Amphioctopus Fangsiao Research Articles

Decabromodiphenyl ethane (DBDPE) inhibited the growth and feeding by disrupting the gut and digestive gland homeostasis in octopus Amphioctopus fangsiao (Mollusca: Cephalopoda)

A novel brominated flame retardant decabromodiphenyl ethane (DBDPE) poses a potential threat to animals, but its effects on cephalopods remain unknown. In this study, Amphioctopus fangsiao, a common octopus in China, was exposed to DBDPE (0, 1, 50, 100, 300 μg/L) for 28 days. Chemical analysis revealed that the digestive gland bore a greater burden of DBDPE compared with other tissues. In addition, accumulated DBDPE could curb the growth and feeding performance of A. fangsiao. The potential effects on the “gut-digestive gland axis” were also elucidated. Specifically, DBDPE in the gut shifted the microorganisms toward a Bacteroidetes-dominated composition, and impaired the intestinal epithelial barrier, thereby triggering oxidative stress and inflammation. Excessive DBDPE also threatens the digestive gland function, including histological damage, immune reaction, oxidative stress, glucolipid metabolism dysfunction, and neurotoxicity. Metabolome plasticity enabled A. fangsiao to develop a DBDPE stress-adaptive metabolic profile via alteration of glucolipid metabolism, immunity, oxidative stress, and signaling molecules. Taken together, we identified a new detoxification mechanism linking the microbiota-gut-digestive gland axis with the growth and food intake of A. fangsiao, which is the first time it has been demonstrated in mollusks. These findings provided important clues for a further mechanism study and risk assessment of DBDPE.

Ontogeny of predatory capacity and prey choice during early life of the holobenthic octopus Amphioctopus fangsiao (d’Orbigny, 1841): switching prey-choice strategy

Ontogeny of predatory capacity and prey choice during early life of the holobenthic octopus Amphioctopus fangsiao (d’Orbigny, 1841): switching prey-choice strategy

Transcriptome-based protein-protein interaction analysis reveals immune gene network elucidating white body immunity mechanisms in response to LPS stimulation in Amphioctopus fangsiao

One of the major challenges in the aquaculture of marine organisms is the susceptibility to pathogens. Immune cells defend bacterial invasion by recognizing pathogen-associated molecular patterns (PAMPs) such as Lipopolysaccharides (LPS) in Gram-negative bacteria. The white body are key organs for immune response and hematopoiesis, and play an important role in understanding the antimicrobial biological response in gold-ringed octopus (Amphioctopus fangsiao). In the present research, transcriptome sequencing and bioinformatics analysis were conducted on A. fangsiao white body tissue after 6 h and 24 h of LPS stimulation. The results showed 2,029 and 73 differentially expressed genes (DEGs) at the 6- and 24-hour time points, respectively. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were beneficial in determining immune-related terms and signaling pathways. Subsequently, a protein-protein interaction (PPI) network was constructed and 20 hub genes, such as AKT3, MAPK14, and PIK3CA, were identified. These genes are involved in a variety of signaling pathways, including the Hippo signaling pathway, chemokine signaling pathway, and leukocyte trans endothelial migration. Using qRT-PCR, the accuracy of the 20 hub genes was verified. The present study provides a substantial theoretical foundation for the research on A. fangsiao white body immunity, enhancing the current understanding of cephalopods' innate immunity.

Transcriptome-based network analysis reveals hub immune genes and pathways of hepatopancreas against LPS in Amphioctopus fangsiao

The hepatopancreas is the biggest digestive organ in Amphioctopus fangsiao (A. fangsiao), but also undertakes critical functions like detoxification and immune defense. Generally, pathogenic bacteria or endotoxin from the gut microbiota would be arrested and detoxified in the hepatopancreas, which could be accompanied by the inevitable immune responses. In recent years, studies related to cephalopods immune have been increasing, but the molecular mechanisms associated with the hepatopancreatic immunity are still unclear. In this study, lipopolysaccharide (LPS), a major component of the cell wall of Gram-negative bacteria, was used for imitating bacteria infection to stimulate the hepatopancreas of A. fangsiao. To investigate the immune process happened in A. fangsiao hepatopancreas, we performed transcriptome analysis of hepatopancreas tissue after LPS injection, and identified 2615 and 1943 differentially expressed genes (DEGs) at 6 and 24 h post-injection, respectively. GO and KEGG enrichment analysis showed that these DEGs were mainly involved in immune-related biological processes and signaling pathways, including ECM-receptor interaction signaling pathway, Phagosome signaling pathway, Lysosome signaling pathway, and JAK-STAT signaling pathways. The function relationships between these DEGs were further analyzed through protein-protein interaction (PPI) networks. It was found that Mtor, Mapk14 and Atm were the three top interacting DEGs under LPS stimulation. Finally, 15 hub genes involving multiple KEGG signaling pathways and PPI relationships were selected for qRT-PCR validation. In this study, for the first time we explored the molecular mechanisms associated with hepatopancreatic immunity in A. fangsiao using a PPI networks approach, and provided new insights for understanding hepatopancreatic immunity in A. fangsiao.

The effectiveness and impact of disinfection with chlorine on embryos and early hatchlings of two Western Pacific octopus species

AbstractReplicating maternal care and incubating octopus embryos artificially is advantageous to improve access to embryonic stages, split and share broods, and reduce the required space to culture large quantities of embryos. However, without female care and adequate rearing systems, the risk of bacterial and fungal infections can be high. This study evaluated the potential effects of low‐concentration sodium hypochlorite (NaOCl) as a disinfectant on embryonic survival, hatchling survival, and growth. Embryos of a holobenthic octopus, Amphioctopus fangsiao, and of a merobenthic octopus, Octopus sinensis, were disinfected with 0.004% NaOCl for 3 min either once at the start (IB), at the start and halfway point (RB), and without disinfection (control). Survival of A. fangsiao embryos differed among treatments initially, but the final number of hatchings did not differ among treatments. No differences in the final survival for O. sinensis embryos were observed among the three treatments. Wet weight and growth for A. fangsiao juveniles did not differ among treatments at any point. In contrast, initial and final dry weight for O. sinensis paralarvae from the control treatment were heavier compared to the IB and RB treatments. These results suggest that NaOCl used as disinfectant has a species‐specific effect on early hatchlings depending on the octopus life history.

Living in a dynamic environment: The effects of multi-ways temperature variation on embryo and newborn juveniles of a shallow-water octopus (Amphioctopus fangsiao)

Shallow waters are characterized by fluctuating environmental conditions, modulating marine life cycles and biological phenomena. Multiple variations in water temperature could affect eggs and embryos during spawning events of many marine invertebrate species, yet most of the findings on embryonic development in invertebrates come from experiments based on the constant temperature. In this study, to examine the effects of temperature variation on octopus embryos, Amphioctopus fangsiao, a common shallow-water octopus along the coast of China, was exposed to the constant temperature (18 °C, in situ temperature of the seawater in Lianyungang), ramping temperatures (from 18 to 24 °C), diel oscillating temperatures (18 °C and 20 °C for 12 h each day), and acute increasing temperatures (the temperature increased sharply from 18 °C to 24 °C at embryonic development stage XIX) for 47 days (from embryogenesis to settlement). The results demonstrated that the temperature variations accelerated the development time of A. fangsiao embryos. Temperature fluctuations could cause embryonic oxidative damage and disorder of glycolipid metabolism, thereby affecting the growth performance of embryos and the survival rate of hatchings. Through transcriptome sequencing, the mechanistic adaption of the embryo to environmental temperature variations was revealed. The pathways involved in the TCA cycle, DNA replication and repair, protein synthesis, cell signaling, and nervous system damage repair were significantly enriched, indicating that the embryo could improve heat tolerance to thermal stress by regulating gene expression. Moreover, acute warming temperatures posed the most detrimental effects on A. fangsiao embryos, which could cause embryos to hatch prematurely from the vegetal pole, further reducing the survival of hatchings. Meanwhile, the diel oscillating temperature was observed to affect the normal morphology of the embryo, resulting in embryo deformities. Thus, the constant temperature is critical for balanced growth and defense status in octopuses by maintaining metabolism homeostasis. For the first time, this study evaluates the effects of multiple temperature fluctuations on embryos of A. fangsiao, providing new insights into the physiological changes and molecular responses of cephalopod embryos following dynamic temperature stress.

Transcriptome sequencing reveals the characteristics of spermatogenesis and testis development in Amphioctopus fangsiao

Spermatogenesis is the production of spermatozoa by the testes, a process that is associated with reproduction in males. Amphioctopus fangsiao is an economically important species from the northern coast of China, and little research has been done on spermatogenesis in this species. In this study, we performed transcriptome analysis of testis and other somatic tissues. The maturing stage of testis development was selected, in which the emergence of spermatozoa was observed. A total of 15,773 differentially expressed genes were identified, including 8,922 upregulated genes and 6,851 downregulated genes. GO and KEGG enrichment analyses were performed to explore the functions of genes related to spermatogenesis, and the interactions between genes were examined by constructing protein interaction networks. Pathways and genes related to spermatogenesis were identified, including the oxytocin signaling pathway, the calcium signaling pathway, the FOXO signaling pathway, the DNAH family, and other key genes. The expression levels of 25 key genes were verified by qRT-PCR. The results for spermatogenesis-related genes will provide insights into the processes of spermatogenesis and testis development in A. fangsiao.

Weighted Gene Co-Expression Network Analysis Based on Stimulation by Lipopolysaccharides and Polyinosinic:polycytidylic Acid Provides a Core Set of Genes for Understanding Hemolymph Immune Response Mechanisms of Amphioctopus fangsiao.

The primary influencer of aquaculture quality in Amphioctopus fangsiao is pathogen infection. Both lipopolysaccharides (LPS) and polyinosinic:polycytidylic acid (Poly I:C) are recognized by the pattern recognition receptor (PRR) within immune cells, a system that frequently serves to emulate pathogen invasion. Hemolymph, which functions as a transport mechanism for immune cells, offers vital transcriptome information when A. fangsiao is exposed to pathogens, thereby contributing to our comprehension of the species' immune biological mechanisms. In this study, we conducted analyses of transcript profiles under the influence of LPS and Poly I:C within a 24 h period. Concurrently, we developed a Weighted Gene Co-expression Network Analysis (WGCNA) to identify key modules and genes. Further, we carried out Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses to investigate the primary modular functions. Co-expression network analyses unveiled a series of immune response processes following pathogen stress, identifying several key modules and hub genes, including PKMYT1 and NAMPT. The invaluable genetic resources provided by our results aid our understanding of the immune response in A. fangsiao hemolymph and will further our exploration of the molecular mechanisms of pathogen infection in mollusks.

Sequencing-based protein–protein interaction analysis provides a core set of immune gene for understanding white body immune response mechanisms against Poly I:C stimulation in Amphioctopus fangsiao

Pathogenic infections significantly limit the culture of marine organisms, such as cephalopods. However, there is a lack of immune information regarding cephalopods to address pathogen problems. Polyriboinosinic polyribocytidylic acid (Poly I:C), a synthetic virus-like molecule, can be recognized by the immune cells as pathogen-associated molecular patterns (PAMPs), and this process is often used to simulate the invasion of viruses. The white body is an immune and hematopoietic organ, and its RNA-seq information obtained from Amphioctopus fangsiao stimulated by Poly I:C is essential for understanding the antiviral immune response in this species. In this research, we performed transcriptome sequencing and bioinformatics analysis of A. fangsiao white body tissue within 24 h stimulated by Poly I:C. A large number of differentially expressed genes (DEGs) were detected in this study, including 2,613 and 8 DEGs at 6 h and 24 h, respectively. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were used for searching immune-related terms and genes. A protein–protein interaction (PPI) network was constructed to identify the relationship between immune genes. Finally, the 20 hub genes including RAC1, MAPK14, PIK3CA and other seventeen hub genes were identified based on the network and pathway analyses, and we validated the accuracy of 20 hub genes using qRT-PCR. These hub genes mainly participated in PI3K-Akt signaling pathway, Chemokine signaling pathway and other signaling pathways, in addition to the ubiquitin family. These research results provided a valuable theoretical basis for researching A. fangsiao white body immunity and significantly improved our cognition about innate immunity of cephalopods.

Preliminary study on toxicological mechanism of golden cuttlefish (Sepia esculenta) larvae exposed to cd

BackgroundCadmium (Cd) flows into the ocean with industrial and agricultural pollution and significantly affects the growth and development of economic cephalopods such as Sepia esculenta, Amphioctopus fangsiao, and Loligo japonica. As of now, the reasons why Cd affects the growth and development of S. esculenta are not yet clear.ResultsIn this study, transcriptome and four oxidation and toxicity indicators are used to analyze the toxicological mechanism of Cd-exposed S. esculenta larvae. Indicator results indicate that Cd induces oxidative stress and metal toxicity. Functional enrichment analysis results suggest that larval ion transport, cell adhesion, and some digestion and absorption processes are inhibited, and the cell function is damaged. Comprehensive analysis of protein-protein interaction network and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was used to explore S. esculenta larval toxicological mechanisms, and we find that among the 20 identified key genes, 14 genes are associated with neurotoxicity. Most of them are down-regulated and enriched to the neuroactive ligand-receptor interaction signaling pathway, suggesting that larval nervous system might be destroyed, and the growth, development, and movement process are significantly affected after Cd exposure.ConclusionsS. esculenta larvae suffered severe oxidative damage after Cd exposure, which may inhibit digestion and absorption functions, and disrupt the stability of the nervous system. Our results lay a function for understanding larval toxicological mechanisms exposed to heavy metals, promoting the development of invertebrate environmental toxicology, and providing theoretical support for S. esculenta artificial culture.

Effects of shelter on the welfare of juvenile Amphioctopus fangsiao: Survival, growth, behavior and physiology

Placing shelters in rearing containers is a method of environmental enrichment that can effectively improve the welfare of aquatic animals. However, a set of standard specifications for shelter use is still absent in the artificial culture of Amphioctopus fangsiao. The purpose of this study was to investigate whether shelter type significantly affects the survival, growth performance, behavior, cortisol level, and brain serotonergic system activity of juvenile A. fangsiao. Juveniles (weight 3.72 ± 0.56 g) were reared under different types of shelters for 3 weeks: no shelter (CT), PVC pipe (PP), shell (SH), and PVC corrugated plate (PC). Behavioral and physiological parameters were determined thereafter. In general, octopuses in PP treatment had the highest survival rate (83.3 ± 17.0%). The weight gain (53.40 ± 6.20%) and specific growth rate (2.03 ± 0.19% d−1) of CT octopuses were higher than those of octopuses from other treatments. CT octopuses exhibited the highest levels of aggressive behavior, cortisol, and brain serotonergic system activity, while PC octopuses showed the lowest levels of these stress-related behavioral and physiological indicators. The hepatosomatic index, coefficient of weight variation, and attachment rate did not differ significantly among treatments. Altogether, these results demonstrated for the first time that shelter type had a major impact on octopus survival, growth performance, behavioral phenotype (aggressive behavior), and stress-related physiological processes, and we suggest that using PVC pipes as shelters might be optimal for octopus rearing.

Transcriptome Profiling Based at Different Time Points after Hatching Deepened Our Understanding on Larval Growth and Development of Amphioctopus fangsiao.

As the quality of life improves, there is an increasing demand for nutrition-rich marine organisms like fish, shellfish, and cephalopods. To address this, artificial cultivation of these organisms is being explored along with ongoing research on their growth and development. A case in point is Amphioctopus fangsiao, a highly valued cephalopod known for its tasty meat, nutrient richness, and rapid growth rate. Despite its significance, there is a dearth of studies on the A. fangsiao growth mechanism, particularly of its larvae. In this study, we collected A. fangsiao larvae at 0, 4, 12, and 24 h post-hatching and conducted transcriptome profiling. Our analysis identified 4467, 5099, and 4181 differentially expressed genes (DEGs) at respective intervals, compared to the 0 h sample. We further analyzed the expression trends of these DEGs, noting a predominant trend of continuous upregulation. Functional exploration of this trend entailed GO and KEGG functional enrichment along with protein-protein interaction network analyses. We identified GLDC, DUSP14, DPF2, GNAI1, and ZNF271 as core genes, based on their high upregulation rate, implicated in larval growth and development. Similarly, CLTC, MEF2A, PPP1CB, PPP1R12A, and TJP1, marked by high protein interaction numbers, were identified as hub genes and the gene expression levels identified via RNA-seq analysis were validated through qRT-PCR. By analyzing the functions of key and core genes, we found that the ability of A. fangsiao larvae to metabolize carbohydrates, lipids, and other energy substances during early growth may significantly improve with the growth of the larvae. At the same time, muscle related cells in A. fangsiao larvae may develop rapidly, promoting the growth and development of larvae. Our findings provide preliminary insights into the growth and developmental mechanism of A. fangsiao, setting the stage for more comprehensive understanding and broader research into cephalopod growth and development mechanisms.

Metatranscriptomics unmasks Mollusca virome with a remarkable presence of rhabdovirus in cephalopods

IntroductionMollusks play a significant role in marine ecosystems and have economic value for aquaculture. Sometimes, unexpected and unexplained mortalities among mollusks have been described. The role of potential pathogens such as viruses remains unknown due to the lack of molluscan cell cultures, which is one of the major drawbacks to determining the viral role in such mortalities. Several oceanographic studies have suggested a high abundance of viruses in the oceans. Virus identification and understanding of viral interaction with organisms in marine ecosystems are in their infancy. Metatranscriptomics could become a useful tool to identify viruses using a shotgun approach and the growing number of viral genomes and sequences deposited in public databases.MethodsIn this work, several bioinformatics approaches were set up to screen Mollusca RNA sequences to find and confirm viral traces in their transcriptomes. This meta-analysis included an extensive search of SRA datasets belonging to mollusks available in the NCBI database, selecting a total of 55 SRA datasets that were further analyzed searching for viral sequences.ResultsTwenty-two bivalves, 19 cephalopods and 16 gastropods from 16 geographical origins and 17 different tissues were considered. The domain search approach was the most productive method to find viral sequences. This virus search showed that Cephalopoda samples (Idiosepius notoides and Amphioctopus fangsiao) exhibited the highest number of virus identifications. Some of the detected viral sequences were similar or identical to others previously identified. However, 33 putative new viruses were identified and analyzed phylogenetically when the RdRp domain was available. Specifically, Cephalopoda samples showed a considerable number of viruses belonging to the Rhabdoviridae family.

Ocean acidification increases copper accumulation and exacerbates copper toxicity in Amphioctopus fangsiao (Mollusca: Cephalopoda): A potential threat to seafood safety

Ocean acidification (OA) and trace metal pollutants coexist to exert combined effects on the functions and services of marine ecosystems. Increasing atmospheric carbon dioxide has caused a decrease in the pH of the ocean, affecting the bioavailability and speciation of trace metals and consequently altering metal toxicity in marine organisms. As an important trace metal functioned in hemocyanin, the richness of Copper (Cu) in octopuses is remarkable. Therefore, the biomagnification and bioaccumulation capacities of Cu in octopuses may be a non-negligible risk of contamination. Here, Amphioctopus fangsiao was continuously exposed to acidified seawater (pH 7.8) and copper (50 μg/L) to investigate the combined effect of ocean acidification and Cu exposure on marine mollusks. Our results showed that A. fangsiao could adapt well to ocean acidification after 21 days of the rearing experiment. However, the accumulation of Cu in A. fangsiao intestine increased significantly in acidified seawater under high levels of Cu stress. In addition, Cu exposure can influence the physiological function of A. fangsiao, including growth and feeding. This study also demonstrated that Cu exposure disturbed glucolipid metabolism and induced oxidative damage to intestine tissue, and ocean acidification further exacerbated these toxic effects. The obvious histological damage and microbiota alterations were also caused by Cu stress and its combined effect with ocean acidification. At the transcription level, we found numerous differentially expressed genes (DEGs) and significantly enriched KEGG pathways, involving glycolipid metabolism, transmembrane transport, glucolipid metabolism, oxidative stress, mitochondrial, protein and DNA damage, all revealing the strong toxicological synergetic effect of Cu and OA exposure and the molecular adaptation mechanism of A. fangsiao. Collectively, this study demonstrated that octopuses may withstand future ocean acidification conditions, however, the complex interactions of future OA and trace metal pollution need to be emphasized. OA can influence the toxicity of trace metals, inducing a potential threat to marine organism safety.

Assessing the Attractive Effects of Floating Artificial Reefs and Combination Reefs on Six Local Marine Species

Artificial reefs (ARs) have been advocated for and implemented as management tools for recreational fisheries, species conservation, and habitat replacement; however, the research that includes attracting marine species of floating ARs remains in its early stages. Here, two types of floating ARs were designed to evaluate the attractive effects using the occurrence rate and attracting index for six commercially important species (Lateolabrax maculatus, Liza haematocheila, Sebastes schlegelii, Acanthopagrus schlegelii, Litopenaeus vannamei, and Amphioctopus fangsiao) in the Bohai Bay of China; their combined ARs were meanwhile compared with two variants of artificial seagrass beds (SA and SB) and the traditional double-frame artificial reef (TD). All of the designed ARs were effective in attracting experimental species. The ARs with higher shelter areas (SB and TD) showed a better attracting effect. The efficiency of the ARs in attracting different species depended on their life histories. The bottom-mounted ARs were more efficient in attracting demersal species, while the floating ARs attracted epipelagic fish. In addition, the combined reefs had a better attractive effect than single ARs did. Overall, floating ARs and their combined ARs show the potential to deploy especially for attracting epipelagic fishes, although further study is needed.

Gene network analyses of larvae under different egg-protecting behaviors provide novel insights into immune response mechanisms of Amphioctopus fangsiao

Amphioctopus fangsiao was a representative economic species in cephalopods, which was vulnerable to marine bacteria. Vibrio anguillarum was a highly infectious pathogen that have recently been found to infect A. fangsiao and inhibit its growth and development. There were significant differences in the immune response mechanisms between egg-protected and egg-unprotected larvae. To explore larval immunity under different egg-protecting behaviors, we infected A. fangsiao larvae with V. anguillarum for 24 h and analyzed the transcriptome data about egg-protected and egg-unprotected larvae infected with 0, 4, 12, and 24 h using weighted gene co-expression networks (WGCNA) and protein-protein interaction (PPI) networks. Network analyses revealed a series of immune response processes after infection, and identified six key modules and multiple immune-related hub genes. Meanwhile, we found that ZNF family, such as ZNF32, ZNF160, ZNF271, ZNF479, and ZNF493 might play significant roles in A. fangsiao immune response processes. We first creatively combined WGCNA and PPI network analysis to deeply explore the immune response mechanisms of A. fangsiao larvae with different egg-protecting behaviors. Our results provided further insights into the immunity of V. anguillarum infected invertebrates, and laid the foundation for exploring the immune differences among cephalopods with different egg protecting behaviors.

Differences of Sucker Formation Processes Depending on Benthic or Pelagic Posthatching Lifestyles in Two Octopus Species.

AbstractMorphologies of animal appendages are highly diversified depending on animal lifestyles. In cephalopods (Mollusca, Cephalopoda), an individual possesses multiple arms that contribute to elaborate behaviors, and suckers on them enable various arm functions. In octopus hatchlings, arm and sucker morphologies can be divided into two different types due to alternative posthatching lifestyles, that is, pelagic or benthic lifestyles, although the underlying developmental differences have yet to be elucidated. In this study, therefore, detailed developmental processes of arms and suckers were observed during embryogenesis in two different octopus species, Octopus parvus and Amphioctopus fangsiao, showing pelagic and benthic posthatching lifestyles, respectively. In O. parvus, sucker formation stopped at a relatively early stage in which three suckers on an arm were produced. In addition, at late embryonic stages, cell proliferation was hardly detected in whole arms, while in A. fangsiao, sucker production continued throughout embryogenesis and cell proliferation also remained active in whole arms even in the late stages. Therefore, although further investigations in other octopus species are required, it is suggested that in octopus evolution, the developmental program of suckers has been modified in accordance with the acquisition of a novel lifestyle.

The morphological characteristics and modification of the oviducal glands adapted for long-term sperm storage and fertilization in Amphioctopus fangsiao

Sperm storage is an important reproductive strategy in Amphioctopus fangsiao. Sperm can be stored in the oviducal glands up to eight months before fertilization. However, studies on the underlying storage mechanisms remain limited. In this study, the morphological structures of the oviductal glands and the changes adapted for sperm storage and fertilization were investigated by Hematoxylin eosin (HE) staining, transmission electron microscopy (TEM), and Alcian blue periodic acid Schiff (AB-PAS) staining. Results revealed that the nucleo-cytoplasmic ratio of the peripheral gland cells decreased and the eosinophilic secretory granules (glycoproteins) appeared in the cytoplasm and lumen during sperm storage. The cytoplasm of the central gland appeared vacuolated and AB pH 2.5 (+) secretions (acid mucins) were dispersed in the cells. These secretions may pass through the central cavity into the oviducts and be involved in zygote packaging. The spermathecae are composed of columnar epithelial ciliated cells with secretory functions. PAS (+) glycoproteins were found in the lumen, providing a microenvironment conducive to sperm survival. Cilia in the oviducts, central cavity, and spermathecae may help with sperm delivery, storage, and release, and egg transport. This study enhances our understanding of the biological reproduction process in A. fangsiao.

Sequencing-based network analysis provides a core set of genes for understanding hemolymph immune response mechanisms against Poly I:C stimulation in Amphioctopus fangsiao

Aquatic viruses can spread rapidly and widely in seawater for their high infective ability. Polyinosinic-polycytidylic acid (Poly I:C), a viral dsRNA analog, is an immunostimulant that has been proved to activate various immune responses of immune cells in invertebrate. Hemolymph is a critical site that host immune response in invertebrates, and its transcriptome information obtained from Amphioctopus fangsiao stimulated by Poly I:C is crucial for understanding the antiviral molecular mechanisms of this species. In this study, we analyzed gene expression data in A. fangsiao hemolymph tissue within 24 h under Poly I:C stimulation and found 1082 and 299 differentially expressed genes (DEGs) at 6 and 24 h, respectively. Union set (1,369) DEGs were selected for subsequent analyses. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment analyses were carried out for identifying DEGs related to immunity. Several significant immune-related terms and pathways, such as toll-like receptor signaling pathways term, inflammatory response term, TNF signaling pathway, and chemokine signaling pathway were identified. A protein–protein interaction (PPI) network was constructed for examining the relationships among immune-related genes. Finally, 12 hub genes, including EGFR, ACTG1, MAP2K1, and other nine hub genes, were identified based on the KEGG enrichment analysis and PPI network. The quantitative RT-PCR (qRT-PCR) was used to verify the expression profile of 12 hub genes. This research provides a reference for solving the problem of high mortality of A. fangsiao and other mollusks and provides a reference for the future production of some disease-resistant A. fangsiao.

Effects of stocking density on juvenile Amphioctopus fangsiao (Mollusca: Cephalopodasca: Cephalopoda): Survival, growth, behavior, stress tolerance and biochemical response

Stocking density determines the welfare, growth performance, and even survival of aquatic organisms in aquaculture. However, how stocking density affects cephalopods are still unclear. Here we selected Amphioctopus fangsiao, a promising cephalopod for mariculture, to examine the effect of stocking density on survival, growth, physiology and behavior. Juvenile A. fangsiao (≈ two months post-hatch, 2.41 g ± 0.03) were reared for 21 days under different stocking density groups: 30, 45, 60 and 75 individual/m2 (ind./m2) (G1, G2, G3 and G4, respectively). Subsequently, the growth performance, aggressive acts, survival rate, stress response, oxidative stress and glucolipid metabolism were analyzed. This study showed that the high stocking density had a negative impact on growth performance. A. fangsiao reared in G3 and G4 had a significantly lower final body weight, weight gain and specific growth rate than those reared in G1 and G2 (p < .05), and hepatosomatic index in different treatment groups have a similar trend of difference (p < .05). Increased stocking density also caused more fights of A. fangsiao, resulting in a high mortality rate for farmed individuals. The individual numbers of released ink after touch and the recovery time of the breathing rate after air emersion showed that the low stocking density lowered the basal stress level and reduced the recovery time after acute stress of juvenile A. fangsiao. In addition, this study demonstrated that high stocking density has caused oxidative stress and lipid disturbance of A. fangsiao. There were significant differences in reactive oxygen species (ROS), lipid peroxidation's level (MDA content), the activities of superoxide dismutase (SOD) and catalase (CAT), pyruvic acid level (Pyr), total cholesterol level (T-CHO) and triglyceride (TG) between the highest density group (G4) and the lowest density group (G1). Overall, high stocking density (G3 and G4) had a negative effect on reared A. fangsiao. However, there were no significant differences between G1 and G2. Therefore, to improve the welfare of reared A. fangsiao and maximize the economic benefits for producers, juveniles are recommended to rear at a medium density (about 45 ind./m2).