Comparative Transcriptome Analysis Reveals the Light Spectra Affect the Growth and Molting of Scylla paramamosain by Changing the Chitin Metabolism.

Light is an essential ecological factor that has been demonstrated to affect aquatic animals' behavior, growth performance, and energy metabolism. Our previous study found that the full-spectrum light and cyan light could promote growth performance and molting frequency of Scylla paramamosain while it was suppressed by violet light. Hence, the purpose of this study is to investigate the underlying molecular mechanism that influences light spectral composition on the growth performance and molting of S. paramamosain. RNA-seq analysis and qPCR were employed to assess the differentially expressed genes (DEGs) of eyestalks from S. paramamosain reared under full-spectrum light (FL), violet light (VL), and cyan light (CL) conditions after 8 weeks trial. The results showed that there are 5024 DEGs in FL vs. VL, 3398 DEGs in FL vs. CL, and 3559 DEGs in VL vs. CL observed. GO analysis showed that the DEGs enriched in the molecular function category involved in chitin binding, structural molecular activity, and structural constituent of cuticle. In addition, the DEGs in FL vs. VL were mainly enriched in the ribosome, amino sugar and nucleotide sugar metabolism, lysosome, apoptosis, and antigen processing and presentation pathways by KEGG pathway analysis. Similarly, ribosome, lysosome, and antigen processing and presentation pathways were major terms that enriched in FL vs. CL group. However, only the ribosome pathway was significantly enriched in up-regulated DEGs in VL vs. CL group. Furthermore, five genes were randomly selected from DEGs for qPCR analysis to validate the RNA-seq data, and the result showed that there was high consistency between the RNA-seq and qPCR. Taken together, violet light exposure may affect the growth performance of S. paramamosain by reducing the ability of immunity and protein biosynthesis, and chitin metabolism.

Recirculating aquaculture system as microbial community and water quality management strategy in the larviculture of Scylla paramamosain.

The structure and function of the water microbial community can change dramatically between different rearing modes. Yet investigations into the relationships between microbial community and water quality remain obscure. We provide the first evidence that rearing modes alter bacterial community and water quality in the rearing water of the mud crab (Scylla paramamosain) larvae. The juveniles in the recirculating aquaculture system (RAS) had a higher viability than those in the water exchange system (WES). RAS had the significantly lower levels of total ammonia nitrogen (TAN), NH3, NO2--N, total nitrogen (TN), total dissolved solids (TDS), and chemical oxygen demand than those of WES. The number of significantly different amplicon sequence variants between rearing modes increased as the larvae developed. NH3, TAN, TDS, NO2--N, and TN were closely related to the late alterations in water bacterial community. Both the FAPROTAX tool and quantitative PCR analysis showed enhanced nitrogen cycling functional potential of water bacterial community of RAS. Random forest analysis identified the enriched water bacteria especially heterotrophic bacteria such as Phaeodactylibacter, Tenacibaculum, and Hydrogenophaga, which were vital in removing nitrogenous compounds via simultaneous nitrification and denitrification. Notably, RAS could save 18.5 m3 of seawater relative to WES in larviculture on the scale of 2.5 m3. Together, these data indicate that RAS could function as microbial community and water quality management strategy in the larviculture of crab.

Performance of the graphite felt flow-through electrode in hexavalent chromium reduction using a single-pass mode.

Flow-through electrodes generally outcompete traditional parallel-plate electrodes in current efficiency and mass transfer. High-performance electrode materials can be costly and complicated to fabricate, hindering their wide application. In this study, we used commercial graphite felt (GF) as the cathode of a flow-through electrochemical cell to investigate its potential in treating Cr(VI) solution through electroreduction. The flow-through design with the porous GF electrode allowed sufficient contact surface with Cr(VI) and single-pass tests demonstrated a high reduction efficiency (95~100%) [117 mg/L~3 mg/L Cr(VI)] under acidic conditions. Slow flow rate and high current promoted electroreduction of Cr(VI). The presence of other metal ions could further improve Cr(VI) reduction at low flow rates due to enhanced conductivity in dilute solutions and generation of low valent ions as reducing agents. At fast flow rates, competition of these ions for reduction decreased Cr(VI) reduction efficiency. Moreover, an acidic environment prevented the coating of an insoluble layer on the GF surface and promoted durable performance, with a lower energy consumption [0.46 kWh for treating 100 L 117 mg/L Cr(VI) solution per unit area of GF]. This work demonstrated the potential of Cr(VI) detoxification using GF cathodes in flow-through electrochemical cell.

Isolation, characterization and virulence of Mesanophrys sp. (Ciliophora: Orchitophryidae) in farmed swimming crab (Portunus trituberculatus) in eastern China.

A disease outbreak occurred in swimming crab (Portunus trituberculatus) farmed in eastern China, with a mortality rate of more than 80%. To further investigate the characteristics and pathogenesis, we reported isolation, characterization and virulence of the causative agent of this disease from 10 sick crabs. Histopathological observation found that multiple tissues, especially haemolymph, contained lots of ciliates. The ciliate was isolated and cultured in vitro, and molecular and morphological studies were done. The results showed that SSU rDNA and LSU rDNA sequences of the ciliate were similar to Mesanophrys ciliates (>96.81%), while ITS1-5.8s-ITS2 sequence was similar to Mesanophrys pugettensis (95.37%) and identical to Orchitophrya stellarum (100%). Furthermore, the results of the morphological study confirmed that the ciliate was similar to Mesanophrys ciliates and O. stellarum cultured in supportive media, but different from O. stellarum cultured in living sperm cells of starfish (Leptasterias spp.). Also, the growth of the ciliate did not interfere with light, which was different from O. stellarum. Accordingly, the ciliate was classified as genus Mesanophrys and temporarily named as Mesanophrys sp. In addition, experimental infection confirmed that Mesanophrys sp. was the pathogen that infected farmed crabs. In summary, Mesanophrys sp. was first isolated and characterized in P. trituberculatus.

16S rRNA gene sequencing reveals the relationship between gut microbiota and ovarian development in the swimming crab Portunus trituberculatus.

Gut microbiota executes many beneficial functions. In this study, the relationship between gut microbiota and ovarian development in the swimming crab P. trituberculatus was explored for the first time. A total of 28 phyla and 422 genera were identified across all samples. However, 105 differential operational taxonomic units, and four differential phyla (Gemmatimonadetes, Actinobacteria, Firmicutes, Marinimicrobia_(SAR406_clade)) were identified. At the genus level, 42 differential genera were identified and 144 bacterial indicators were identified. A key finding was that the relative abundance of 139 indicator bacteria detected in the anisomycin-2 mg/kg group (AK group) was higher than that of blank group (BK group), control group (CK group), SP600125-15 mg/kg group (SK group). In addition, the relative abundance of three indicator bacteria (OTU_236, OTU_1395, OTU_552) detected in the SK group was higher than that of the BK, CK and AK groups. It was also found that the relative abundance of 20 differential genera (Methyloversatilis, Coprococcus_1, Erysipelotrichaceae_UCG_003, Rikenella, Corynebacterium, Ruminiclostridium, Fusicatenibacter, [Eubacterium]_ruminantium_group, Rikenellaceae_RC9_gut_group, Bifidobacterium, Lachnospiraceae_NK4A136_group, Ruminococcaceae_UCG_014, Christensenellaceae_R_7_group, uncultured_Bacteroidales_bacterium, Coprococcus_2, Desulfovibrio, Aggregatibacter, Ambiguous_taxa, Alloprevotella and Ruminococcaceae_NK4A214_group) in the SK, BK, CK, and AK group samples were increasing. These differential genera may reveal the relationship between gut microbial communities and ovarian development in P. trituberculatus after injection with the JNK pathway inhibitor SP600125 or the activator anisomycin. In summary, this study provides a new understanding into the relationship between gut microbiota and ovarian development in response to stimulation with inhibitor or activator.

Effects of Elevated pCO2 on the Survival and Growth of Portunus trituberculatus.

Identifying the response of Portunus trituberculatus to ocean acidification (OA) is critical to understanding the future development of this commercially important Chinese crab species. Recent studies have reported negative effects of OA on crustaceans. Here, we subjected swimming crabs to projected oceanic CO2 levels (current: 380 µatm; 2100: 750 µatm; 2200: 1500 µatm) for 4 weeks and analyzed the effects on survival, growth, digestion, antioxidant capacity, immune function, tissue metabolites, and gut bacteria of the crabs and on seawater bacteria. We integrated these findings to construct a structural equation model to evaluate the contribution of these variables to the survival and growth of swimming crabs. Reduced crab growth shown under OA is significantly correlated with changes in gut, muscle, and hepatopancreas metabolites whereas enhanced crab survival is significantly associated with changes in the carbonate system, seawater and gut bacteria, and activities of antioxidative and digestive enzymes. In addition, seawater bacteria appear to play a central role in the digestion, stress response, immune response, and metabolism of swimming crabs and their gut bacteria. We predict that if anthropogenic CO2 emissions continue to rise, future OA could lead to severe alterations in antioxidative, immune, and metabolic functions and gut bacterial community composition in the swimming crabs through direct oxidative stress and/or indirect seawater bacterial roles. These effects appear to mediate improved survival, but at the cost of growth of the swimming crabs.

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.

JNK signaling pathway regulates the development of ovaries and synthesis of vitellogenin (Vg) in the swimming crab Portunus trituberculatus.

The development of Portunus trituberculatus egg cells is directly related to the nutritional status of the fertilized egg, which affects the key production stages of offspring hatching. Vitellogenin plays a key role in the nutrient supply required for the development of the egg cells. The c-Jun N-terminal kinase (JNK) is an important member of the mitogen-activated protein kinase (MAPK) superfamily and plays an important role in cell proliferation, transformation, differentiation, and apoptosis. At present, there are no reports on the involvement of the JNK signaling pathway in the reproductive regulation of P. trituberculatus. In this study, rapid amplification of complementary DNA ends amplification technology was used to clone the full length of JNK complementary DNA, which has a length of 2094 bp, including an open reading frame (ORF) of 1266 bp encoding a 421-amino acid protein. The protein includes the S_TKC conserved domain with a TPY phosphorylation site, which is a typical feature of the JNK gene family. Observing tissue sections found the oocytes in the inhibitor group developed slowly, while the oocytes in the activated group showed accelerated development. Meanwhile, Portunus trituberculatus JNK and vitellogenin (Vg) genes exhibited the same trend in the hepatopancreas and ovaries, and the expression of the SP600125 group was downregulated (P < 0.05), while the anisomycin group was upregulated (P < 0.05). In addition, JNK enzyme activity and vitellin (Vn) content in the ovarian tissue showed that the JNK activity of the SP600125 group decreased, while activity increased in the anisomycin group. The accumulation of Vn content in the SP600125 group decreased, and that in the anisomycin group increased. In summary, after injection with inhibitor or activator, the JNK signaling pathway of P. trituberculatus was inhibited or activated, the accumulation of Vn in the ovary was reduced or increased, and ovarian development was inhibited or accelerated, respectively. These results indicated that the JNK signaling pathway is involved in the regulation of Vg synthesis and ovarian development in P. trituberculatus. The results of this study further add to the knowledge of the breeding biology of P. trituberculatus and provide a theoretical reference for the optimization of breeding techniques in aquaculture production systems.

Characterization of PHB in the gonadal development of the swimming crab Portunus trituberculatus.

Prohibitin (PHB) is an evolutionarily conserved multifunctional protein with ubiquitous expression. In this study, we cloned the PHB gene from the testis of the swimming crab Portunus trituberculatus (PtPHB) and analyzed the deduced amino acid sequence. The expression level of phb mRNA in larvae was analyzed using qRT-PCR. The expression level of phb mRNA and PHB protein in different tissues were analyzed using qRT-PCR and Western blot respectively. Enzyme-linked immunosorbent assay analyses of the PHB protein were conducted with the testis and ovaries from P. trituberculatus specimens at different developmental stages. PHB was localized with mitochondria and ubiquitin in the testis and ovaries. The PtPHB gene was found to contain an open reading frame of 825 bp, encoding a predicted peptide with 275 amino acids, sharing between 65.9% and 96.7% similarity with that of other species. The qRT-PCR and Western blot results showed that the phb gene and PHB protein both expressed less in the testis and ovary than in other tissues, and the phb gene presented the lowest expression in the Z1 stage. Furthermore, the phb gene and PHB protein expression were different in the testis and ovaries at different developmental stages. PHB was mainly found to be co-localized with mitochondria and ubiquitin in cytoplasm and acrosome complex during spermatogenesis and in follicular cells during oogenesis. Interestingly, PHB-mitochondria signals and ubiquitin signal were also found in oocytes. These results indicated that PHB might play important roles during spermatogenesis and oogenesis by regulating mitochondrial activities.

Characterisation and functional analysis of an L-type lectin from the swimming crab Portunus trituberculatus.

L-type lectins are involved in glycoprotein secretion and are associated with immune responses. Herein, an L-type lectin was identified in swimming crab (Portunus trituberculatus). The 1347 bp PtLTL cDNA includes a 26 bp 5'-untranslated region (UTR), a 547 bp 3'-UTR with a poly(A) tail, and a 774 bp open reading frame encoding a 257 amino acid protein with a putative 21 residue signalling peptide. The protein includes an L-type lectin carbohydrate recognition domain containing four conserved cysteines. The 714 bp cDNA fragment encoding the mature peptide of PtLTL1 was recombined into pET-21a (+) with a C-terminally hexa-histidine tag fused in-frame and expressed in Escherichia coli Origami (DE3). Recombinant PtLTL1 caused agglutination of all three Gram-positive and Gram-negative bacterial strains tested. In addition, erythrocyte agglutination and LPS-binding activity were observed. PtLTL1 mRNA transcripts were most abundant in P. trituberculatus hepatopancreas and hemocytes, and expression was up-regulated in hemocytes challenged with Vibrio alginolyticus, suggesting PtLTL functions in the immune response against bacterial pathogens.

iTRAQ-based proteomic analysis identifies proteins involved in limb regeneration of swimming crab Portunus trituberculatus.

The swimming crab (Portunus trituberculatus) has a striking capacity for limb regeneration, which has drawn the interest of many researchers. In this study, isobaric tag for relative and absolute quantitation (iTRAQ) approach was utilised to investigate protein abundance changes during limb regeneration in this species. A total of 1830 proteins were identified, of which 181 were significantly differentially expressed, with 94 upregulated and 87 downregulated. Our results highlight the complexity of limb regeneration and its regulation through cooperation of various biological processes including cytoskeletal changes, extracellular matrix (ECM) remodelling and ECM-receptor interactions, protein synthesis, signal recognition and transduction, energy production and conversion, and substance transport and metabolism. Additionally, real-time PCR confirmed that mRNA levels of differentially expressed genes were correlated with protein levels. Our results provide a basis for studying the regulatory mechanisms associated with crab limb regeneration.

[Function of luxS gene in sulfurmetabolism of Streptococcus mutans].

OBJECTIVE: To investigate the function of luxS in sulfurmetabolism of Streptococcus mutans (Sm). METHODS: The growth with absorbency (A) of the standards and mutant strains was measured and analyzed in the sulfur-limited defined medium at different periods. The laser scanning confocal microscopy (LSCM) was used to observe and compare the biofilm thickness of the two kinds of strains at different culture conditions. RESULTS: The significant increases in the thickness of mutant strain biofilm and its growth were observed after the addition of cysteine, but did not reach the standards strain levels (P < 0.05). The growth and the biofilm thickness of the mutant strains were (1.301 ± 0.009) and (45.009 ± 0.429) µm. When methionine and S-adenosylhomocysteine of certain concentrations were respectively added, the biofilm thickness and the growth of mutant strain were raised but did not reach the level of the standards strain at 24 h (P < 0.05), but at 48 h they did. When the methionine was added in the mutant strains for 24 h, the biofilm thickness and the growth of mutant strain were (0.448 ± 0.028) and (37.068 ± 2.392) µm, as for the adding of S-adenosylhomocysteine were (0.460 ± 0.005) and (27.343 ± 1.107) µm. When adding the supernatant fluid of standard strains, the biofilm thickness and the growth levels of mutant strain were much higher than those of the standards strain. The biofilm thickness and growth of both kinds of strains decreased after the addition of S-adenosylmethionine. CONCLUSIONS: luxS gene plays not only a role in quorum sensing but also a role in sulfurmetabolism.

[Application of Cre-loxP(*) system in constructing the markerless double-gene-deletion strain in Streptococcus mutans].

OBJECTIVE: To construct double gene deletions at the htrA and clpP loci on the chromosome of Streptococcus mutans (Sm) and to remove the antibiotic resistance markers with the Cre-loxP(*) site-specific recombination system. METHODS: The htrA gene was cloned into the pGEM-T-Easy TA cloning vector and then inactivated via the insertion of a kanamycin resistance cassette (lox71-Km-lox66), yielding pGEM-T-ΔhtrA/Km for deleting the htrA gene. Using the same method, the pGEM-T-ΔclpP/Sp was constructed for deleting the clpP gene. Following the transformation of pGEM-T-ΔhtrA/Km in Sm, the homologous recombination event was selected. One such mutant was transformed with a cre expression plasmid (pCrePA). The kanamycin resistance gene was then excised. The pCrePA was then easily eliminated at nonpermissive temperatures, resulting in a mutant strain (MSΔhtrA) carrying a deletion at the htrA loci without a selectable marker. This mutant was verified by PCR and DNA sequencing. Then, the clpP and spectinomycin resistance gene were deleted from MSΔhtrA, yielding markerless mutant strain lacking clpP and htrA. RESULTS: The deletion of htrA, clpP and antibiotic resistance markers were confirmed by PCR analysis and DNA sequencing. CONCLUSIONS: A mutant of Sm was constructed successfully which contained a deletion of the htrA and clpP gene without selectable marker. The Cre-loxP(*) system can be applied to Sm, which provides experimental evidence for generating markerless multiple gene deletion mutants.

Monte Carlo renormalization: the triangular Ising model as a test case.

We test the performance of the Monte Carlo renormalization method in the context of the Ising model on a triangular lattice. We apply a block-spin transformation which allows for an adjustable parameter so that the transformation can be optimized. This optimization purportedly brings the fixed point of the transformation to a location where the corrections to scaling vanish. To this purpose we determine corrections to scaling of the triangular Ising model with nearest- and next-nearest-neighbor interactions by means of transfer-matrix calculations and finite-size scaling. We find that the leading correction to scaling just vanishes for the nearest-neighbor model. However, the fixed point of the commonly used majority-rule block-spin transformation appears to lie well away from the nearest-neighbor critical point. This raises the question whether the majority rule is suitable as a renormalization transformation, because the standard assumptions of real-space renormalization imply that corrections to scaling vanish at the fixed point. We avoid this inconsistency by means of the optimized transformation which shifts the fixed point back to the vicinity of the nearest-neighbor critical Hamiltonian. The results of the optimized transformation in terms of the Ising critical exponents are more accurate than those obtained with the majority rule.