Amphioctopus Fangsiao Research Articles

The gold-ringed octopus (Amphioctopus fangsiao) genome and cerebral single-nucleus transcriptomes provide insights into the evolution of karyotype and neural novelties

BackgroundColeoid cephalopods have distinctive neural and morphological characteristics compared to other invertebrates. Early studies reported massive genomic rearrangements occurred before the split of octopus and squid lineages (Proc Natl Acad Sci U S A 116:3030-5, 2019), which might be related to the neural innovations of their brain, yet the details remain elusive. Here we combine genomic and single-nucleus transcriptome analyses to investigate the octopod chromosome evolution and cerebral characteristics.ResultsWe present a chromosome-level genome assembly of a gold-ringed octopus, Amphioctopus fangsiao, and a single-nucleus transcriptome of its supra-esophageal brain. Chromosome-level synteny analyses estimate that the chromosomes of the ancestral octopods experienced multiple chromosome fission/fusion and loss/gain events by comparing with the nautilus genome as outgroup, and that a conserved genome organization was detected during the evolutionary process from the last common octopod ancestor to their descendants. Besides, protocadherin, GPCR, and C2H2 ZNF genes are thought to be highly related to the neural innovations in cephalopods (Nature 524:220–4, 2015), and the chromosome analyses pinpointed several collinear modes of these genes on the octopod chromosomes, such as the collinearity between PCDH and C2H2 ZNF, as well as between GPCR and C2H2 ZNF. Phylogenetic analyses show that the expansion of the octopod protocadherin genes is driven by a tandem-duplication mechanism on one single chromosome, including two separate expansions at 65 million years ago (Ma) and 8–14 Ma, respectively. Furthermore, we identify eight cell types (i.e., cholinergic and glutamatergic neurons) in the supra-esophageal brain of A. fangsiao, and the single-cell expression analyses reveal the co-expression of protocadherin and GPCR in specific neural cells, which may contribute to the neural development and signal transductions in the octopod brain.ConclusionsThe octopod genome analyses reveal the dynamic evolutionary history of octopod chromosomes and neural-related gene families. The single-nucleus transcriptomes of the supra-esophageal brain indicate their cellular heterogeneities and functional interactions with other tissues (i.e., gill), which provides a foundation for further octopod cerebral studies.

Polystyrene microplastic ingestion induces the damage in digestive gland of Amphioctopus fangsiao at the physiological, inflammatory, metabolome and transcriptomic levels

Microplastics are ubiquitous in the aquatic and terrestrial ecosystem, increasingly becoming a serious concern for aquatic organism health. However, information regarding the effects of microplastics on cephalopods is remain limited to date. Amphioctopus fangsiao, an important economic species in cephalopods, can serve as a potential indicator of environmental pollution due to its short life expectancy and high metabolic rates. Here, to explore the toxic effects during the microplastic stress response, we analyzed the growth performance, histopathological damage, oxidative stress biomarkers, metabolomic and transcriptomic response in digestive gland of A. fangsiao under different concentrations (0, 100 and 1000 μg/L) of commercial polystyrene microplastics (MPS) exposure (5 μm, sphere) for 21 days. The results showed that MPS exerted a huge influence on the growth performance of A. fangsiao. The oxidative stress and inflammation in digestive gland of A. fangsiao were also detected after exposure to MPS. In addition, most of the altered metabolites observed in the metabolic analysis were related to inflammation, oxidative stress and glucolipid metabolism. Transcriptome analysis detected the differentially expressed genes (DEGs) and the significantly enriched KEGG pathways associated with glycolipid metabolism, inflammation and DNA damage. Collectively, our results indicate that excessive environmental microplastic exposure will cause toxicity damage and then initiate the detoxification mechanism in A. fangsiao digestive gland to maintain homeostasis. This study revealed that microplastic can cause adverse consequences on cephalopods, providing novel insights into the toxicological effect of microplastic exposure.

Transcriptome Profile Based on Protein-Protein Interaction Networks Provides a Set of Core Genes for Understanding the Metabolic Mechanisms of the Egg-Protecting Behavior in Amphioctopus fangsiao

Transcriptome Profile Based on Protein-Protein Interaction Networks Provides a Set of Core Genes for Understanding the Metabolic Mechanisms of the Egg-Protecting Behavior in Amphioctopus fangsiao

Toxic effects of polystyrene microplastics on the intestine of Amphioctopus fangsiao (Mollusca: Cephalopoda): From physiological responses to underlying molecular mechanisms

Microplastics are broadly used and among the most studied environmental pollutants due to their potential impacts on organisms and human health. Amphioctopus fangsiao (Cephalopoda: Octopodidae) is an important commercial species in the Pacific Northwest and is very popular among consumers owing to its rich nutritional value and fresh flavor. However, the toxic effects of microplastic exposure on A. fangsiao, including phenotypical effect and underlying molecular mechanism, remain limited. In this study, the octopus A. fangsiao were exposed to microplastics (polystyrene microplastics, Micro-PS) at concentrations of 100 and 1000 μg/L for 21 days, and then the physiological response, histopathological analysis, biomarkers of oxidative stress and glycolipid metabolism, microbiome perturbations and transcriptomic profiles in the intestines were performed. Results demonstrated that Micro-PS exposure had distinct adverse effects on the food intake of A. fangsiao. Histological analysis revealed that Micro-PS exposure has resulted in histopathological damage, thus causing early inflammation of the intestine. Oxidative stresses, metabolic disorders and microbiome perturbations were also detected in the intestine of A. fangsiao based on physiological biomarkers and microbiome analyses. Moreover, transcriptome analysis detected the differentially expressed genes (DEGs) and significantly enriched KEGG pathways in response to oxidative stress, glycolipid metabolism, DNA damage and transmembrane transport of intestinal cells, revealing distinct toxic effects at the molecular level. In summary, Micro-PS exposure has a strong impact on the intestines of A. fangsiao. For the first time, this study uses multiple approaches based on the physiological and biochemical response as well as transcriptional regulation analysis. The first assessment of the toxic impact of this species under Micro-PS exposure is also reported.

Effects of Environmentally Relevant Concentrations of Antipsychotic Drugs (Sulpiride and Clozapine) on Serotonergic and Dopaminergic Neurotransmitter Systems in Octopus Brain Tissue

Pharmaceuticals and personal care products (PPCPs) from specific activities often enter surface and groundwater, adversely affecting the physiological functions of non-target organisms and posing a serious threat to a wide range of aquatic organisms. Therefore, the aim of this study was to investigate the effects of environmentally relevant concentrations of the antipsychotic drugs sulpiride and clozapine on dopaminergic (DAergic) and serotonergic (5-hydroxytryptaminergic, i.e., 5-HTergic) neurotransmitter systems in the brain of a short-arm octopus (Octopus ochellatus). Octopus ochellatus adults were exposed to environmentally relevant concentrations of sulpiride, clozapine, or a mixture of sulpiride and clozapine. The effects of the drug on the transcription and expression levels of major functional molecules in the DAergic and 5-HTergic systems of the brain were analyzed. By antagonizing the dopamine receptor D2 (DRD2) or 5-hydroxytryptamine receptor 2A (5-HTR2A), the two drugs induced abnormal transcription and expression levels of important functional molecules in the brain’s DA and 5-HT signaling pathways. In addition, dose-dependent adverse reactions were observed with these antipsychotics. Our results suggest that sulpiride and clozapine interfere with DAergic and 5-HTergic neurotransmitter systems in the brain of Amphioctopus fangsiao (O. ochellatus), possibly affecting brain functions, such as reproduction, predation, camouflage, learning, and memory. As a result, they pose a serious threat to the health of Amphioctopus fangsiao.

Transcriptome profiling based on protein–protein networks provides a core set of genes for understanding blood immune response mechanisms against LPS stress in Amphioctopus fangsiao

Gram-negative bacteria are significant pathogens in the ocean, posing serious threats to marine organisms. Lipopolysaccharide (LPS) is a characteristic chemical constituent in Gram-negative bacteria that can be recognized by the pattern recognition receptor (PRR) of immune cells. This system is often used to simulate the invasion of bacteria. Blood is a transport channel for immune cells, and its transcriptome information obtained from Amphioctopus fangsiao stimulated by LPS is essential for understanding the antibacterial biological mechanisms of this species. In this study, we analyzed the gene expression profiles of A. fangsiao blood within 24h under LPS stress and found 778 and 561 differentially expressed genes (DEGs) at 6 and 24h, respectively. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment analyses were performed to search for immune-related DEGs. The relationships among immune genes were examined by constructing a protein–protein interaction (PPI) network. Finally, 16 hub genes were identified based on the PPI network and KEGG enrichment analysis. The expression profiles of these genes were verified using quantitative RT-PCR (qRT-PCR). This research provides valuable resources for the healthy culture of A. fangsiao and helps us understand the molecular mechanisms of innate immunity.

Change in cephalopod species composition in the overexploited coastal China seas with a closer look on Haizhou Bay, Yellow Sea

Climate change and intensive fishing have affected not only population abundance, but also species composition. Cephalopods have been increasing in abundance in the world ocean under climate change due to their flexible life-history traits, including the over-exploited China Seas. Despite the increasing importance of coastal cephalopods in the China Seas, there have been no reports of changes in either species composition, nor the ecological roles of species with different life-history traits. Thus, this study first presents the changes in species composition of coastal cephalopods throughout the China Seas as summarized from fishery-independent survey reports over the last six decades. This is followed by an investigation of species composition of cephalopods in Haizhou Bay in the Yellow Sea. The ecological roles of two currently targeted cephalopods, Amphioctopus fangsiao and Loliolus spp. (Loliolus beka and Loliolus japonicus), are evaluated using an ecosystem model. The species composition of coastal cephalopods in the China Seas has changed since the 1960s, from species of large size and high value to small-size, low-value species. Cephalopod species composition in Haizhou Bay shows great seasonality, which is probably due largely to the characteristics of their life cycle. The population abundance of A. fangsiao and Loliolus spp. appear to be affected by ambient water temperature, and population distribution of Loliolus spp. seems to correlate with water depth. Occupying the highest trophic level in this ecosystem, A. fangsiao potentially displays strong top-down control over other organisms. Loliolus spp. are keystone species showing higher keystoneness in the autumn, owing to a low abundance of fish species which normally prey on them. The species-specific life-history traits and ecological roles of cephalopods are therefore important factors to consider in order to manage them effectively.

Transcriptome profiling based on larvae at different time points after hatching provides a core set of gene resource for understanding the immune response mechanisms of the egg-protecting behavior against Vibrio anguillarum infection in Amphioctopus fangsiao

Mollusks have recently received increasing attention because of their unique immune systems. Mollusks such as Amphioctopus fangsiao are economically important cephalopods, and the effects of their egg-protecting behavior on the larval immune response are unclear. Meanwhile, little research has been done on the resistance response of cephalopod larvae infected with pathogenic bacteria such as Vibrio anguillarum. In this study, V. anguillarum was used to infect the primary hatching A. fangsiao larvae under different egg-protecting behaviors for 24 h, and a total of 7156 differentially expressed genes (DEGs) were identified at four time points after hatching based on transcriptome analysis. GO and KEGG enrichment analyses showed that multiple immune-related GO terms and KEGG signaling pathways were enriched. Protein–protein interaction networks (PPI networks) were used to search functional relationships between immune-related DEGs. Finally, 20 hub genes related to multiple gene functions or involved in multiple signaling pathways were identified, and their accuracy was verified using quantitative RT-PCR. PPI networks were first used to study the effects A. fangsiao larvae after infection with V. anguillarum under different egg-protecting behaviors. The results provide significant genetic resources for exploring invertebrate larval immune processes. The data lays a foundation for further study the immune response mechanisms for invertebrates after infection.

DNA Barcoding Reveals High Hidden Species Diversity of Chinese Waters in the Cephalopoda

Species delimitation by traditional morphological methods is challenging in cephalopods due to their flexible bodies, changeable pigment traits, and sometimes high morphological homoplasy. Molecular information provides important assistance for taxonomic decision. In this study, we applied three species delimitation methods on 132 cephalopods from 7 families along Chinese waters, and we found anunderestimated species diversity in these taxa. A maximum of 56 molecular operational taxonomic units was detected, and possible cryptic diversities were revealed in Loliolus beka, Uroteuthis edulis, Octopus minor, Amphioctopus fangsiao, and Hapalochlaena lunulate. This study provides molecular evidence for the hidden species diversity along Chinese waters, and it provides insight into further taxonomic research on these morphologically variable taxa.

Differential gene expression analysis related to sperm storage in spermathecas of Amphioctopus fangsiao

Sperm storage in the female body is an important strategy in animal reproductive behavior. Amphioctopus fangsiao is an economically important cephalopod that has a sperm storage period of up to seven months. There are few studies concerning the mechanism of sperm storage in A. fangsiao. In this study, we performed transcriptome gene expression profiling of the oviductal glands at different phases (presence and absence of sperm storage). In total, 7943 differentially expressed genes (DEGs) comprising 4737 upregulated and 3206 downregulated genes were identified. GO and KEGG enrichment analyses were used to search for sperm storage-related genes. A protein interaction network was constructed to examine the interactions between genes. Nineteen genes associated with immunity, apoptosis, and autophagy were obtained and verified by qRT-PCR. This is the first comprehensive analysis of sperm storage-related genes in A. fangsiao. The results provide basic insights into the complex sperm storage mechanism of A. fangsiao.

Aggregata sinensis n. sp. (Apicomplexa: Aggregatidae), a new coccidian parasite from Amphioctopus fangsiao and Octopus minor (Mollusca: Octopodidae) in the Western Pacific Ocean.

A new species of parasitic Aggregata, Aggregata sinensis n. sp., is described from the octopuses Amphioctopus fangsiao (d'Orbigny, 1839-1841) and Octopus minor (Sasaki, 1920) (Mollusca: Octopodidae) in China based on morphological data and 18S ribosomal RNA (rRNA) gene sequences. The sexual gamogonic and sporogonic stages of the parasite were observed under light microscope. The sizes of oocysts were 207.35-618.74μm in length and were 136.31-420.68μm in width. Oocysts were fully sporulated and were shaped spherically or irregularly. Mature sporocysts within oocysts were spherical or slightly subovoid and were 19.42-22.32μm in length and 19.12-21.55μm in width. Sporozoites were typically spiral within sporocysts and were 17.81-24.90μm long and 2.05-3.42μm wide. Molecular phylogenetic analysis constructed using the 18S rRNA gene sequences supports the morphological classification that our species is placed within the genus Aggregata. Moreover, the sequence belonged to the genus Aggregata. However, the 18S rRNA gene sequence of Ag. sinensis was significantly different from that of other Aggregata available currently. Ag. sinensis is the first two-host parasitic species of Aggregata reported from definitive host Am. fangsiao and O. minor and is the only species of the genus Aggregata known in Western Pacific waters.

Ontogenetic variation of trophic habitat for sympatric benthic octopods in East China Sea derived from isotopic analysis on beaks

Sympatry is commonly observed in marine species, and always accompanied by high competition. Octopus consists of large amount of species and also widely distributed in the neritic area of the sea. As typical benthic species, octopus tend to live in the similar habitat with less movement during its lifetime. It is important to understand the trophic ecology of sympatric octopod species, which play an irreplaceable role in marine ecosystem. In this study, four neritic octopus species in the East China Sea (Amphioctopus fangsiao, Amphioctopus ovulum, Octopus minor and Octopus sinensis) were chose, we used stable isotope analysis (SIA) to analyze the variation of δ13C and δ15N derived from beak to assess the trophic variation among species and maturity stages. O. sinensis has the largest body and beak size than other species, and this species also had highest mean δ13C, δ15N value among species. The result of ANOVA showed that significant differences existed among four species for isotopic values. The overlaps among species were also calculated by standard ellipse area (SEAc). Isotopic overlap was decreased from immature stage to mature stage. The trophic niches were shrunk from immature to mature for all the four octopods species except for A. ovulum, which presented an expansion tendency. Trophic overlaps between specific two species in different groups were also calculated with distribution and mean values based on Bayesian model. The highest and lowest isotopic overlap occurred between O. minor and A. ovulum in immature group, A. fangsiao and O. sinensis in mature group, respectively. The four octopods species had very different trophic niches during ontogeny.

Growth and Distribution of Amphioctopus fangsiao (d’Orbigny, 1839–1841) in Haizhou Bay, Yellow Sea

Octopus fisheries have prospered in many areas of the world ocean over the last six decades. Despite degradation and overexploitation of Chinese coastal ecosystems, octopus has become a stable Chinese domestic fishery species. Among the octopus species in the Bohai and Yellow, East and South China Seas, Amphioctopus fangsiao (d’Orbigny [in Ferussac & d’Orbigny], 1839–1841) plays an increasingly important role both economically and ecologically. However, no systematic research has been conducted on its biology and population dynamics in the China Seas. In this study, we characterized the growth and distribution of A. fangsiao in Haizhou Bay, Yellow Sea based on four years of trawl survey data. As the results, A. fangsiao is characterized by allometric growth and an overall sex ratio of 0.97 with a slight male bias. Small octopus can be observed through a whole year, suggesting that A. fangsiao may have an extended or continuous spawning season. A. fangsiao may migrate short distances along with seasonal changes and coastal currents, considering they overwinter in the offshore water and spawn in the inshore water. In addition to fishing pressure, annual variations in the population structure and biomass appeared to be influenced by ambient water temperature. These results provide basic biological information for a better understanding of the population dynamics and the ecological importance of A. fangsiao.

Embryonic development of Amphioctopus fangsiao under elevated temperatures: Implications for resource management and conservation

Amphioctopus fangsiao is an important fishery resource in northern coastal China, and this species has been developed for commercial fishing and aquacultural purposes. However, its embryos are vulnerable to ambient temperature, which leads to challenges in resource management and conservation. The present study investigated how high temperatures could affect the embryonic development (e.g., morphology, development time, and viability) of A. fangsiao. The results showed that the embryonic developmental rate was accelerated as the temperature increased. In addition, temperatures higher than the thermal threshold detrimentally affected the morphology and viability of eggs. Embryos maintained at 18, 21, and 24 ℃ successfully hatched at 40, 30, and 24 d after spawning, respectively. However, no embryos hatched at 27 ℃. Elevated temperatures altered the trajectory of normal embryonic development and increased the percentage of eggs with abnormal morphology. Embryos maintained at 24 ℃ failed to undergo the second inversion during embryonic development and remained at the animal pole until stage XVII (15 d). Upon the completion of organogenesis at stage XIX, an elevated temperature, as the external stimulus, could promote the early hatching of embryos with poor physiological features (e.g., small size and large yolk sac). The results indicate that the thermal range for A. fangsiao embryonic development is 18–24 ℃. Our findings on the relationship between embryonic stage and temperature might help fishery managers predict the emergence time of the hatchling population and promote the conservation of this species when establishing related fishery policies.

Development of Amphioctopus fangsiao (Mollusca: Cephalopoda) from eggs to hatchlings: indications for the embryonic developmental management

Amphioctopus fangsiao is an important fishery resource in northern coastal China, and there is an interest to develop its commercial cultivation under controlled conditions. In this study, we describe the embryonic development of A. fangsiao from eggs to hatchlings, focusing on the formation of the mantle, eyes and arms. Results indicate that this species has several favorable characteristics for aquaculture, such as a short incubation period and a large body size of hatchlings. During the organogenesis, the primordia of organs, including the optic sac and arm crown, first appeared at stage VIII; then the mantle primordium, eye primordia and arm crown become visible at stage IX–X, and the rudiment of embryo (mantle, eyes and arms) was completely outlined at stage XI. The retinal pigment appeared yellowish at stage X–XI and gradually deepens in pigmentation in the subsequent stages, turning from yellowish to yellow, red, dark-red and eventually black. At stage XV, the primary lid fold, with only a circular opening remaining, covered the eye, and the dorsal ocular edge did not reach the ventral side until hatching (day 1–7). Afterwards, about 24 days after hatching, both the dorsal and ventral sides of eyelid could contract freely, with an oval and elongate slit remaining that was a miniature version of the adult morphology. The chromatophores and ink sac appeared at stage XIV. These observations provide a foundation for the reproductive biology and aquaculture of A. fangsiao and also offer an important reference for the research of embryonic development in related species.

Genetic structure of Amphioctopus fangsiao (Mollusca, Cephalopoda) in Chinese waters inferred from variation in three mtDNA genes (ATPase 6, ND2, and ND5)

Amphioctopus fangsiao is an economically important resource found in Chinese coastal waters. Three mitochondrial DNA genes were used to assess the genetic structure among nine populations spanning the northern to southern regions of the Chinese coast. Fragments of 575, 639, and 640 bp in length representing three genes (ATPase 6, ND2, and ND5) were amplified from 183, 170, and 167 individuals, respectively. Overall, ATPase 6, ND2, and ND5 showed high haplotype diversity and low nucleotide diversity (HD: 0.683–0.896; −π: 0.021–0.033). All three mtDNA genes revealed high molecular variance among populations and low variance within populations. ΦST values obtained for ATPase 6 (ΦST = 0.000 to 0.997), ND2 (ΦST = 0.000 to 0.997), and ND5 (ΦST = 0.125 to 0.983) showed differentiation among the populations. The constructed phylogeographic tree and haplotype network separated the nine populations into two clades representing the northern and southern populations. Low salinity in the Changjiang River estuary may act as a barrier to promote the differentiation between the two clades. These results enhance our understanding of the genetic structure of A. fangsiao and can promote the management of its genetic resources.

Octopicola huanghaiensis n. sp. (Copepoda: Cyclopoida: Octopicolidae), a new parasitic copepod of the octopuses Amphioctopus fangsiao (d’Orbigny) and Octopus minor (Sasaki) (Octopoda: Octopodidae) in the Yellow Sea

A new species of parasitic copepod, Octopicola huanghaiensis n. sp., collected from the octopuses Amphioctopus fangsiao (d'Orbigny) and Octopus minor (Sasaki) (Octopoda: Octopodidae) in the Yellow Sea (off Qingdao, Shandong Province, China), is described. The new species is most similar to O. superba Humes, 1957, but can be distinguished from the latter by: (i) the third antennal segment having a different ornamentation; (ii) the fourth antennal segment of females much shorter than that in O. superba (49 vs 94 μm); (iii) males much smaller than females (mean body length 1.3 vs 2.0 mm, respectively) (vs similar male and female body size in O. superba, 1.9 mm and 1.8 mm respectively); and (iv) the presence of a spike at the posterior tip of each labrum flap. Octopicola huanghaiensis n. sp. is the first species of Octopicola Humes, 1957 reported from A. fangsiao and O. minor and is the only species of the family Octopicolidae Humes & Boxshall, 1996 known in North Pacific waters.

Beak identification of four dominant octopus species in the East China Sea based on traditional measurements and geometric morphometrics

Octopus is the most abundant genus in the family Octopodidae and accounts for more than half of the total cephalopod landing in neritic fisheries. A taxonomic problem still exists due to synonymous scientific names and limited genetic information. The cephalopod beak is a stable structure that allows an effective solution to the problem of the species and stock identification. Beak shape variation has been more widely considered than beak measurements in recent years. In this study, with the beak as the experimental material, we combined geometric morphometrics (GM) with machine learning methods and compared the discrimination results obtained by traditional and GM methods in four Chinese neritic octopus species (Amphioctopus fangsiao, Amphioctopus ovulum, Octopus minor and Octopus sinensis). According to our analyses, Octopus sinensis has the larger beak size [both upper beak (UB) and lower beak (LB)] than other species. The results of ANOVA showed that all beak measurements differed significantly among the four species. Significant differences in both UB and LB shapes among four species were identified in MANOVA analysis based on the GM method. The results of GM-based discriminant analysis were better than those of traditional measurements, and machine learning methods also showed the higher correct classification rates than linear discriminant analysis. GM is a useful method to reconstruct the shape cephalopod beak and can also effectively distinguish different species. We should improve classification accuracy with machine learning methods for determining species structure in the future.

Construction of a normalized full-length cDNA library of cephalopod Amphioctopus fangsiao and development of microsatellite markers

Amphioctopus fangsiao is one of the most economically important species and has been considered to be a candidate for aquaculture. In order to facilitate its fine-scale genetic analyses, we constructed a normalized full-length library successfully and developed a set of microsatellite markers in this study. The normalized full-length library had a storage capacity of 6.9×105 independent clones. The recombination efficiency was 95% and the average size of inserted fragments was longer than 1000 bp. A total of 3440 high quality ESTs were obtained, which were assembled into 1803 unigenes. Of these unigenes, 450 (25%) were assigned into 33 Gene Ontology terms, 576 (31.9%) into 153 Kyoto Encyclopedia of Genes and Genomes pathways, and 275 (15.3%) into 22 Clusters of Orthologous Groups. Seventy-six polymorphic microsatellite markers were identified. The number of alleles per locus ranged from 4 to 17, and the observed and expected heterozygosities varied between 0.167 and 0.967 and between 0.326 and 0.944, respectively. Twelve loci were significantly deviated from Hardy-Weinberg equilibrium after Bonferroni correction and no linkage disequilibrium was found between different loci. This study provided not only a useful resource for the isolation of the functional genes, but also a set of informative microsatellites for the assessment of population structure and conservation genetics of A. fangsiao.

Comparison of the genetic relationship between nine Cephalopod species based on cluster analysis of karyotype evolutionary distance.

Karyotype analysis was carried out on gill cells of three species of octopods using a conventional air-drying method. The karyotype results showed that all the three species have the same diploid chromosome number, 2n=60, but with different karyograms as 2n=38M+6SM+8ST+8T, FN (fundamental number)=104 (Cistopus chinensis Zheng et al., 2012), 2n=42M+6SM+4ST+8T, FN=108 (Octopus minor (Sasaki, 1920)) and 2n=32M+16SM+12T, FN=108 (Amphioctopus fangsiao (d’Orbigny, 1839–1841)). These findings were combined with data from earlier studies to infer the genetic relationships between nine species via cluster analysis using the karyotype evolutionary distance (De) and resemblance-near coefficient (λ). The resulting tree revealed a clear distinction between different families and orders which was substantially consistent with molecular phylogenies. The smallest intraspecific evolutionary distance (De=0.2013, 0.2399) and largest resemblance-near coefficient (λ=0.8184, 0.7871) appeared between O. minor and C. chinensis, and Sepia esculenta Hoyle, 1885 and S. lycidas Gray, 1849, respectively, indicating that these species have the closest relationship. The largest evolutionary gap appeared between species with complicated karyotypes and species with simple karyotypes. Cluster analysis of De and λ provides information to supplement traditional taxonomy and molecular systematics, and it would serve as an important auxiliary for routine phylogenetic study.