neurotoxin, tachykinin, this was absent from H.maculosa, perhaps reflecting the acquisition of a potent nonproteinaceous neurotoxin, tetrodotoxin (TTX) produced by bacteria in the salivary glands of that species. The dispersal factor, hyaluronidase was particularly abundant in H.maculosa. Chitinase was abundant in both species and is believed to facilitate envenomation in chitinous prey such as crustaceans. Cephalopods represent a largely unexplored source of novel proteins distinct from all other venomous taxa and are of interest for further inquiry, as novel proteinaceous toxins derived from venoms may contribute to pharmaceutical design.

Nocturnal mating behaviour and dynamic male investment of copulation time in the southern blue-ringed octopus, Hapalochlaena maculosa (Cephalopoda: Octopodidae)

The southern blue-ringed octopus,Hapalochlaena maculosaHoyle (1883), is a nocturnal species that exhibits a mating system in which females hold sperm from multiple males over a one to two month breeding window before laying a single egg clutch. Contrary to most studied animal mating systems where anisogamy exists, gamete package production is limited for both males and females of this species (approx. 50 spermatophores/eggs). This presents an animal model for studying aspects of sperm competition and dynamic mate choice behaviours. The present study reports on the mating behaviour ofH. maculosaobserved under laboratory conditions using infrared closed-circuit television video footage. Rates of male copulation attempts increased with male size, while female receptivity to mating attempts increased with female size, resulting in larger animals of both sexes gaining more copulations and spending more time per day in copulation. There was some evidence of female preference of larger males, but no male preference of females based on measured morphological traits. Both sexes terminated copulations in equal frequencies but male-terminated copulations were significantly shorter in duration. Males were more likely to terminate copulation early with females they had previously mated with, however were less likely to do so if the female had recently mated with a different male. Among male-terminated copulations, males mated for longer with females that had previously mated with other males in the trial. Male–male mounts were as common as male–female mounts, suggesting that maleH. maculosaare not able to discriminate the sex of conspecifics. These findings suggest male strategic allocation of spermatophores based female mating history is an important factor influencing mating behaviours of this species.

Tentacles of Venom: Toxic Protein Convergence in the Kingdom Animalia

The origin and evolution of venom in many animal orders remain controversial or almost entirely uninvestigated. Here we use cDNA studies of cephalopod posterior and anterior glands to reveal a single early origin of the associated secreted proteins. Protein types recovered were CAP (CRISP, Antigen 5 [Ag5] and Pathogenesis-related [PR-1]), chitinase, peptidase S1, PLA(2) (phospholipase A(2)), and six novel peptide types. CAP, chitinase, and PLA(2) were each recovered from a single species (Hapalochlaena maculosa, Octopus kaurna, and Sepia latimanus, respectively), while peptidase S1 transcripts were found in large numbers in all three posterior gland libraries. In addition, peptidase S1 transcripts were recovered from the anterior gland of H. maculata. We compare their molecular evolution to that of related proteins found in invertebrate and vertebrate venoms, revealing striking similarities in the types of proteins selected for toxic mutation and thus shedding light on what makes a protein amenable for use as a toxin.

Distribution of tetrodotoxin in the body of the blue-ringed octopus ( Hapalochlaena maculosa)

Tetrodotoxin (TTX) was quantitatively assayed in six specimens of semi- adult blue-ringed octopus, Hapalochlaena maculosa, by a post-column fluorescent-HPLC system. TTX was found to be present in all body parts, e.g. in high concentrations in the arms followed by the abdomen and cephalothorax. The toxin is not associated exclusively with the posterior salivary gland.

Inking in a Blue-Ringed Octopus, Hapalochlaena lunulata, with a Vestigial Ink Sac

Inking in a Blue-Ringed Octopus, Hapalochlaena lunulata, with a Vestigial Ink Sac Christine L. Huffard and R. L. Caldwell Abstract Here we report for the first time that adult Hapalochlaena lunulata (Quoy & Gaimard, 1832), which has a vestigial ink sac, is capable of inking. Ink was released under three different agonistic conditions: female-female aggression, rejection of mating attempt, and when attacked by a predator. We observed no apparent reaction to the ink by the other animals involved in these interactions. Ink sacs are found in most shallow-water coleoid cephalopods (octopuses, squids, and cuttlefish), but the function of inking as a defensive behavior of cephalopods rarely has been investigated. Released ink is believed to (1) provide a smoke screen for escape; (2) serve as a pseudomorph; or possibly (3) block olfactory or taste receptors (Schäfer 1956, Moynihan and Rodaniche 1977, Hanlon and Messenger 1996, Boletzky 1997). Cephalopod ink elicits jetting in squids (Gilly and Lucero 1992) and increases ventilation rate in cuttlefish (Boal and Golden 1999). This may indicate its function as an alarm pheromone to warn conspecifics, but such behaviors also could be elicited if the ink was a general irritant. In fact, some predators may be attracted to ink (Grüninger 1997). In octopuses, the reduction or loss of an ink sac may have evolved independently in at least three shallow-water taxa that have developed other effective defenses. Ameloctopus littoralis Norman, 1992, a small, intertidal octopus, lacks an ink sac but readily autotomizes its long arms (Norman 1992). The undescribed "Wonderpus," another long-armed, shallow-water octopus capable of arm autotomy, has a severely reduced ink sac (M. D. Norman and F. G. Hochberg, pers. comm.) and does not ink despite considerable disturbance (C.L.H., pers. obs.). Blue-ringed octopuses, Hapalochlaena spp., are known best for their potent, tetrodotoxin-laden venom (Norman 2000). The genus is diagnosed by the presence of multiple iridescent blue rings or lines on the body and arms and marked reduction of the ink sac in adults (Robson 1929). Although Robson did not speculate on the vestigial ink sac in adults, he did note its drastic reduction in size in the type species, H. lunulata (Quoy & Gaimard, 1832), compared with Hapalochlaena maculosa (Hoyle, 1883). Hatchling Hapalochlaena are able to expel ink: H. fasciata (Hoyle, 1886) (Tranter and Augustine 1973 [taxonomy according to Stranks and Lu 1991]); H. maculosa (Stranks and Lu 1991); H. lunulata (R.L.C., pers. obs.). However, H. fasciata does not ink after they are 4 weeks old (Tranter and Augustine 1973). Juvenile (5- to 6-mm mantle length) H. maculosa expel ink, but the reservoir degenerates with age (Stranks and Lu 1991) until neither production nor release of ink occurs in adult H. maculosa (Roper and Hochberg 1988, Stranks and Lu 1991). Tranter and Augustine (1973) also reported that H. fasciata does not eject ink as adults. Recently, two undescribed species of Hapalochlaena were noted to ink as adults (sp. 3 and sp. 5 of Norman 2000). We therefore can no longer assume that degeneracy of the reservoir in Hapalochlaena spp. obligates the loss of the ability to ink. During laboratory studies on H. lunulata obtained from Indonesia, we recorded seven [End Page 255] Click for larger view View full resolution Figure 1. Line drawing taken from a frame of video of two female Hapalochlaena lunulata interlocking arms while engaged in an aggressive interaction. The animal on the right released the diffuse cloud of ink (top, right). instances when adults released a single, poorly defined cloud of brown ink. In each case, the small puff of ink diffused in seconds. The octopuses ejecting the ink did not jet from the inked area. We observed no apparent direct reaction to the ink by the other animals involved in these situations. Two adult octopuses (one male and one female) inked as they attempted to escape from an attack by a stomatopod, Odontodactylus scyllarus (Linnaeus, 1758). Another male inked as it jetted away from a nearby stomatopod. A female inked as she rejected the approach of a male with which she had previously mated, and a brooding female inked while a male mounted her, attempting to mate. An...

A "Good-Sperm" Model Can Explain the Evolution of Costly Multiple Mating by Females

A "Good-Sperm" Model Can Explain the Evolution of Costly Multiple Mating by Females

Sphero-echinocytosis of human red blood cells caused by snake, red-back spider, bee and blue-ringed octopus venoms and its inhibition by snake sera

It was found that bee (Apis mellifera) venom, red-back spider (Latrodectus mactans) venom, blue-ringed octopus (Hapalochlaena maculosa) venom, ten different snake venoms, phospholipase A2 and four snake toxins caused sphero-echinocytosis of human red blood cells at 200 ng/ml. Most venoms and toxins lost the ability to deform human red blood cells when their components of less than mol. wt 10,000 were applied. In a number of cases the sphero-echinocytotic effect was also inhibited by blood sera of Notechis scutatus and Pseudonaja textilis.

Occurrence of a tetrodotoxin-like compound in the eggs of the venomous blue-ringed octopus ( Hapalochlaena maculosa)

A lethal toxin was isolated and partly purified from the eggs of the blue-ringed octopus, Hapalochlaena maculosa. Examination of the toxin by thin layer chromatography, isoelectric focusing and its effects upon the compound nerve action potentials of the toad sciatic nerve gave results that were indistinguishable from those displayed by authentic tetrodotoxin, the toxin present in the venom glands of the octopus.

A Comparison of Anaerobic Energy Metabolism in Mantle and Tentacle Muscle of the Blue-Ringed Octopus, Hapalochlaena maculosa, During Swimming

The blue-ringed octopus Hapalochlaena maculosa uses both the mantle and tentacles for swimming. Activities of octopine dehydrogenase. lactate dehydrogenase and alpha-glycerophosphate dehydrogenase in mantle and tentacle muscles indicate that both tissues depend on anaerobic glycolysis during swimming. with octopine rather than lactate accumulating as an end product. Following swimming. both mantle and tentacles show a decrease in arginine phosphate, an increase in octopine. and a fall in energy charge. On the basis of ATP equivalents per grain of muscle obtained from arginine phosphate and anaerobic glycolysis, the two tissues are similar, but when the relative muscle weights are taken into account ATP production is at least 10-fold greater in the tentacles than in the mantle.

Maculotoxin: a neurotoxin from the venom glands of the octopus Hapalochlaena maculosa identified as tetrodotoxin.

Maculotoxin, a potent neurotoxin isolated from the posterior salivary glands of the blue-ringed octopus. Hapalochlaena maculosa, has now been identified as tetrodotoxin. This is the first reported case in which tetrodotoxin has been found to occur in a venom.

Histological structure of the posterior salivary glands in the blue ringed octopus Hapalochlaena maculosa Hoyle

The histology of the posterior salivary glands in adults of the blue ringed octopus Hapalochlaena maculosa is described and compared with the glands in 5 day, 15 day and 42 day old juvenile H. maculosa. A brief comparison is also made to the posterior salivary glands in Octopus australis. In H. maculosa the salivary glands have a compound tubuloacinar exocrine structure while in O. australis the glands are a mixture of tubular and tubuloacinar elements. On the basis of the histological structure it is suggested that transport of “saliva” is accomplished by muscular contractions of the gland rather than by cilial action as is common in other molluscs. The glands are located in the intestinal blood space. The possible evolutionary significance of variation in gland structure shown by octopuses is discussed.

An octopus toxin, maculotoxin, selectively blocks sodium current in squid axons.

1. A low molecular weight, stable, cationic neurotoxin (maculotoxin, MTX) extracted from the posterior salivary glands of the octopus Hapalochlaena maculosa, blocked sodium current in voltage-clamped squid axons without affecting potassium current. 2. The effectiveness of MTX was increased by repetitive, brief, depolarizing pulses but not by a single prolonged depolarization. 3. The potency of MTX decreased at pHs from 8 to 9. Effectiveness could be restored be restored by lowering the pH to 7-1 again. It was concluded that MTX is active in its cationic form. 4. MTX affected sodium conductance kinetics, slowing the turn-on of sodium current. This effect was most noticeable with small deploarizations but became progressively less with larger depolarizations. Neither the turn-off of sodium current nor sodium inactivation kinetics were affected by the toxin. 5. MTX inhibited sodium current without inhibiting sodium gating current. 6. The effectiveness of MTX was not detectably changed when calcium concentration was varied from 50 to 10 mM, or sodium concentration was varied from 225 to 750 mM.

On the nature of “Maculotoxin”, a toxin from the blue-ringed octopus ( Hapalochlaena maculosa)

Two lethal fractions were prepared from the posterior salivary glands of Hapalochlaena maculosa by methanol extraction and chromatography on a silica gel column. These two fractions were compared with the lethal water extract of the glands, previously named maculotoxin (MTX), by thin layer and ion exclusion chromatography. It was concluded that the two methanol fractions contained the same active principle, that this was the same material as MTX, and that it closely resembled tetrodotoxin. No evidence for the existence of a second toxin in effective amounts was found.