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.

Effects of low temperature and highlight stress on lipid accumulation and cell structure of Tropidoneis maxima.

Tropidoneis maxima is a marine diatom with a rapid growth rate that produces high levels of lipids. To explore whether the lipid content could be further enhanced, cultures were first incubated under optimal conditions and then stressed under low temperature (10°C), a high light intensity level (80 µmol/m2 ·s), and the two factors together (interaction treatment). The results indicated that high light intensity and the temperature-light interaction exhibited greater impacts on lipid synthesis of T. maxima than low temperature. The two stress treatments increased lipid content by 17.16% and 16.6% compared to the control. In particular, higher biomass concentration was obtained with high light intensity (1.082 g L-1 ) and low temperature (1.026 g L-1 ). Moreover, high light intensity (9.06%) and interaction (10.3%) treatments yielded lower starch content compared to low temperature (14.27%) at the end of the stress culture. After 3 days of stress culture, the high light intensity treatment resulted in a 97.01% increase in cell wall thickness and an 18.46% decrease in cell diameter. The results suggest that high light intensity stress on T. maxima would open a new approach to cost-effective biolipid production.

Embryonic exposure to environmentally relevant levels of tributyltin affects embryonic tributyltin bioaccumulation and the physiological responses of juveniles in cuttlefish (Sepia pharaonis).

Tributyltin (TBT) is a typical organic pollutant that persists in aquatic sediments due to its wide usage as an antifouling fungicide during the past few decades. Despite increased awareness of the serious negative consequences of TBT on aquatic species, studies on the effects of TBT exposure on cephalopod embryonic development and juvenile physiological performance are scarce. To investigate the lasting effects of TBT toxicity on Sepia pharaonis from embryo to hatchling, embryos (gastrula stage, 3-5 h post fertilization) were exposed to four levels of TBT until hatching: 0 (control), 30 (environmental level), 60, and 120 ng/L. Subsequently, juvenile growth performance endpoints and behavioral alterations were assessed over 15 days post-hatching. Egg hatchability was significantly reduced and embryonic development (i.e., premature hatching) was accelerated in response to 30 ng/L TBT exposure. Meanwhile, TBT-induced alterations in embryonic morphology primarily included yolk-sac lysis, embryonic malformations, and uneven pigment distributions. During the pre-middle stage of embryonic development, the eggshell serves as an effective barrier to safeguard the embryo from exposure to 30-60 ng/L TBT, according to patterns of TBT accumulation and distribution in the egg compartment. However, even environmental relevant levels of TBT (30 ng/L) exposure during embryonic development had a negative impact on juvenile behavior and growth, including slowing growth, shortening eating times, causing more irregular movements, and increasing inking times. These findings indicate that after TBT exposure, negative long-lasting effects on S. pharaonis development from embryo to hatchling persist, suggesting that long-lasting toxic effects endure from S. pharaonis embryos to hatchlings.

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.

Combined metabolomics and histological analysis of the tissues from cuttlefish Sepia pharaonis exposed to inking stress.

Inking is part of a defensive stress response in cephalopods (cuttlefish, squid, and octopus). Some individual cuttlefish (Sepia pharaonis) die after continued stress and inking; however, the physiological effects of cephalopods in response to stress and inking remain unknown. The present study investigated the metabolic profile and discussed the physiological roles of S. pharaonis tissues in response to continuous inking using the 1H NMR spectroscopy coupled with multivariate data analysis. A total of 50 metabolites, including amino acids, organic osmolytes, nucleotides, energy storage compounds, and obvious tissue-specific metabolites induced by inking stress, were identified in S. pharaonis tissues. Exposure to inking stress had different effects on the levels of the studied metabolites, for example, the levels of isoleucine, trimethylamine-N-oxide, and betaine increased, but those of arginine and ATP decreased in the liver; inosine and lactate were accumulated whereas glutamate and choline were depleted in the gill; the levels of lactate and isoleucine were elevated but those of arginine and glycogen were depleted in the muscle tissue. Furthermore, the corresponding metabolic pathways of the characteristic metabolites indicated major changes in the functions of these metabolites. Histological changes in the studied tissues revealed liver lobule damage immediately after inking, with the presence of disordered epithelial cells and partial cell necrosis in the gill. Our results demonstrated that a combination of metabolomics and histological analyses could provide molecular-level insights for elucidating the defense response of cuttlefish against predators.

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.

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.

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 temperature on the embryonic development and larval growth of Sepia lycidas].

A single-factor experiment was conducted to study the effects of different temperature (15, 18, 21, 24, 27, 30, and 33 degrees C) on the embryonic development and larval growth of Sepia lycidas, aimed to search for the optimum temperature for the development and growth of S. lycidas. The results showed that temperature had significant effects on the embryonic development and larval growth of S. lycidas (P < 0.05). The suitable temperature for hatching ranged from 21 degrees C to 30 degrees C, and the optimum temperature was 24 degrees C. At the optimum temperature, the hatching rate was (93.3 +/- 2.9)%, incubation period was (24.33 +/- 0.58) d, hatching period was (6.00 +/- 1.00) d, completely absorked rate of yolk sac was (96.4 +/- 3.1)%, and newly hatched larvae mass was (0.258 +/- 0.007) g. The effective accumulated temperature model was N = 284.42/(T-12.57). The suitable temperature for the larval survival and growth ranged from 21 degrees C to 30 degrees C, and the optimum temperature was from 24 degrees C to 27 degrees C. At the optimum temperature, the survival rate ranged from 70.0% to 73.3%, and the specific growth rate was from 2.4% to 3.8%.