Asymmetry in the frequency and proportion of arm truncation in three sympatric California Octopus species.

Octopuses have eight radially symmetrical arms that surround the base of a bilaterally symmetrical body. These numerous appendages, which explore the environment, handle food, and defend the animal against predators, are highly susceptible to truncation or loss. Here, we used scaling relationships specific to the arms of three sympatric octopus species of the genus Octopus, to calculate the proportion of arm truncation. We then compared the frequency and proportion of arm losses between different body locations. Truncated arms were found in 59.8 % of specimens examined, with individuals bearing one to as many as seven injured arms. We found a significant left side bias for greater proportion of arm truncation for all species and sexes except in O. bimaculatus males. We also found that sister species O. bimaculatus and O. bimaculoides had a greater proportion of their anterior arms (pairs 1 and 2) truncated, while in O. rubescens, posterior arms (pairs 3 and 4) were more truncated. The mean percent of arm that was truncated was 28.1 % overall but varied between species and by sex and was highest in O. rubescens females (56 %). The arms of O. rubescens also exhibited the steepest scaling patterns, and showed a positive correlation between body size and number of truncated arms. Overall, we show that arm injuries in our sampling of three intertidal species are frequent and asymmetrical, and that when injured, octopus on average lose a considerable proportion of their arm. Through quantifying the variation in arm truncation, this study provides a new foundation to explore behavioral compensation for arm loss in cephalopods.

Chromosome-level genome assembly of the East Asian common octopus (Octopus sinensis) using PacBio sequencing and Hi-C technology.

The Cephalopoda are a group of highly diverse marine species in the phylum Mollusca, which are distributed worldwide. They have evolved some vertebrate-like biological traits and exhibit complicated behavioural repertoires. Thus, they are interesting species for studying the mechanisms of evolutionary convergence, innovational functional structures and evolutionary adaptation to a highly active, predatory lifestyle in diverse marine environments. Despite the evolutionary placement and biological significance of cephalopods, genomic data on these organisms remain limited. Here, we assembled a chromosome-level genome of a female East Asian common octopus (Octopus sinensis) by combining Pacific Bioscience (PacBio) single-molecule real-time sequencing, Illumina paired-end sequencing and Hi-C technology. An O. sinensis genome of 2.72 Gb was assembled from a total of 245.01 Gb high-quality PacBio sequences. The assembled genome represents 80.2% completeness (BUSCO) with a contig N50 of 490.36 Kb and a scaffold N50 of 105.89 Mb, showing a considerable improvement compared with other sequenced cephalopod genomes. Hi-C scaffolding of the genome resulted in the construction of 30 pseudochromosomes in Cephalopoda, representing 96.41% of the assembled sequences. The genome contained 42.26% repeat sequences and 5,245 noncoding RNAs. A total of 31,676 protein-coding genes were predicted, of which 82.73% were functionally annotated. The comparative genomic analysis identified 17,020 orthologous gene families, including 819 unique gene families and 629 expanded gene families. This genomic information will be an important molecular resource for further investigation of biological function and evolutionary adaptations in octopuses, and facilitate research into their population genetics and comparative evolution.

Phylogeography of the insular populations of common octopus, Octopus vulgaris Cuvier, 1797, in the Atlantic Macaronesia.

Exploited, understudied populations of the common octopus, Octopus vulgaris Cuvier, 1797, occur in the northeastern Atlantic (NEA) throughout Macaronesia, comprising the Azores, Madeira and Canaries, and also the Cabo Verde archipelago. This octopus species, found from the intertidal to shallow continental-shelf waters, is largely sedentary, and the subject of intense, frequently unregulated fishing effort. We infer connectivity among insular populations of this octopus. Mitochondrial control region and COX1 sequence datasets reveal two highly divergent haplogroups (α and ß) at similar frequencies, with opposing clinal distributions along the sampled latitudinal range. Haplogroups have different demographic and phylogeographic patterns, with origins related to the two last glacial maxima. FST values suggest a significant differentiation for most pairwise comparisons, including insular and continental samples, from the Galicia and Morocco coasts, with the exception of pairwise comparisons for samples from Madeira and the Canaries populations. Results indicate the existence of genetically differentiated octopus populations throughout the NEA. This emphasizes the importance of regulations by autonomous regional governments of the Azores, Madeira and the Canaries, for appropriate management of insular octopus stocks.

Surveying cephalopod diversity of the Amazon reef system using samples from red snapper stomachs and description of a new genus and species of octopus.

The cephalopod fauna of the southwestern Atlantic is especially poorly-known because sampling is mostly limited to commercial net-fishing operations that are relatively inefficient at obtaining cephalopods associated with complex benthic substrates. Cephalopods have been identified in the diets of many large marine species but, as few hard structures survive digestion in most cases, the identification of ingested specimens to species level is often impossible. Samples can be identified by molecular techniques like barcoding and for cephalopods, mitochondrial 16S and COI genes have proven to be useful diagnostic markers for this purpose. The Amazon River estuary and continental shelf are known to encompass a range of different substrates with recent mapping highlighting the existence of an extensive reef system, a type of habitat known to support cephalopod diversity. The present study identified samples of the cephalopod fauna of this region obtained from the stomachs of red snappers, Lutjanus purpureus, a large, commercially-important fish harvested by fisheries using traps and hook-and-line gear that are capable of sampling habitats inaccessible to nets. A total of 98 samples were identified using molecular tools, revealing the presence of three squid species and eight MOTUs within the Octopodidae, representing five major clades. These include four known genera, Macrotritopus, Octopus, Scaeurgus and Amphioctopus, and one basal group distinct from all known octopodid genera described here as Lepidoctopus joaquini Haimovici and Sales, new genus and species. Molecular analysis of large predatory fish stomach contents was found to be an incredibly effective extended sampling method for biodiversity surveys where direct sampling is very difficult.

The evolution and origin of tetrodotoxin acquisition in the blue-ringed octopus (genus Hapalochlaena).

Tetrodotoxin is a potent non-proteinaceous neurotoxin, which is commonly found in the marine environment. Synthesised by bacteria, tetrodotoxin has been isolated from the tissues of several genera including pufferfish, salamanders and octopus. Believed to provide a defensive function, the independent evolution of tetrodotoxin sequestration is poorly understood in most species. Two mechanisms of tetrodotoxin resistance have been identified to date, tetrodotoxin binding proteins in the circulatory system and mutations to voltage gated sodium channels, the binding target of tetrodotoxin with the former potentially succeeding the latter in evolutionary time. This review focuses on the evolution of tetrodotoxin acquisition, in particular how it may have occurred within the blue-ringed octopus genus (Hapalochlaena) and the subsequent impact on venom evolution.

Ancient horizontally transferred genes in the genome of California two-spot octopus, Octopus bimaculoides.

Horizontal gene transfer (HGT), a mechanism that shares genetic material between the host and donor from separated offspring branches, has been described as a means of producing novel and beneficial phenotypes for the host organisms. However, in molluscs, the second most diverse group, the existence of HGT is still controversial. In the present study, 12 HGT genes were identified from California two-spot octopus Octopus bimaculoides based on a similarity search, phylogenetic construction, gene composition analysis and PCR (Polymerase Chain Reaction) validation. Based on the phylogenetic topologies, ten HGT genes were identified to have been transferred into the possible molluscan ancestor, possibly before its radiation. Furthermore, most of the donor organisms were predicted to be familiar bacteria in marine environments. These horizontally transferred genes were under a strong negative selection and could be transcribed in octopus functionally. The predicted biochemical functions of these genes include metabolism, neurotransmission, immune defense and tissue integrity. Seven Zn-metalloproteinases were validated as the main type of HGT genes in octopus with divergent motif composition, intron presence and phylogenetic relationship to the endogenous ones. Furthermore, the functions of Zn-metalloproteinase were predicted to be responsible for immune defense and tissue remolding. Three HGT genes were distributed mainly in the nervous system and were predicted to regulate the neurotransmission through glia-neuronal interactions. The results collectively indicated the existence of HGT in molluscs and its potential contribution to the evolution of octopus with regards to functional innovation and adaptability.

Complete mitochondrial genome and phylogenetic relationship analyses of Amphioctopus aegina (Gray, 1849) (Cephalopoda: Octopodidae).

In this paper, the circular mitochondrial genome of Amphioctopus aegina (Cephalopoda: Octopodidae) was sequenced. The whole mitogenome of A. aegina was 15 545 base pairs (bp) in length with a base composition of 42.53% A, 33.26% T, 16.70% C, and 7.51% G. The complete mitogenome contained 13 protein-coding genes (PCGs), 2 ribosomal RNA genes, 22 transfer RNA genes, and a major non-coding region. The gene arrangements of A. aegina showed remarkable similarity to other Octopodidae species reported. The phylogenetic relationships were reconstructed with the concatenated sequences of the 13 PCGs of the mitochondrial genome, and illustrated that A. aegina had the closest genetic relatives to A. fangsiao.

The complete mitochondrial genome of Octopus bimaculatus Verrill, 1883 from the Gulf of California.

The complete mitochondrial genome of Octopus bimaculatus is 16 085 bp in length and includes 13 protein-codes genes, 2 ribosomal RNA genes, 22 transfers RNA genes, and a control region. The composition of genome is A (40.9%), T (34.7%), C (16.9%), and G (7.5%). The control region of O. bimaculatus contains a VNTR locus not present in the genomes from other octopus species. A phylogenetic analysis shows a closer relationship between the mitogenomes from O. bimaculatus and O. vulgaris.

A new species of pouched octopus, Cistopus Gray, 1849 (Cephalopoda: Octopodidae) from the southwest coast of India.

Octopuses of the genus Cistopus Gray, 1849 are commercially valuable catches in the cephalopod fisheries of India. The primary and unique diagnostic character of this genus is the possession of eight small mucous pouches embedded in the oral faces of the webs between the bases of each arm. Historically only a single species of Cistopus, C. indicus, had been reported from Indian waters. In reviewing the octopod fauna off the Kerala coast, we have detected three species of Cistopus, of which one is described here as a new species. Cistopus platinoidus sp. nov. is distinct from Cistopus species described to date (C. indicus, C. taiwanicus and C. chinensis) on the basis of sucker counts, the number and position of enlarged suckers in males, and presence/absence of a calamus. Our studies of catch composition of Kerala octopod fisheries indicate a higher diversity of target species than previously suspected, including a number of undescribed species. Taxonomic resolution and collation of biological and distributional data are required for effective monitoring and management of these valuable fisheries.

Genetic differentiation of Octopus minor (Mollusca, Cephalopoda) off the northern coast of China as revealed by amplified fragment length polymorphisms.

Octopus minor (Sasaki, 1920) is an economically important cephalopod that is found in the northern coastal waters of China. In this study, we investigated genetic differentiation in fishery populations using amplified fragment length polymorphisms (AFLPs). A total of 150 individuals were collected from five locations: Dalian (DL), Yan-tai (YT), Qingdao (QD), Lianyungang (LY), and Zhoushan (ZS), and 243 reproducible bands were amplified using five AFLP primer combinations. The percentage of polymorphic bands ranged from 53.33 to 76.08%. Nei's genetic identity ranged from 0.9139 to 0.9713, and the genetic distance ranged from 0.0291 to 0.0900. A phylogenetic tree was constructed using the unweighted pair group method with arithmetic mean, based on the genetic distance. The DL and YT populations originated from one clade, while the QD, LY, and ZS populations originated from another. The results indicate that the O. minor stock consisted of two genetic populations with an overall significantly analogous FST value (0.1088, P < 0.05). Most of the variance was within populations. These findings will be important for more sustainable octopus fisheries, so that this marine resource can be conserved for its long-term utilization.

The octopus genome and the evolution of cephalopod neural and morphological novelties.

Coleoid cephalopods (octopus, squid and cuttlefish) are active, resourceful predators with a rich behavioural repertoire. They have the largest nervous systems among the invertebrates and present other striking morphological innovations including camera-like eyes, prehensile arms, a highly derived early embryogenesis and a remarkably sophisticated adaptive colouration system. To investigate the molecular bases of cephalopod brain and body innovations, we sequenced the genome and multiple transcriptomes of the California two-spot octopus, Octopus bimaculoides. We found no evidence for hypothesized whole-genome duplications in the octopus lineage. The core developmental and neuronal gene repertoire of the octopus is broadly similar to that found across invertebrate bilaterians, except for massive expansions in two gene families previously thought to be uniquely enlarged in vertebrates: the protocadherins, which regulate neuronal development, and the C2H2 superfamily of zinc-finger transcription factors. Extensive messenger RNA editing generates transcript and protein diversity in genes involved in neural excitability, as previously described, as well as in genes participating in a broad range of other cellular functions. We identified hundreds of cephalopod-specific genes, many of which showed elevated expression levels in such specialized structures as the skin, the suckers and the nervous system. Finally, we found evidence for large-scale genomic rearrangements that are closely associated with transposable element expansions. Our analysis suggests that substantial expansion of a handful of gene families, along with extensive remodelling of genome linkage and repetitive content, played a critical role in the evolution of cephalopod morphological innovations, including their large and complex nervous systems.

Octopus lipid and vitamin E composition: interspecies, interorigin, and nutritional variability.

Octopus vulgaris, Octopus maya, and Eledone cirrhosa from distinct marine environments [Northeast Atlantic (NEA), Northwest Atlantic (NWA), Eastern Central Atlantic, Western Central Atlantic (WCA), Pacific Ocean, and Mediterranean Sea] were characterized regarding their lipid and vitamin E composition. These species are those commercially more relevant worldwide. Significant interspecies and interorigin differences were observed. Unsaturated fatty acids account for more than 65% of total fatty acids, mostly ω-3 PUFA due to docosahexaenoic (18.4-29.3%) and eicosapentanoic acid (11.4-23.9%) contributions. The highest ω-3 PUFA amounts and ω-3/ω-6 ratios were quantified in the heaviest specimens, O. vulgaris from NWA, with high market price, and simultaneously in the lowest graded samples, E. cirrhosa from NEA, of reduced dimensions. Although having the highest cholesterol contents, E. cirrhosa from NEA and O. maya from WCA have also higher protective fatty acid indexes. Chemometric discrimination allowed clustering the selected species and several origins based on lipid and vitamin E profiles.

The complete mitochondrial genomes of two octopods Cistopus chinensis and Cistopus taiwanicus: revealing the phylogenetic position of the genus Cistopus within the order Octopoda.

In the present study, we determined the complete mitochondrial DNA (mtDNA) sequences of two species of Cistopus, namely C. chinensis and C. taiwanicus, and conducted a comparative mt genome analysis across the class Cephalopoda. The mtDNA length of C. chinensis and C. taiwanicus are 15706 and 15793 nucleotides with an AT content of 76.21% and 76.5%, respectively. The sequence identity of mtDNA between C. chinensis and C. taiwanicus was 88%, suggesting a close relationship. Compared with C. taiwanicus and other octopods, C. chinensis encoded two additional tRNA genes, showing a novel gene arrangement. In addition, an unusual 23 poly (A) signal structure is found in the ATP8 coding region of C. chinensis. The entire genome and each protein coding gene of the two Cistopus species displayed notable levels of AT and GC skews. Based on sliding window analysis among Octopodiformes, ND1 and DN5 were considered to be more reliable molecular beacons. Phylogenetic analyses based on the 13 protein-coding genes revealed that C. chinensis and C. taiwanicus form a monophyletic group with high statistical support, consistent with previous studies based on morphological characteristics. Our results also indicated that the phylogenetic position of the genus Cistopus is closer to Octopus than to Amphioctopus and Callistoctopus. The complete mtDNA sequence of C. chinensis and C. taiwanicus represent the first whole mt genomes in the genus Cistopus. These novel mtDNA data will be important in refining the phylogenetic relationships within Octopodiformes and enriching the resource of markers for systematic, population genetic and evolutionary biological studies of Cephalopoda.

Phylogenetic relationships among Octopodidae species in coastal waters of China inferred from two mitochondrial DNA gene sequences.

Octopus in the family Octopodidae (Mollusca: Cephalopoda) has been generally recognized as a "catch-all" genus. The monophyly of octopus species in China's coastal waters has not yet been studied. In this paper, we inferred the phylogeny of 11 octopus species (family Octopodidae) in China's coastal waters using nucleotide sequences of two mitochondrial DNA genes: cytochrome c oxidase subunit I (COI) and 16S rRNA. Sequence analysis of both genes revealed that the 11 species of Octopodidae fell into four distinct groups, which were genetically distant from one another and exhibited identical phylogenetic resolution. The phylogenies indicated strongly that the genus Octopus in China's coastal waters is also not monophyletic, and it is therefore clear that the Octopodidae systematics in this area requires major revision. It is demonstrated that partial sequence information of both the mitochondrial genes 16S rRNA and COI could be used as diagnostic molecular markers in the identification and resolution of the taxonomic ambiguity of Octopodidae species.

Rapid method for controlling the correct labeling of products containing common octopus (Octopus vulgaris) and main substitute species (Eledone cirrhosa and Dosidicus gigas) by fast real-time PCR.

The TaqMan real-time PCR has the highest potential for automation, therefore representing the currently most suitable method for screening, allowing the detection of fraudulent or unintentional mislabeling of species. This work describes the development of a real-time polymerase chain reaction (RT-PCR) system for the detection and identification of common octopus (Octopus vulgaris) and main substitute species (Eledone cirrhosa and Dosidicus gigas). This technique is notable for the combination of simplicity, speed, sensitivity and specificity in an homogeneous assay. The method can be applied to all kinds of products; fresh, frozen and processed, including those undergoing intensive processes of transformation. This methodology was validated to check how the degree of food processing affects the method and the detection of each species. Moreover, it was applied to 34 commercial samples to evaluate the labeling of products made from them. The methodology herein developed is useful to check the fulfillment of labeling regulations for seafood products and to verify traceability in commercial trade and for fisheries control.

Development of microsatellite markers to genetically differentiate populations of Octopus minor from Korea and China.

Of the more than 300 octopus species, Octopus minor is one of the most popular and economically important species in Eastern Asia, including Korea, along with O. vulgaris, O. ocellatus, and O. aegina. We developed 19 microsatellite markers from Octopus minor and eight polymorphic markers were developed to analyze the genetic diversity and relationships among four octopus populations from Korea and three from China. The number of alleles per locus varied from 10 to 49, and allelic richness per locus ranged from 2 to 16.4 across all populations. The average allele number among the populations was 11.1, with a minimum of 8.3 and a maximum of 13.6. The mean allelic richness was 8.7 in all populations. The Hardy-Weinberg equilibrium (HWE) test revealed significant deviation in 19 of the 56 single-locus sites, and null alleles were presumed in five of eight loci. The pairwise F ( ST ) values between populations from Korea and China differed significantly in all pairwise comparisons. The genetic distances between the China and Korea samples ranged from 0.161 to 0.454. The genetic distances among the populations from Korea ranged from 0.033 to 0.090, with an average of 0.062; those among populations from China ranged from 0.191 to 0.316, with an average of 0.254. The populations from Korea and China formed clearly separated into clusters via an unweighted pair group method with arithmetic mean dendrogram. Furthermore, a population from muddy flats on the western coast of the Korean Peninsula and one from a rocky area on Jeju Island formed clearly separated subclusters. An assignment test based on the allele distribution discriminated between the Korean and Chinese origins with 96.9 % accuracy.

Venom on ice: first insights into Antarctic octopus venoms.

The venom of Antarctic octopus remains completely unstudied. Here, a preliminary investigation was conducted into the properties of posterior salivary gland (PSG) extracts from four Antarctica eledonine (Incirrata; Octopodidae) species (Adelieledone polymorpha, Megaleledone setebos, Pareledone aequipapillae, and Pareledone turqueti) collected from the coast off George V's Land, Antarctica. Specimens were assayed for alkaline phosphatase (ALP), acetylcholinesterase (AChE), proteolytic, phospholipase A(2) (PLA(2)), and haemolytic activities. For comparison, stomach tissue from Cirroctopus sp. (Cirrata; Cirroctopodidae) was also assayed for ALP, AChE, proteolytic and haemolytic activities. Dietary and morphological data were collected from the literature to explore the ecological importance of venom, taking an adaptive evolutionary approach. Of the incirrate species, three showed activities in all assays, while P. turqueti did not exhibit any haemolytic activity. There was evidence for cold-adaptation of ALP in all incirrates, while proteolytic activity in all except P. turqueti. Cirroctopus sp. stomach tissue extract showed ALP, AChE and some proteolytic activity. It was concluded that the AChE activity seen in the PSG extracts was possibly due to a release of household proteins, and not one of the secreted salivary toxins. Although venom undoubtedly plays an important part in prey capture and processing by Antarctica eledonines, no obvious adaptations to differences in diet or morphology were apparent from the enzymatic and haemolytic assays. However, several morphological features including enlarged PSG, small buccal mass, and small beak suggest such adaptations are present. Future studies should be conducted on several levels: Venomic, providing more detailed information on the venom compositions as well as the venom components themselves; ecological, for example application of serological or genetic methods in identifying stomach contents; and behavioural, including observations on capture of different types of prey.

The inkless octopuses (Cephalopoda: Octopodidae) of the southwest Atlantic.

Three inkless octopodids are described from the continental shelf off southeastern South America. These octopuses are a non-commercial by-catch in the Falkland Islands fishery. Muusoctopus eureka (Robson, 1929) is one of two common inkless octopuses and is of medium size, with orange-pink skin and a distinctive pattern of irregular dark markings, interspersed with white spots visible only in living or freshly dead specimens. The second common inkless octopus is M. longibrachus akambei, a new subspecies of the Chilean species Muusoctopus longibrachus ( Ibáñez, Sepúlveda and Chong, 2006 ). It has slender arms and is much larger at full maturity than M. eureka. It is a plain orange color when alive, pinkish cream when preserved. Muusoctopus bizikovi, sp. nov., is a smaller, rarer species, colored wine-red whether alive or preserved, and has a vestigial ink duct between the digestive gland and the anus. Relations with other species are discussed. This group of octopuses has often been associated with the genus Benthoctopus Grimpe, 1921 , which is a junior synonym of Bathypolypus Grimpe (a genus of small species characterized by much shorter arms and males with a robust copulatory organ bearing transverse lamellae). It is argued that the misleading characterization of the so-called Benthoctopus group of species as "smooth skinned" is based upon the artefactual appearance of specimens fixed and preserved suboptimally following a detrimental freeze-thaw cycle of fisheries material previously frozen while at sea.

Genetic identification of Southern Ocean octopod samples using mtCOI.

East Antarctic octopods were identified by sequencing mtCOI and using four analytical approaches: Neighbor-joining by Kimura-2-Parameter-based distances, character-based, BLAST, and Bayesian Inference of Phylogeny. Although the distance-based analytical approaches identified a high proportion of the sequences (99.5% to genus and 88.1% to species level), these results are undermined by the absence of a clear gap between intra- and interspecific variation. The character-based approach gave highly conflicting results compared to the distance-based methods and failed to identify apomorphic characters for many of the species. While a DNA independent approach is necessary for validation of the method comparisons, crude morphological observations give early support to the distance-based results and indicate extensive range expansions of several species compared to previous studies. Furthermore, the use of distance-based phylogenetic methods nevertheless group specimens into plausible species clades that are highly useful in non-taxonomical or non-systematic studies.

Molecular phylogeny of the benthic shallow-water octopuses (Cephalopoda: Octopodinae).

Octopus has been regarded as a "catch all" genus, yet its monophyly is questionable and has been untested. We inferred a broad-scale phylogeny of the benthic shallow-water octopuses (subfamily Octopodinae) using amino acid sequences of two mitochondrial DNA genes: Cytochrome oxidase subunit III and Cytochrome b apoenzyme, and the nuclear DNA gene Elongation Factor-1alpha. Sequence data were obtained from 26 Octopus species and from four related genera. Maximum likelihood and Bayesian approaches were implemented to estimate the phylogeny, and non-parametric bootstrapping was used to verify confidence for Bayesian topologies. Phylogenetic relationships between closely related species were generally well resolved, and groups delineated, but the genes did not resolve deep divergences well. The phylogenies indicated strongly that Octopus is not monophyletic, but several monophyletic groups were identified within the genus. It is therefore clear that octopodid systematics requires major revision.