[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.

[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.

Metabolic responses and arginine kinase expression of juvenile cuttlefish (Sepia pharaonis) under salinity stress.

The pharaoh cuttlefish Sepia pharaonis is particularly sensitive to environmental changes in its breeding environment. The breeding of S. pharaonis larvae was carried out in different salinities for 48h, and the changes in survival rate, histological structure, energy metabolism, and anti-oxidative stress parameters were investigated and correlated with arginine kinase (AK) expression changes in muscle and liver tissues. The suitable salinity for larvae cultivation ranged from 24 to 30‰, and the survival rate showed a significant decline at 21‰ salinity. Histological observations of muscle and liver showed that changes in salinity and osmotic pressure had an adverse effect on tissue structure. Measurements of glycogen and lactic acid levels suggested that S. pharaonis could dynamically adjust energy metabolism to provide additional energy under unsuitable salinity. The protein levels and enzyme activities of AK in muscle significantly increased at 21‰ salinity. The results were consistent with prompt replenishment of phosphoarginine stores during salinity stress to maintain a dynamic ATP balance, suggesting that AK plays an important role in the regulation of energy metabolism. This study provides insight into metabolic changes during salinity stress and sheds light on the functional role of AK in S. pharaonis.

Kinetics for Cu(2+) induced Sepia pharaonis arginine kinase inactivation and aggregation.

Arginine kinase plays an important role in cellular energy metabolism and is closely related to the environmental stress response in marine invertebrates. We studied the Cu(2+)-mediated inhibition and aggregation of Sepia pharaonis arginine kinase (SPAK) and found that Cu(2+) markedly inhibited the SPAK activity along with mixed-type inhibition against the arginine substrate and noncompetitive inhibition against the ATP cofactor. Spectrofluorimetry results showed that Cu(2+) induced a tertiary structure change in SPAK, resulting in exposure of the hydrophobic surface and increased aggregation. Cu(2+)-mediated SPAK aggregation followed first-order kinetics consistent with monophasic and a biphasic processes. Addition of osmolytes, including glycine and proline, effectively blocked SPAK aggregation and restored SPAK activity. Our results demonstrated the effects of Cu(2+) on SPAK catalytic function, conformation, and aggregation, as well as the protective effects of osmolytes on SPAK folding. This study provided important insights into the role of Cu(2+) as a negative effector of the S. pharaonis metabolic enzyme AK and the possible responses of cephalopods to unfavorable environmental conditions.

Kinetics for Zinc Ion Induced Sepia Pharaonis Arginine Kinase Inactivation and Aggregation.

Arginine kinase is an essential enzyme which is closely related to energy metabolism in marine invertebrates. Arginine kinase provides a significant role in quick response to environmental change and stress. In this study, we simulated a tertiary structure of Sepia pharaonis arginine kinase (SPAK) based on the gene sequence and conducted the molecular dynamics simulations between SPAK and Zn(2+). Using these results, the Zn(2+) binding sites were predicted and the initial effect of Zn(2+) on the SPAK structure was elucidated. Subsequently, the experimental kinetic results were compared with the simulation results. Zn(2+) markedly inhibited the activity of SPAK in a manner of non-competitive inhibitions for both arginine and ATP. We also found that Zn(2+) binding to SPAK resulted in tertiary conformational change accompanying with the hydrophobic residues exposure. These changes caused SPAK aggregation directly. We screened two protectants, glycine and proline, which effectively prevented SPAK aggregation and recovered the structure and activity. Overall, our study suggested the inhibitory effect of Zn(2+) on SPAK and Zn(2+) can trigger SPAK aggregation after exposing large extent of hydrophobic surface. The protective effects of glycine and proline against Zn(2+) on SPAK folding were also demonstrated.

Identification and analysis of an intracellular Cu/Zn superoxide dismutase from Sepiella maindroni under stress of Vibrio harveyi and Cd2+.

Superoxide dismutases (SODs) are ubiquitous family of metalloenzymes involved in protecting organisms from excess reactive oxygen species damage. In this paper, a novel intracellular Cu/ZnSOD from Sepiella maindroni (designated as SmSOD) was identified and characterized. The full-length cDNA sequence of SmSOD (GenBank accession No. KF908850) was 709 bp containing an open reading frame (ORF) of 459 bp, encoding 153 amino acid residues peptide with predicted pI/MW (6.02/15.75 kDa), a 131 bp-5'- and 116 bp-3'- untranslated region (UTR). BLASTn analysis and phylogenetic relationship strongly suggested that the sequence shared high similarity with known Cu/Zn SODs. Several highly conserved motifs, including two typical Cu/Zn SOD family domains, two conserved Cu-/Zn-binding sites (H-47, H-49, H-64, H-120 for Cu binding, and H-64, H-72, H-81, D-84 for Zn binding) and intracellular disulfide bond (C-58 and C-146), were also identified in SmSOD. Time-dependent mRNA expression of SmSOD in hepatopancreas was recorded by quantitative real-time RT-PCR after Vibrio harveyi injection and Cd(2+) exposure. The results indicated that SmSOD was an acute-phase protein involved in the immune responses against pathogens and biological indicator for metal contaminants in aquatic environment.

An autoinhibitory helix in the C-terminal region of phospholipase C-ß mediates Gαq activation.

The enzyme phospholipase C-ß (PLCß) is a crucial regulator of intracellular calcium levels whose activity is controlled by heptahelical receptors that couple to members of the Gq family of heterotrimeric G proteins. We have determined atomic structures of two invertebrate homologs of PLCß (PLC21) from cephalopod retina and identified a helix from the C-terminal regulatory region that interacts with a conserved surface of the catalytic core of the enzyme. Mutations designed to disrupt the analogous interaction in human PLCß3 considerably increase basal activity and diminish stimulation by Gαq. Gαq binding requires displacement of the autoinhibitory helix from the catalytic core, thus providing an allosteric mechanism for activation of PLCß.

Elevated seawater PCO2 differentially affects branchial acid-base transporters over the course of development in the cephalopod Sepia officinalis.

The specific transporters involved in maintenance of blood pH homeostasis in cephalopod molluscs have not been identified to date. Using in situ hybridization and immunohistochemical methods, we demonstrate that Na(+)/K(+)-ATPase (soNKA), a V-type H(+)-ATPase (soV-HA), and Na(+)/HCO(3)(-) cotransporter (soNBC) are colocalized in NKA-rich cells in the gills of Sepia officinalis. mRNA expression patterns of these transporters and selected metabolic genes were examined in response to moderately elevated seawater Pco(2) (0.16 and 0.35 kPa) over a time course of 6 wk in different ontogenetic stages. The applied CO(2) concentrations are relevant for ocean acidification scenarios projected for the coming decades. We determined strong expression changes in late-stage embryos and hatchlings, with one to three log2-fold reductions in soNKA, soNBCe, socCAII, and COX. In contrast, no hypercapnia-induced changes in mRNA expression were observed in juveniles during both short- and long-term exposure. However, a transiently increased ion regulatory demand was evident during the initial acclimation reaction to elevated seawater Pco(2). Gill Na(+)/K(+)-ATPase activity and protein concentration were increased by ~15% during short (2-11 days) but not long-term (42-days) exposure. Our findings support the hypothesis that the energy budget of adult cephalopods is not significantly compromised during long-term exposure to moderate environmental hypercapnia. However, the downregulation of ion regulatory and metabolic genes in late-stage embryos, taken together with a significant reduction in somatic growth, indicates that cephalopod early life stages are challenged by elevated seawater Pco(2).

Evidence of early nervous differentiation and early catecholaminergic sensory system during Sepia officinalis embryogenesis.

Within Mollusca, cephalopods exhibit a particularly complex nervous system. The adult brain is formed from the fusion of several "typical" molluscan ganglia but it remains poorly understood how these ganglia emerge, migrate, and differentiate during embryogenesis. We studied the development of both central and peripheral nervous system by antibodies raised against alpha-tubulin and tyrosine hydroxylase (TH) in Sepia officinalis embryos to visualize neurites and catecholamine-containing neurons, respectively. In early embryos, when organs start delineating, some ganglia already exhibited a significant fiber network. TH-like immunoreactivity was detected in these fibers and in some primary sensory neurons in the embryo periphery. These data attest to the occurrence of an early embryonic sensory nervous system, likely effective, transient in part, and in relation to the perception of external cues. Concerning the peripheral nervous network, the stellate ganglia emerged as a plexus of numerous converging axons from TH-like immunoreactive sensory cells, first at the mantle edge, and then in the whole mantle surface. Later, TH-immunopositive motor fibers, originating from the stellate ganglia, penetrated the circular muscles of the mantle. These patterns reveal the setup of a mantle midline with likely attractive and repulsive properties. Our findings seem to challenge the widespread, still accepted, view of a late differentiation of cephalopod ganglia, and provides significant data for further investigations about axonal guidance during cephalopod development.

Phenoloxidase activation in the embryo of the common cuttlefish Sepia officinalis and responses to the Ag and Cu exposure.

The prophenoloxidase (proPO) system catalyzing the melanin production is considered as implicated in the innate immune system in invertebrates. The phenoloxidase (PO)-like activity was detected in the cuttlefish embryo sampled at the end of the organogenesis and few hours before hatching. Various modulators of the PO activity were used to assess the triggering of the proPO activating system. The results demonstrated the evidence of a true PO activity in the cuttlefish embryo. However, SDS and LPS granted contrasting effects on the PO-like activity between the developmental stages suggesting a progressive maturation of the proPO system from the embryonic to the juvenile stages. In eggs exposed to dissolved trace metals all along the embryonic development, Ag (1.2 microg L(-1)) inhibited the PO-like activity in the cuttlefish embryo except at hatching time, suggesting the synthesis of a new "juvenile" form of the PO enzyme. In similar conditions as for Ag, Cu (230 microg L(-1)) stimulated and then inhibited the PO-like activity according to a progressive metal accumulation within the egg and suggesting the occurrence of a threshold, above which the toxicity of the essential metal reduced the PO activity.

Comparative expression and tissue distribution analyses of astacin-like squid metalloprotease in squid and cuttlefish.

Astacin-like squid metalloprotease (ALSM) is a member of the astacin family of metalloproteases. In the present study, we investigated the expression and tissue distribution of ALSM in bigfin reef squid (Sepioteuthis lessoniana) and golden cuttlefish (Sepia esculenta). Myosin heavy chain hydrolysis tests showed ALSM-I-like activity in both species. We isolated partial cDNA clones showing high sequence similarity to ALSM-I and -III, suggesting that ALSM is common to squid and cuttlefish. Phylogenetic analysis showed that ALSMs are classified into two clades: ALSM-I forms one clade, and ALSM-II and -III form the other. ALSM was expressed in several tissues in bigfin reef squid, though expression was confined to the liver in cuttlefish. ALSMs are distributed in digestive organs but not in mantle muscle of squid and cuttlefish. Immunofluorescence analysis further showed that cellular localization of ALSM is evident not only in hepatic cells but also in pancreatic cells of bigfin reef squid. Thus, ALSM is commonly expressed in squid and cuttlefish, but its expression levels and distribution are distinct.

Location of intrinsic and inducible phenoloxidase activity in molluscan hemocyanin.

The phenoloxidase (PO) activity of the hemocyanins (Hcs) from two molluscan species, the gastropod Helix pomatia (Hp) and the cephalopod Sepia officinalis (So), was studied. With catechol as substrate the Hcs showed a weak o-diPO activity, which was moderately enhanced on limited proteolysis with subtilisin. The sites in the Hc molecules mainly responsible for this activity were identified. The highest intrinsic o-diPO activity and also by far the highest level of induction were found in the functional units (FUs) Hp f and So g, isolated from Hp beta-Hc and So Hc (subunit 2), respectively. The results thus support the earlier conclusion, made on the basis of sequence homology between molluscan Hcs, that Hp f and So g are functional and structural analogues. The subtilisin treatment of Hp f also induced monoPO activity, considered to be at the origin of browning of the sample.

Nitric oxide synthase in the nervous system and ink gland of the cuttlefish Sepia officinalis: molecular cloning and expression.

Nitric oxide (NO) signaling is involved in numerous physiological processes in mollusks, e.g., learning and memory, feeding behavior, neural development, and defence response. We report the first molecular cloning of NOS mRNA from a cephalopod, the cuttlefish Sepia officinalis (SoNOS). SoNOS was cloned using a strategy that involves hybridization of degenerate PCR primers to highly conserved NOS regions, combined with RACE procedure. Two splicing variants of SoNOS, differing by 18 nucleotides, were found in the nervous system and the ink gland of Sepia. In situ hybridization shows that SoNOS is expressed in the immature and mature cells of the ink gland and in the regions of the nervous system that are related to the ink defence system.