Comparative transcriptomic analysis of the different developmental stages of ovary in the cuttlefish Sepia pharaonis.

The cuttlefish, Sepia pharaonis, is characterized by rapid growth and strong disease resistance, making it an important commercially farmed cephalopod species in the southeastern coastal regions of China. However, in the reproductive process of S. pharaonis, there are challenges such as a low output of eggs, poor quality, and low survival rates of newly hatched juveniles. Therefore, there is an urgent need to study the molecular mechanisms underlying ovarian development in this species. In this study, we conducted the first transcriptomic analysis of the ovary at four developmental stages: the undeveloped stage, developing stage, nearly-ripe stage, and ripe stage, and compared the transcriptomics among these four stages using Illumina sequencing technology. The total numbers of clean reads of the four stages ranged from 40,890,772 to 52,055,714 reads. A total of 136,829 DEGs were obtained, GC base ratios of raw data were between 38.44 and 44.59%, and the number of uniquely mapped reads spanned from 88.08 to 95.90%. The Pearson correlation coefficient demonstrated a strong correlation among different samples within the same group, PCA and Anosim analysis also revealed that the grouping of these four stages was feasible, and each stage could be distinguished from the others. GO enrichment analysis demonstrated that ovarian follicle growth, sex differentiation, and transforming growth factor beta receptor, played a foreshadowing role at the early ovarian development stage, and the terms of small molecule metabolic process, peptide metabolic process, and catalytic activity were prominent at the mature stage. Meanwhile, KEGG analysis showed that the early ovarian development of S. pharaonis was mainly associated with the cell cycle, DNA replication, and carbon metabolism, while the mid-late ovarian development was involved with the signal transduction, endocrine system, and reproduction pathway. RT-qPCR further confirmed the consistent expression patterns of genes such as 17ß-HSD, GH, VGS, NFR, and NYR in the ovaries of S. pharaonis, exhibiting elevated levels of expression during the maturation stage. Conversely, ER and OM exhibited high expression levels during the early stages of ovarian development. These transcriptomic data provide insights into the molecular mechanisms of S. pharaonis ovarian development. The findings of this study will contribute to improving the reproduction and development of cuttlefish and enriching the bioinformatics knowledge of cephalopods.

Comparison of Cd, Cu, Se, and Zn Concentration in the Muscle and Hepatopancreas of Sepia pharaonis and Uroteuthis duvauceli in the North of Persian Gulf (Iran).

The objective of this research was to examine and contrast the levels of cadmium (Cd), copper (Cu), zinc (Zn), and selenium (Se) in the muscle and hepatopancreas tissues of two species, namely pharaoh cuttlefish (Sepia pharaonis) and Indian squid (Uroteuthis duvauceli), from the Persian Gulf. A total of thirty individuals of each species were gathered in January 2009 from the northern waters of the Persian Gulf. The metal concentrations were significantly higher in muscle tissue (p < 0.05) than in other tissues. S. pharaonis had higher metal concentrations than U. duvauceli. In the muscle and hepatopancreas samples of S. pharaonis, the highest mean concentrations were found to be for Zn (58.45 ± 0.96 µg/g dw) and Cu (1541.47 ± 192.15 µg/g dw), respectively. In U. duvauceli, the highest concentration of measured elements was seen for Zn in both muscle (36.52 ± 0.56 µg/g dw) and hepatopancreas (60.94 ± 2.65 µg/g dw). Se had the lowest concentration among the elements measured in both species. There was a negative and significant correlation between Cu and biometrical factors (total body length and weight) in both muscle and hepatopancreas samples of S. pharaonic and only in the muscle samples of U. duvauceli (p < 0.01, R2 = - 052; p < 0.01, R2 = - 0.055). However, there was a strong correlation between Zn and biometrical factors in hepatopancreas samples of both species. The comparison of metal concentrations with standards revealed that only Cd levels in S. pharaonis exceeded the ESFA and WHO standards, whereas other metals were below the standards.

Comparative proteomic analysis of the protein profile in the cuttlefish Sepia pharaonis associated with skin ulceration syndrome.

Skin ulceration syndrome (SUS) is becoming a severe problem in the breeding and culturing process of the cuttlefish Sepia pharaonis. However, limited knowledge is available about the occurrence of this devastating disease. In this study, proteomic analysis was used to identify the differentially expressed proteins (DEPs) and the biological pathways enriched in SUS-diseased S. pharaonis. Both the healthy group and diseased group were analyzed in triplicate, with 4 cuttlefish in each replicate. The results showed that 85 DEPs were identified between the two groups, including 36 upregulated proteins and 49 downregulated proteins in the diseased group compared to the healthy group. GO enrichment analysis revealed that the DEPs were mainly enriched in cellular component organization or biogenesis, nucleus and ion binding processes. The results of the KEGG pathway analysis indicated that extracellular matrix (ECM)-receptor interaction was the most enriched upregulated pathway. Real-time reverse transcriptase PCR was used to identify the expression of two differentially expressed matrix metalloproteinases (MMPs), and the results showed that the mRNA expression of MMP14 and MMP19 was significantly upregulated in the skin tissue of the diseased group. Furthermore, the protease activity of the diseased group was higher than that of the healthy group. Our results offer basic knowledge on the changes in protein profiles during the occurrence of SUS in the cuttlefish S. pharaonis.

Newly described antioxidant disecolactonic ergosteroids from marine cuttlefish Sepia pharaonis: Pharaonoids A-B as prospective carbohydrate digestive enzyme inhibitors.

Biochemical investigation of crude solvent extract of pharaoh cuttlefish Sepia pharaonis (family Sepiidae) led to the isolation of two undescribed disecolactonic ergosteroids, pharaonoids A-B. The compounds were characterized as 11ß-acteoxy-7α-hydroxy-19-Nor-1,10:9,10-disecoergosta-3-ene-61-oxa-1-one (pharaonoid A) and 11ß-hydroxy-19-Nor-1,10:9,10-disecoergosta-3-ene-61-oxa-1-one (pharaonoid B) in conjunction with spectroscopic analysis encompassing one and two-dimensional nuclear magnetic resonance and mass spectrometric analyses. Pharaonoid A, bearing an acetoxy and hydroxyl groups, respectively at C-11 and C-7 positions exhibited considerably greater inhibition potential against carbohydrate hydrolysing enzymes α-amylase (IC50 1.14 mM) and α-glucosidase (IC50 1.23 mM) than those displayed by pharaonoid B (IC50 1.49/1.38 mM), and was proportionate with those exhibited by standard drug acarbose (IC50 0.60 and 0.40 mM, respectively), thereby recognizing the anti-hyperglycemic potential of pharaonoid A. Promising anti-oxidant property for pharaonoid A (IC50 âˆ¼ 1 mM) could conceivably corroborate its attenuation potential against carbohydrate digestive enzymes. Greater electronic parameters along with optimum lipophilic-hydrophobic balance of pharaonoid A were directly corroborated to the anti-carbolytic properties occurring via transcellular mechanism. Greater binding energies (-9.50 kcal mol-1) and inhibition constant (Ki 48.21 nM) at the active site of α-amylase enzyme were displayed by pharaonoid A than those exhibited by its B analogue. Promising bioactive properties of the disecolactonic steroids isolated from the marine pharaoh cuttlefish are anticipated to be utilized as functional food components and potential nutraceuticals against oxidative stress and hyperglycemic disorders.

Effects of ammonia toxicity on the histopathology, detoxification, oxidative stress, and immune response of the cuttlefish Sepia pharaonis and the mitigation of γ-aminobutyric acid.

In this study, γ-aminobutyric acid (GABA) was examined as an additional supplement to improve the ammonia stress resistance of S. pharaonis. Specifically, we added different doses of GABA (0, 20, 40, 60, 80, and 100 mg/kg) to food, cultivated S. pharaonis in regular seawater for 8 weeks and then in 8.40 mg/L ammonia seawater for 48 h and then investigated the accumulation of ammonia (the hepatic ammonia content), ammonia detoxification process (the urea content), antioxidant enzymes (superoxide dismutase (SOD) and catalase (CAT) enzyme activities), immune response (the serum haemolytic complement (C3) and lysozyme (LYZ) contents), membrane lipid peroxidation (malondialdehyde (MDA)) and histopathology of the liver. The results showed that ammonia poisoning could induce ammonia and MDA accumulation and subsequently lead to oxidative stress (decreases in SOD and CAT activities), immunosuppression (reductions in the haemolytic C3 and LYZ content), and histopathological injury in the liver. The application of GABA had a significant effect on alleviating the adverse effect of ammonia poisoning, and 80-100 mg/kg treatment exerted the best effect. This treatment significantly reduced the ammonia and MDA contents, significantly increased the urea content, increased the SOD, CAT, C3 and LYZ activities, reduced the MDA content, suppressed membrane lipid peroxidation, and significantly improved the histopathological injury to the liver. In summary, the results could provide a new method for mitigating liver damage, alleviating the physiological and metabolic disorders caused by ammonia stress in cuttlefish, and provide a theoretical basis for the application of GABA in alleviating ammonia poisoning.

Marine cuttlefish derived 2H-benzochromenone: Pharachromenone as a dual inhibitor of pro-inflammatory 5-lipoxygenase and cyclooxygenase-2.

Cephalopod cuttlefish, Sepia pharaonis, has been considered as a commercially important resource, which is widely regarded as nutritious food in the southwest of Indian and Mediterranean coasts. Chemical analysis of the crude extract of S. pharaonis resulted in the isolation of an undescribed 2H-benzochromenone, pharachromenone, which was characterized as methyl-2″-(7-hydroxy-4-(5'-methylpent-5'-en-1'-yl-oxy-methyl)-2-oxo-2H-benzo[h]chromen-5-yl-methyl)-butanoate by mass and nuclear magnetic resonance spectral experiments. Pharachromenone revealed effective biopotency against 5-lipoxygenase (IC50 1.85 mM) and cyclooxygenase-2 (IC50 0.52 mM) than that displayed by nonsteroidal anti-inflammatory drug ibuprofen (IC50 4.36 mM, p < .05). Promising antioxidant property for pharachromenone (IC50 1.42-1.61 mM) compared with those exhibited by antioxidative agents butylated hydroxyl anisole (BHA) and α-tocopherol (IC50 1.40-1.90 mM) could conceivably validate its dual inhibition potential against 5-lipoxygenase and cyclooxygenase-2. Greater electronic parameters, lesser steric bulkiness, along with acceptable lipophilic-hydrophobic balance significantly contributed toward its promising anti-inflammatory activities. Molecular docking studies showing significantly greater inhibition constant (Ki) 8.24 nM and binding energy (-11.03 kcal/mol) of pharachromenone than the standard ibuprofen (Ki 4.65 µM, binding energy -7.27 kcal/mol) at the binding site of 5-lipoxygenase recognized its noncompetitive binding, which could describe the promising anti-inflammatory potential. Pharachromenone could be developed as a functional food component against oxidative stress-related inflammatory disorders. PRACTICAL APPLICATIONS: The cuttlefish Sepia pharaonis (family Sepiidae) comprises a major share in the global fishery sector due to its culinary delicacy and nutritionally valued high-quality meat. Furthermore, cephalopod mollusks are gaining pharmaceutical acceptance as resources to derive bioactive compounds with therapeutic significance. Bioassay-guided chromatographic fractionation of crude extract of S. pharaonis could result in the isolation of a 2H-benzochromenone derivative, pharachromenone exhibiting potent antioxidant and anti-inflammatory properties. This study recognized the therapeutic potential of a marine cuttlefish-originated food constituent against inflammatory conditions, and could be anticipated as a high-value functional food lead to minimize oxidative stress-related inflammatory disorders.

Antioxidant spiropharanone, an undescribed variant of trans-decalin spiro-γ-lactone, from pharaoh cuttlefish Sepia pharaonis: Twin inhibitors of inflammatory 5-lipoxygenase and serine protease dipeptidyl peptidase-4.

Marine pharaoh cuttlefish Sepia pharaonis (family Sepiidae) is regarded as an economically important class of cephalopod in the coastal Mediterranean and Asian regions. Bioassay-guided chromatographic purification of solvent extract of S. pharaonis led to the identification of a trans-decalin based spirolactone, spiropharanone, which was characterized as 1-hydroxy-7-(4'-methoxy-3-methylbut-2-enyl)-3,9,15-trimethyl-8-oxo-octahydro-5H-spiro[furan-8,9-naphtho]-8-yl-acetate by spectroscopic techniques. Spiropharanone exhibited significantly greater anti-inflammatory activity by attenuating pro-inflammatory 5-lipoxygenase (IC50 1.02 mM) than the non-steroidal drug ibuprofen (IC50 4.61 mM, p ≤ .05). Superior antioxidant properties of spiropharanone against free radicals (EC50 ~1.20 mM) and other oxidants (hydroxyl [EC50 0.97 mM] and superoxide [EC50 1.47 mM] scavenging) also reinforced its promising anti-inflammatory activity. The studied spiropharanone also exhibited significant attenuation toward insulin secretion regulating enzyme dipeptidyl peptidase-4 (IC50 0.92 mM) recognizing its anti-hyperglycemic potential. Significantly higher electronic properties (topological polar surface area ~100) combined with balanced hydrophilic-lipophilic properties (partition coefficient of logarithmic octanol-water ~3) and lesser docking parameters of spiropharanone demonstrated that the compound could be utilized as an important bioactive lead against oxidative stress, inflammation, and hyperglycemic-related ailments. PRACTICAL APPLICATIONS: Nutritionally rich edible marine pharaoh cuttlefish Sepia pharaonis occupies a prominent place among seafood fisheries owing to the presence of bioactive nutrients and functional food ingredients. These marine cuttlefish are widely distributed along the Asian and Mediterranean coasts, and consumed as culinary delicacy for decades. An undescribed trans-decalin spirolactone, spiropharanone was isolated from the organic extract of S. pharaonis based on bioactivity-assisted sequential chromatographic fractionation. Spiropharanone displayed promising antioxidant potential along with attenuation properties against inducible pro-inflammatory 5-lipoxygenase and insulin secretion regulating enzyme dipeptidyl peptidase-4. This study established the ameliorating potential of a naturally derived marine food constituent against inflammatory and diabetic ailments, and thus anticipated as functional food lead in pharmaceutical formulations towards inflammation and maintaining glucose homeostasis.

Visual Attack on the Moving Prey by Cuttlefish.

Visual attack for prey capture in cuttlefish involves three well characterized sequential stages: attention, positioning, and seizure. This visually guided behavior requires accurate sensorimotor integration of information on the target's direction and tentacular strike control. While the behavior of cuttlefish visual attack on a stationary prey has been described qualitatively, the kinematics of visual attack on a moving target has not been analyzed quantitatively. A servomotor system controlling the movement of a shrimp prey and a high resolution imaging system recording the behavior of the cuttlefish predator, together with the newly developed DeepLabCut image processing system, were used to examine the tactics used by cuttlefish during a visual attack on moving prey. The results showed that cuttlefish visually tracked a moving prey target using mainly body movement, and that they maintained a similar speed to that of the moving prey right before making their tentacular strike. When cuttlefish shot out their tentacles for prey capture, they were able to either predict the target location based on the prey's speed and compensate for the inherent sensorimotor delay or adjust the trajectory of their tentacular strike according to the prey's direction of movement in order to account for any changes in prey position. These observations suggest that cuttlefish use the various visual tactics available to them flexibly in order to capture moving prey, and that they are able to extract direction and speed information from moving prey in order to allow an accurate visual attack.

Transcriptome analysis of the Sepia pharaonis: Identification of low salinity stress-related information and microsatellite markers.

Sepia pharaonis has great commercial value for aquaculture. However, it is sensitive to salinity fluctuations and lacking in genomic information. The present work utilized high-throughput transcriptome sequencing to assess the effect of low salinity (22.0 ppt) on gills of S. pharaonis. 6153 genes were identified as differentially expressed (p < 0.05), of which 3340 were increased and 2813 were decreased in low salinity group (22.0 ppt) relative to the control group (29.0 ppt). Subsequently, these DEGs were allocated to 226 KEGG pathways and 491 GO terms. Analysis of the transcriptome sequences and DEGs identified several unigenes and pathways involved in salt stress regulation. Moreover, the S. pharaonis carried 101,576 simple sequence repeats (SSRs). This is the first time osmoregulation in S. pharaonis has been explored by transcriptome sequencing. The data presented here reveals key insights into the genetic markers of salt stress in S. pharaonis.

1H NMR-based metabolomic analysis of cuttlefish, Sepia pharaonis (Ehrenberg, 1831) exposed to hypoxia stresses and post-anoxia recovery.

Oxygen deficiency (hypoxia and anoxia) is an emerging concern in estuarine and coastal ecosystems worldwide. Previous studies on Mollusca Cephalopoda have focused on the effects of hypoxia stress on physiological performance and survival, but there are few reports on the molecular mechanism, and the application of metabolomics in cephalopods remains unknown. In this study, a 1H nuclear magnetic resonance (NMR) based metabolomics approach was applied to investigate the metabolites profiles of Sepia pharaonis (Ehrenberg, 1831) during hypoxia and post-anoxia recovery. The results revealed that obvious tissue-specific metabolic responses were induced by hypoxia stresses. Hypoxia exposure influenced the levels of many metabolites (e.g. BCAAs, lactate, and betaine strongly accumulated in the hepatic tissue while arginine and ATP significantly reduced; lactate and adenosine significantly increased in gills whereas arginine and choline significantly decreased; GABA, taurine and adenosine levels increased in brain but a significant depletion of N-Acetylaspartate and glycogen was found), disturbed energy and amino acid metabolism, and broke the balance of neurotransmitters and osmoregulators. Notably, almost all metabolites returned to pre-exposure levels after acute hypoxia recovery. However, we noted a pronounced depletion of the amino acid pool (arginine, glutamine, and alanine) in hepatic and gills after recovery, as well as organic osmolytes fluctuations (choline, betaine, and taurine). This work highlights the potential of metabolomics methods to elucidate the response of cuttlefish to hypoxia stress, as well as to provide knowledge on metabolic changes in cephalopods under the influences of environmental stress.

An FMRFamide Neuropeptide in Cuttlefish Sepia pharaonis: Identification, Characterization, and Potential Function.

Neuropeptides are released by neurons that are involved in a wide range of brain functions, such as food intake, metabolism, reproduction, and learning and memory. A full-length cDNA sequence of an FMRFamide gene isolated from the cuttlefish Sepia pharaonis (designated as SpFMRFamide) was cloned. The predicted precursor protein contains one putative signal peptide and four FMRFamide-related peptides. Multiple amino acid and nucleotide sequence alignments showed that it shares 97% similarity with the precursor FMRFamides of Sepiella japonica and Sepia officinalis and shares 93% and 92% similarity with the SpFMRFamide gene of the two cuttlefish species, respectively. Moreover, the phylogenetic analysis also suggested that SpFMRFamide and FMRFamides from S. japonica and S. officinalis belong to the same sub-branch. Tissue expression analysis confirmed that SpFMRFamide was widely distributed among tissues and predominantly expressed in the brain at the three development stages. The combined effects of SpFMRFamide+SpGnRH and SpFLRFamide+SpGnRH showed a marked decrease in the level of the total proteins released in the CHO-K1 cells. This is the first report of SpFMRFamide in S. pharaonis and the results may contribute to future studies of neuropeptide evolution or may prove useful for the development of aquaculture methods for this cuttlefish species.

Continuous Inking Affects the Biological and Biochemical Responses of Cuttlefish Sepia pharaonis.

Several marine mollusks, including cephalopods (cuttlefish, squid, and octopus) and gastropods (e.g., sea hares), can release a colored ink secretion when chased by predators or stimulated. Ink release is part of a defensive response, but the threshold for the biochemical responses caused by stimulation is unknown. The present study aimed to reveal antipredator responses of cuttlefish, such as escaping via inking and/or jetting, and to investigate its biological and biochemical responses to continuous ink release. Results showed that the behavioral responses to continuous ink release mainly manifested as blazing body pattern changes. Cuttlefish escaped from predators covered by jetting/inking and warned the potential threats by displaying a unique body pattern. Moreover, persistent inking in the presence of an overt stimulus caused uncontrollable ink release from the ink duct/anal canal (loss of control). This study first verified the characteristics of the cuttlefish ink solution, prepared a standard curve of ink solution concentrations, and fitted the relationship function between the release frequency and the released ink weight. Biological statistics indicated that cuttlefish has the ability to continuously release ink (releasing ∼90% of the ink from the ink sac) and that the individuals adapted well during the recovery period. However, re-releasing ink would result in "overexploitation" and high mortality. Hexokinase (HK), pyruvate kinase (PK), and superoxide dismutase (SOD) activities, as well as malondialdehyde (MDA) concentration increased or remained stable in different tissues after releasing ink. The expression of heat shock protein 90 and arginine kinase (AK) were upregulated by stimuli in all tissues. Biochemical changes indicated that continuous inking not only consumed considerable energy but also damaged the tissues. In summary, cuttlefish released almost 90% of their ink for active defense against predators, and it took ∼30 days for the ink sac to be refilled, but "overexploitation" resulted in serious physiological damage. These findings will be helpful to further study the defense and ink release mechanisms and to consider animal health and welfare when using cephalopods as experimental animals and for aquaculture practices.

[Effects of light intensity on growth, survival, metabolism and related enzyme activities of Sepia pharaonis.] / å ‰ç §å¼ºåº¦å¯¹è™Žæ–‘ä¹Œè´¼ç”Ÿé•¿ã€å­˜æ´»ã€ä»£è°¢åŠç›¸å ³é ¶æ´»æ€§çš„å½±å“.

An experiment with single-factor design was conducted to investigate the effects of light intensity on growth and survival of cuttlefish (Sepia pharaonis). The specific growth rate, survival rate, oxygen consumption rate, ammonia excretion rate, lactic acid content in muscle, respiratory metabolic enzymes (including hexokinase, pyruvate kinase, and lactate dehydrogenase), supero-xide dismutase, and malondialdehyde in liver were measured in five constant light intensity treatments (10, 30, 50, 70, 90 µmol·m-2·s-1). The main results were as follows: The specific growth rate and survival rate remained steady initially and then decreased gradually with the increases of light intensity. There was no significant difference between groups 10 and 30 µmol·m-2·s-1, but they were significantly higher than those of the other groups. Exposed to light intensities of 10 and 30 µmol·m-2·s-1, the specific growth rates were (8.43±0.22)%·d-1 and (8.47±0.17)%·d-1, and the survival rates were (79.2±5.9)% and (80.0±4.9)%, respectively. Oxygen consumption rates and ammonia excretion rates increased first slowly and then sharply, and reached the maximum value when light intensity was 90 µmol·m-2·s-1, which was significantly higher than those of the other groups. Lactic acid content in muscle firstly decreased and then increased, with the minimum value at 30 µmol·m-2·s-1. The acid content of 10 µmol·m-2·s-1 was significantly lower than those of the other groups except 30 and 50 µmol·m-2·s-1. With the increases of light intensity, the activities of HK and PK in gills remained steady initially and then decreased gradually, and reached the highest level when exposed to 10 and 30 µmol·m-2·s-1, which were significantly higher than those of the other groups. LDH activity in muscle had the lowest level at the light intensity of 10 and 30 µmol·m-2·s-1, which was significantly lower than those of the other groups. SOD activity in liver firstly increased and then decreased, and reached the highest level ((104.93±4.17) U·mg-1 pro) when exposed to 70 µmol·m-2·s-1, which was significantly higher than those of the other groups. MDA content in liver first remained steady and then increased gradually, and reached the highest level ((5.06±0.35) nmol·mg-1 pro) when exposed to 90 µmol·m-2·s-1, which was significantly higher than those of the other groups. In conclusion, the optimum light intensities for growth, survival and metabolism of S. pharaonis were 10 and 30 µmol·m-2·s-1, beyond which S. pharaonis would be under stress. Therefore, sunproof measures should be taken to keep weak light condition in culture practice.

A difference in timing for the onset of visual and chemosensory systems during embryonic development in two closely related cuttlefish species.

Embryos perceive environmental stimuli, thanks to their almost mature sensory systems. In cuttlefish, the embryonic development of Sepia officinalis and Sepia pharaonis is similar but the egg capsule transparency is different. S. officinalis' eggs are black (ink), which provide protection from predators. Conversely, those of S. pharaonis are translucent. The aim of this study was to test the visual and chemosensory perception abilities of these two cuttlefish embryos by observation of the ventilation rate (VR) before and after stimulation. Our results show that S. pharaonis responds to light at stage 22 and S. officinalis at stage 24. Conversely, S. pharaonis responds to predator odor at stage 23 and S. officinalis at stage 22. Both species are able to respond to these stimuli before hatching but do not have the same developmental schedule. Neither are the responses of the two cuttlefish exactly the same. In S. officinalis, VR increases after stimulations. In S. pharaonis, VR increases after light stimulation and decreases following the odor stimulation after stage 25. This result could reveal an ability to recognize stimuli at stage 25. The decrease could be identified as freezing-like behavior which would be more adaptive than an increase, since the embryos are visible.

Isolation and characterization of a virulence related Vibrio alginolyticus strain Wz11 pathogenic to cuttlefish, Sepia pharaonis.

Vibrio alginolyticus is a ubiquitous marine opportunistic pathogen that can infect various hosts in marine environment. In the present study, V. alginolyticus strain Wz11 was isolated from diseased cuttlefish, Sepia pharaonis, with 20% of promoted death and high survival capacity in skin mucus and tissue liquid. Its growth, siderophore production, and expressions of haemolysin and swarming related genes were characterized under iron limited conditions. The minimal inhibitory concentration (MIC) of 2,2'-dipyridyl (DP) to V. alginolyticus strain Wz11 was 640 µM. While growth of V. alginolyticus strain Wz11 was inhibited by DP, production of iron-seizing substances, haemolytic activity and swarming motility were increased. Moreover, expressions of haemolysin related genes tlh, tdh and vah and flagellar related genes flgH, fliC, fliD and fliS were also characterized using real-time reverse transcriptase PCR. Expression of tdh was up-regulated to 7.7-fold, while expressions of tlh and vah were down-regulated to 0.016-fold and 0.03-fold, respectively. The expression of fliC, flgH, fliD and fliS was up-regulated to 4.9-, 3.8-, 8.6- and 4.5-fold, respectively. Concluded from our results suggested that V. alginolyticus strain Wz11 was considered as a potential pathogen of S. pharaonis, and iron level played an important role in the production of iron-seizing substances, and activities of haemolysin and bacterial swarming as well as their related gene expressions.

[Effects of light intensity and photoperiod on the embryonic development of Sepia pharaonis.] / å ‰ç §å¼ºåº¦ä¸Žå ‰å‘¨æœŸå¯¹è™Žæ–‘ä¹Œè´¼èƒšèƒŽå‘è‚²çš„å½±å“.

We investigated the effects of lumination on hatching of fertilized eggs of Sepia pharaonis, to reveal the best light conditions for its embryonic development. A single-factor experiment was carried out to examine the effects of different light intensities (10, 30, 50, 70, 90 µmol·m-2·s-1) and different photoperiod L:D (24 h:0 h, 18 h:6 h, 12 h:12 h, 6 h:18 h, 0 h:24 h) on the embryonic development. The results showed that the effects of light intensity on the hatching rate, fractured yolk sac rate, incubation period, mass of newly hatched larvae and mantle length was significant. There was no significant effect on hatching period and survival rate after hatching 7 days. With the increases of light intensity, the hatching rate, incubation period, mass of newly hatched larvae and mantle length first increased and then decreased, while the fractured yolk sac rate gradually increased. The optimum light intensity was 30 µmol·m-2·s-1. Exposed to this light intensity, the hatching rate, fractured yolk sac rate, incubation period, hatching period, mass of newly hatched larvae, mantle length and survival rate after hatching 7 days were (90.0±4.1)%, (7.3±1.5)%, (25.50±0.35) d, (8.10±0.89) d, (0.213±0.011) g, (1.013±0.022) cm, (97.1±4.0)%, respectively. The effects of photoperiod on the hatching rate, incubation period, hatching period were significant, but there was no significant effect on fractured yolk sac rate, mass of newly hatched larvae, mantle length and survival rate after hatching 7 days. With the increases of illumination time, the hatching rate and hatching period first increased and then decreased. The optimum photoperiod was L:D (12 h:12 h). When exposed to this photoperiod environment, the hatching rate, fractured yolk sac rate, incubation period, hatching period, mass of newly hatched larvae, mantle length and survival rate after hatching 7 days were (88.7±1.8)%, (8.7±1.8)%, (25.00±0.50) d, (7.00±3.20) d, (0.209±0.005) g, (0.998±0.026) cm, (96.8±7.1)%, respectively. In conclusion, embryo hatchability of S. pharaonis preferred to low light intensity (30 µmol·m-2·s-1) and normal photoperiod L:D (12 h:12 h). In production practice, sunproof measures should be taken to keep the eggs in weak light condition.

Isolation and partial characterization of collagen from outer skin of Sepia pharaonis (Ehrenberg, 1831) from Puducherry coast.

Type I collagen from outer skin of Sepia pharaonis was extracted and partially characterized. Yield of Acid Soluble Collagen (ASC) and Pepsin Soluble Collagen (PSC) were calculated as 1.66% and 3.93% and the total protein content of ASC and PSC were found as 18.4% and 48.6%. FT-IR spectrum of ASC and PSC recorded 12 and 14 peaks, respectively. 1H NMR spectrum of ASC showed singlets at 1.23 ppm, 3.1 ppm, 3.55 ppm and 3.7 ppm and PSC at 1.23 ppm and 2.08 ppm. The molecular weight for ASC was calculated as 102 kDa and for PSC as 110, 108 and 102 kDa through SDS-PAGE. Differential Scanning Calorimetry (DSC) results supported that PSC withstand high thermal stability (82.85 °C) than ASC (73.13 °C). Higher denaturation temperature with high molecular weight well support the property of type I collagen from skin of S. pharaonis and it could be used as another potent source for the extraction of collagen.

First isolation of Miamiensis avidus (Ciliophora: Scuticociliatida) associated with skin ulcers from reared pharaoh cuttlefish Sepia pharaonis.

In the winter of 2015, a skin ulcer disease outbreak occurred in a pharaoh cuttlefish Sepia pharaonis population cultured on a land-based fish farm in China. Affected cuttlefish (about 60% of the population) were characterized as having developed ulcers on the dorsal skin, fin fringe, or distal mantle tip. Masses of a ciliated protozoan were isolated from skin ulcers. The ciliate was identified as Miamiensis avidus based on the morphological features of living and protargol-impregnated specimens. This identification was also supported by high sequence similarity of the small subunit ribosomal RNA gene (100%) and another ribosomal DNA region (including the 2 internal transcribed spacers and the 5.8S gene; 99%) with published sequences of fish parasitic M. avidus strains. M. avidus is known to be a histophagous marine fish parasite. This report describes the first case of M. avidus associated with skin ulcers in a cephalopod mollusk (Mollusca, Cephalopoda). This finding suggests that M. avidus may infect a phylogenetically broader range of hosts than what has previously been reported. Furthermore, M. avidus may pose a health risk to hatchery-reared cephalopods.

A transcriptome resource for pharaoh cuttlefish (Sepia pharaonis) after ink ejection by brief pressing.

Ink ejection is one of the most important defense mechanisms against external stimuli for pharaoh cuttlefish (Sepia pharaonis). The molecular changes during this process remain unknown. To understand the transcriptome changes after ink ejection by brief pressing, two cDNA libraries of pharaoh cuttlefish, from the inkjet group and control group were sequenced using Illumina HiSeq™ 2000. A total of 9,255,502,440nt bases were obtained and by de novo assembly, 73,298 unigenes were generated, which first provided numerous expressed sequence tags from pharaoh cuttlefish. By comparing the expression levels between the two groups, we identified 7064 up-regulated and 2024 down-regulated genes after ink ejection. These differentially-expressed genes included genes related to immunity, cancer, and blood coagulation, which indicated the various effects after ink ejection by brief pressing. These results provide new valuable resources for functional genomic and genetic studies on pharaoh cuttlefish.

[Oxygen consumption rate of Sepia pharaonis embryos.] / 虎斑乌贼的胚胎耗氧率.

This research was conducted to unravel the variation of oxygen consumption rate during different developmental stages and the effects of different ecological factors on embryonic oxygen consumption rate of Sepia pharaonis. The oxygen consumption rates were measured at twelve deve-lopmental stages by the sealed volumetric flasks, and four embryonic developmental periods (oosperm, gastrula, the formation of organization, endoskeleton) were selected under various ecological conditions, such as salinity (21, 24, 27, 30, 33), water temperature (18, 21, 24, 27, 30 ℃) and pH (7.0, 7.5, 8.0, 8.5, 9.0). The results showed that the oxygen consumption rate rose along with the developmental progress, and distinctly differed from each other. The oxygen consumption rate was 0.082 mg·(100 eggs)-1·h-1 during oosperm period, and rose to 0.279 mg·(100 eggs)-1·h-1 during gastrula period, which was significantly higher than that of blastula period. Finally, the oxygen consumption rate rose to 1.367 mg·(100 eggs)-1·h-1 during hatching period. The salinity showed a significant effect on oxygen consumption rate during the formation of organization and endoskeleton formation stage (P<0.05), but no significant effect during oosperm and gastrula periods (P>0.05). The oxygen consumption rates of four studied embryonic stages all rose and then declined along with the increase of salinity, and reached the highest values [0.082, 0.200, 0.768 and 1.301 mg·(100 eggs)-1·h-1, respectively] at salinity 30. The water temperature had a significant effect on the embryo oxygen consumption rates of gastrula, and the formation of organization and endoskeleton formation stage (P<0.05), with the exception of oosperm (P>0.05). The oxygen consumption rates of four studied embryonic stages all rose and then declined along with the increase of temperature, and reached the highest values at 27 ℃ [0.082, 0.286, 0.806 and 1.338 mg·(100 eggs)-1·h-1, respectively]. The pH had no significant effect on the oxygen consumption rates of four embryonic stages (P>0.05). The oxygen consumption rates of four studied embryonic stages all rose and then declined along with the increase of pH. The oxygen consumption rates of gastrula, the formation of organization, endoskeleton reached the according highest values [0.281, 0.799 and 1.130 mg·(100 eggs)-1·h-1] at pH 8.5, but that during oosperm period occurred at pH 8.0 [0.116 mg·(100 eggs)-1·h-1].