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.

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.

Characterisation, evolution and expression analysis of the interferon regulatory factor (IRF) family from olive flounder (Paralichthys olivaceus) in response to Edwardsiella tarda infection and temperature stress.

Interferon regulatory factor (IRF) family involves in the transcriptional regulation of type I Interferons (IFNs) and IFN-stimulated genes (ISGs) and plays a critical role in cytokine signaling and immune response. However, systematic identification of the IRF gene family in teleost has been rarely reported. In this study, twelve IRF members, named PoIRF1, PoIRF2, PoIRF3, PoIRF4a, PoIRF4b, PoIRF5, PoIRF6, PoIRF7, PoIRF8, PoIRF9, PoIRF10 and PoIRF11, were identified from genome-wide data of olive flounder (Paralichthys olivaceus). Phylogenetic analysis indicated that PoIRFs could be classified into four clades, including IRF1 subfamily (PoIRF1, PoIRF11), IRF3 subfamily (PoIRF3, PoIRF7), IRF4 subfamily (PoIRF4a, PoIRF8, PoIRF9, PoIRF10) and IRF5 subfamily (PoIRF5, PoIRF6). They were evolutionarily related to their counterparts in other fish. Gene structure and motif analysis showed that PoIRFs protein sequences were highly conserved. Under normal physiological conditions, all PoIRFs were generally expressed in multiple developmental stages and healthy tissues. After E. tarda attack and temperature stress, twelve PoIRFs showed significant and different changes in mRNA levels. The expression of PoIRF1, PoIRF3, PoIRF4a, PoIRF5, PoIRF7, PoIRF8, PoIRF9, PoIRF10 and PoIRF11 could be markedly induced by E. tarda, indicating that they played a key role in the process of antibacterial immunity. Besides, temperature stress could significantly stimulate the expression of PoIRF3, PoIRF5, PoIRF6 and PoIRF7, indicating that they could transmit signals rapidly when the temperature changes. In conclusion, this study reported the molecular properties and expression analysis of PoIRFs, and explored their role in immune response, which laid a favorable foundation for further studies on the evolution and functional characteristics of the IRF family in teleost fish.

Ajpacifastin-like is involved in the immune response of Apostichopus japonicus challenged by Vibrio splendidus.

Pacifastin proteins are previously found to regulate the phenoloxidase system in invertebrates and arthropods. In this study, the immune response that was regulated by Ajpacifastin-like in the sea cucumber Apostichopus japonicus was determined. RNA interference was used to knock down the expression of the Ajpacifastin-like gene in A. japonicus, followed by challenge with Vibrio splendidus, and the colony count showed that the survival of V. splendidus in the si-Ajpacifastin group increased 4.64-fold compared to that of the control group. The purified recombinant Ajpacifastin-like showed an inhibitory effect on the extracellular protease activity of the supernatant collected from the V. splendidus culture. Consequently, a comparative transcriptome analysis of the coelomocytes from the control group and the si-Ajpacifastin group was performed to explore the global regulatory effect of the Ajpacifastin-like. A total of 1486 differentially expressed genes (DEGs) were identified, including 745 upregulated genes and 741 downregulated genes. GO enrichment showed that the DEGs were mainly enriched in translation, cytosolic ribosomal subunit and structural constituent of ribosome. KEGG analysis showed that the DEGs were significantly enriched in the retinoic acid-inducible gene I (RIG-I)-like receptor signaling pathway, antigen processing and presentation, toll-like receptor signaling pathway, mitogen-activated protein kinase signaling pathway, nuclear factor-kappa B signaling pathway and other immune-related pathways. Furthermore, real-time reverse transcriptase PCR was used to determine the RNA levels of six DEGs, i.e., cathepsinB, CYLD, caspase8, TRAF6, hsp90 and FADD, to verify the RNA-seq results. Overall, our results specified the immune response and pathways of A. japonicus in which Ajpacifastin-like was involved in.

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.

Proteomic analysis reveals the adaptation of Vibrio splendidus to an iron deprivation condition.

Vibrio splendidus is a ubiquitous Gram-negative marine bacterium that causes diseases within a wide range of marine cultured animals. Since iron deprivation is the frequent situation that the bacteria usually encounter, we aimed to explore the effect of iron deprivation on the proteomic profile of V. splendidus in the present study. There were 425 differentially expressed proteins (DEPs) responded to the iron deprivation condition. When the cells were grown under iron deprivation condition, the oxidation‒reduction processes, single-organism metabolic processes, the catalytic activity, and binding activity were downregulated, while the transport process, membrane cell component, and ion binding activity were upregulated, apart from the iron uptake processes. Kyoto Encyclopedia of Genes and Genomes analysis showed that various metabolism pathways, biosynthesis pathways, energy generation pathways of tricarboxylic acid cycle, and oxidative phosphorylation were downregulated, while various degradation pathways and several special metabolism pathways were upregulated. The proteomic profiles of cells at a OD600 ≈ 0.4 grown under iron deprivation condition showed high similarity to that of the cells at a OD600 ≈ 0.8 grown without iron chelator 2,2'-bipyridine. Correspondingly, the protease activity, the activity of autoinducer 2 (AI-2), and indole content separately catalyzed by LuxS and TnaA, were measured to verify the proteomic data. Our present study gives basic information on the global protein profiles of V. splendidus grown under iron deprivation condition and suggests that the iron deprivation condition cause the cell growth enter a state of higher cell density earlier. KEY POINTS: • Adaptation of V. splendidus to iron deprivation was explored by proteomic analysis. • GO and KEGG of DEPs under different iron levels or cell densities were determined. • Iron deprivation caused the cell enter a state of higher cell density earlier.

Contrasting Mechanisms Determine the Microeukaryotic and Syndiniales Community Assembly in a Eutrophic bay.

Syndiniales is a diverse parasitic group, increasingly gaining attention owing to its high taxonomic diversity in marine ecosystems and inhibitory effects on the dinoflagellate blooms. However, their seasonal dynamics, host interactions, and mechanisms of community assembly are largely unknown, particularly in eutrophic waters. Here, using 18S rRNA gene amplicon sequencing, we intended to elucidate the interactions between Syndiniales and microeukaryotes, as well as community assembly processes in a eutrophic bay. The results showed that Syndiniales group II was dominating throughout the year, with substantially higher abundance in the winter and spring, whereas Syndiniales group I was more abundant in the summer and autumn. Temperature and Dinoflagellata were the most important abiotic and biotic factors driving variations of the Syndiniales community, respectively. The assembly processes of microeukaryotes and Syndiniales were completely different, with the former being controlled by a balance between homogeneous selection and drift and the latter being solely governed by drift. Network analysis revealed that Syndiniales group II had the largest number of interactions with microeukaryotes, and they primarily associated with Dinoflagellata in the winter, while interactions with Chlorophyta and Bacillariophyta increased dramatically in summer and autumn. These findings provide significant insights in understanding the interactions and assembly processes of Syndiniales throughout the year, which is critical in revealing the roles of single-celled parasites in driving protist dynamics in eutrophic waters.

Genome-wide characterization of the cytosolic sulfotransferase 1B member 1 (SULT1B1) family and its expression responses to sulfide stress in the razor clam Sinonovacula constricta.

The razor clam (Sinonovacula constricta), a typical burrowing organism in the intertidal zones, is often exposed to sulfide environment and shows strong sulfide tolerance. Located downstream of the sulfur metabolism pathway, cytosolic sulfotransferase family 1B member 1 (SULT1B1) is a key enzyme catalysing the sulfonation reaction, and plays an important role in the biotransformation of endogenous substances such as thyroid hormones (THs). To investigate their roles in sulfide resistance, a systematic analysis of S. constricta SULT1B1s (ScSULT1B1s), including genomic distribution, phylogenetic relationships, gene structure, conserved motifs, and expression profiles under sulfide stress, was performed. A total of 10 ScSULT1B1 genes were found in the S. constricta genome. Sequence analysis showed that ScSULT1B1 gene family encoded 155-425 amino acids, containing four catalytic active sites (K, N, H, and S), one PAPS binding domain at the N-terminus, and one PAPS binding and dimerization domain at the C-terminus. The spatial-temporal expression patterns of ScSULT1B1s were further estimated by quantitative real-time PCR (qRT-PCR). Among them, partial ScSULT1B1s showed significantly high expression in the gill, hepatopancreas, and siphon. Furthermore, the response expression of certain ScSULT1B1s significantly fluctuated under sulfide stress. Together, our results suggest that ScSULT1B1s, by mediating the sulfonation reaction, may regulate THs levels to maintain basic metabolic and immune functions, making S. constricta highly sulfide tolerant.

Cloning and functional analysis of a pacifastin-like protein from the sea cucumber, Apostichopus japonicus.

Pacifastin family proteins play a crucial role in regulating innate immune responses such as phagocytosis in invertebrates. However, the function of the Ajpacifastin-like counterpart in the sea cucumber Apostichopus japonicus remains elusive. In this study, the pacifastin gene of A. japonicus was cloned, characterized and named Ajpacifastin-like. The open reading frame of Ajpacifastin-like is 1497 bp in length and encodes a polypeptide containing 498 amino acid residues. Structural analysis revealed that the protein encoded by Ajpacifastin-like contains two pacifastin light chain domains (amino acids 287-322 and amino acids 376-407). Real-time reverse transcriptase PCR showed that Ajpacifastin-like mRNA is ubiquitously expressed in all tissues examined, with the highest expression in muscle. Ajpacifastin-like mRNA expression was significantly upregulated to 3.27-fold after challenge with Vibrio splendidus for 24 h. To explore the function of the Ajpacifastin-like protein in the immune response of A. japonicus, dsRNA interference with Ajpacifastin-like expression and with the expression of its postulated target gene was performed. Flow cytometry analysis showed that the rate of phagocytosis by coelomocytes increased to 1.21-fold in individuals treated with specific Ajpacifastin-like siRNA. However, rate of phagocytosis by coelomocytes decreased to 86% in individuals treated with Ajphenoloxidase siRNA. These results show that the Ajpacifastin-like gene is ubiquitously expressed in almost all tissues and that Ajpacifastin-like protein acts as an immunomodulatory factor via phenoloxidase to mediate phagocytosis by coelomocytes in pathogen-challenged A. japonicus.

Disentangling the Mechanisms Shaping the Prokaryotic Communities in a Eutrophic Bay.

Eutrophication occurring in coastal bays is prominent in impacting local ecosystem structure and functioning. To understand how coastal bay ecosystem function responds to eutrophication, comprehending the ecological processes associated with microbial community assembly is critical. However, quantifying the contribution of ecological processes to the assembly of prokaryotic communities is still limited in eutrophic waters. Moreover, the influence of these ecological processes on microbial interactions is poorly understood. Here, we examined the assembly processes and co-occurrence patterns of prokaryotic communities in a eutrophic bay using 156 surface seawater samples collected over 12 months. The variation of prokaryotic community compositions (PCCs) could be mainly explained by environmental factors, of which temperature was the most important. Under high environmental heterogeneity conditions in low-temperature seasons, heterogeneous selection was the major assembly process, resulting in high ß-diversity and more tightly connected co-occurrence networks. When environmental heterogeneity decreased in high-temperature seasons, drift took over, leading to decline in ß-diversity and network associations. Microeukaryotes were found to be important biological factors affecting PCCs. Our results first disentangled the contribution of drift and microbial interactions to the large unexplained variation of prokaryotic communities in eutrophic waters. Furthermore, a new conceptual model linking microbial interactions to ecological processes was proposed under different environmental heterogeneity. Overall, our study sheds new light on the relationship between assembly processes and co-occurrence of prokaryotic communities in eutrophic waters. IMPORTANCE A growing number of studies have examined roles of microbial community assembly in modulating community composition. However, the relationships between community assembly and microbial interactions are not fully understood and rarely tested, especially in eutrophic waters. In this study, we built a conceptual model that links seasonal microbial interactions to ecological processes, which has not been reported before. The model showed that heterogeneous selection plays an important role in driving community assembly during low-temperature seasons, resulting in higher ß-diversity and more tightly connected networks. In contrast, drift became a dominant force during high-temperature seasons, leading to declines in the ß-diversity and network associations. This model could function as a new framework to predict how prokaryotic communities respond to intensified eutrophication induced by climate change in coastal environment.

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.

Characteristics of the iron uptake-related process of a pathogenic Vibrio splendidus strain associated with massive mortalities of the sea cucumber Apostichopus japonicus.

Under low iron bioavailability environment, many bacteria acquire iron for growth and survival through siderophore-mediated iron acquisition systems. However, until now, little research on the growth, siderophore production and siderophore receptors of Vibrio splendidus Vs under iron limited conditions has been reported. In our present study, V. splendidus Vs could survive in media supplemented with 160 µM 2,2'-dipyridyl (DIP), but 74.5% of the growth was suppressed at 48 h, while the siderophore production of V. splendidus Vs increased by 35.9%. As the OD600 of V. splendidus Vs decreased when the concentration of DIP was increased from 40 to 80 µM, the siderophore production of V. splendidus Vs increased from 34.0% to 43.4% at 24 h, and it was further determined to be a hydroxamate siderophore. To explore the potential siderophore receptors anchored on the outer membrane of V. splendidus Vs, outer membrane proteins from cells grown with and without 80 µM DIP were extracted and the differentially expressed proteins were identified by SDS-PAGE and MALDI-TOF/TOF MS. Five proteins, aerobactin siderophore receptor IutA, enterobactin receptor protein FepA, ATP synthase subunit A, ATP synthase subunit B and the ATP synthase F0F1 subunit beta were identified. Real-time reverse transcriptase PCR showed that mRNA levels of iutA, fepA, atpA, atpB and atpß-F0F1 were upregulated 271.5-, 15.1-, 1.1-, 2.5- and 67.9-fold respectively, after 6 h in cells treated with 80 µM DIP. In addition, the promoters of the siderophore receptor genes of iutA and fepA had apparent ferric uptake regulator (Fur) binding sites. Combined with the simultaneous production of both the hydroxamate siderophore and its corresponding aerobactin siderophore receptor IutA, these results suggested that there might be a hydroxamate siderophore-IutA mediated iron uptake pathway in V. splendidus Vs.

A new species of Philocheras (Decapoda: Caridea: Crangonidae) from Western Australia, with a key to all species of this genus worldwide.

One new species and three new distribution records for the family Crangonidae (Crustacea: Decapoda) are reported from Western Australia. A new species, Philocheras gardenensis sp. nov. is described and illustrated based on specimens collected from the Western Australia. The new species closely resembles Philocheras fasciatus (Risso, 1816), but can be distinguished easily by the absence of lobe-like folds on either side of the middle line of the carapace, the presence of a lateral carina extending from the branchiostegal spine, the longer middorsal carina and distolateral tooth of the antennal scale. A key for the genus Philocheras is provided, including currently, a total of 58 species and subspecies reported worldwide. Range extensions of three Crangonid shrimps are reported within Australia, and they are Philocheras obliquus (Fulton Grant, 1902), Pontocaris arafurae (Bruce, 1988) and Vercoia gibbosa Baker, 1904.

Quorum sensing-disrupting coumarin suppressing virulence phenotypes in Vibrio splendidus.

In the present study, the effects of an environmental friendly natural reagent coumarin, on the growth and potential virulence factors, as well as its ability to interfere the infection of Vibrio splendidus (Vs), were determined. Coumarin showed no effects on the maximal growth of Vs, and biofilm formation of Vs, while it significantly decreased protease activity and hemolytic activity by 43 and 80%, respectively. Correspondingly, coumarin exhibited an obviously protective effect, with a relative percent survival of 60% upon Apostichopus japonicus from infection by Vs. To preliminarily investigate the mechanism underlining the inhibitory effects, regulation of genes Vsm and Vsh respectively related to protease activity and hemolytic activity by supernatant and supernatant extract containing acyl-homoserine lactones (AHLs) and coumarin was determined. Cell-free supernatant from higher density and its ethyl acetate extract containing AHL signal molecules could respectively upregulate the mRNA level of Vsm by 17.4- and 2.3-fold and Vsh by 7.2- and 5.0-fold, when Vs was at lower cell density. However, coumarin could reduce the stimulatory effects of both the supernatant and its ethyl acetate extract. Combining all the results in our study, it was suggested that coumarin could be considered as an alternative to be used for controlling infection of Vs, downregulating the expression of potential virulence factors through interfering the AHL-mediated pathways.

Conditions optimising on the yield of biomass, total lipid, and valuable fatty acids in two strains of Skeletonema menzelii.

Polyunsaturated fatty acids (PUFA) and eicosapentaenoic acid (EPA) are essential for the health of aquaculture organisms and human beings. A total of 9 species of diatoms were screened and two strains of Skeletonema menzelii were selected for further study due to their high growth rates, PUFA and EPA contents. The culture conditions for the yield of biomass, total lipid, EPA and PUFA were optimised. The quickest growth rates (0.28-0.41d(-1)) were achieved with moderate supplement of nitrogen, phosphorus, iron and silicon. The accumulation of total lipid (17.85-22.70% of dry weight) benefitted from deprivation of nitrogen, phosphorus and silicon, but a moderate iron supplement. Highest PUFA (38.26-50.48% of total fatty acids) or EPA (14.26-18.39% of total fatty acids) contents were observed under high nitrogen and phosphorus supplement, moderate available iron but deprivation of silicon. These findings make tangible contributions to culture S. menzelii for commercial production of PUFA or EPA.

Three members in JAK/STAT signal pathway from the sea cucumber Apostichopus japonicus: Molecular cloning, characterization and function analysis.

The JAK/STAT signal transduction pathway plays a critical role in host defense against bacterial infections. In the present study, we firstly cloned the full-length cDNAs of three molecules in JAK/STAT cascade, STAT5, FOXP and SOCS2, from sea cucumber Apostichopus japonicus (denoted as AjSTAT5, AjFOXP, AjSOCS2, respectively) and investigated their immune functions towards Vibrio splendidus infection and LPS exposure. The AjSTAT5 cDNA was composed of 2643 bp consisting of 787 amino acid residues which included protein interaction domain, STAT-α domain, DNA binding domain and SH2 domain. The putative AjFOXP contained a ZnF_C2H2 domain, the leucine zipper-like domain and FH domain, all of which were thought to be the representative characteristics of FOXP subfamily. The deduced amino acids sequence of AjSOCS2 included an SH2 domain and SOCS box domain similar to vertebrate SOCS counterparts. Phylogenetic trees further supported that all these three identified proteins belonged to novel members of JAK/STAT signal pathway in sea cucumber. Tissue specific expression analysis showed that three genes were ubiquitously expressed in all examined tissues. AjSTAT5 and AjFOXP were both dominantly expressed in intestine, tentacle and respiratory tree, and weak in muscle. In contrary, the peak expression of AjSOCS2 was observed in muscle and lowest in respiratory tree. The V. splendidus challenge and LPS exposure could both significantly up-regulate the mRNA expression of three genes, in which AjSOCS2 showed opposite expression trends to those of AjSTAT5 and AjFOXP. Silencing the AjSTAT5 by siRNA depressed the AjFOXP expression, but induced the expression level of AjSOCS2, revealing that AjSTAT5 might directly modulate AjFOXP, and AjSOCS2 function primarily by acting as a potent inhibitor involve in JAK/STAT pathway. The present study would expand our understanding on JAK/STAT signaling transduction pathway in modulating the innate immune responses of sea cucumber.