The interaction between 20-hydroxyecdysone and AMPK through PI3K activation in Chinese mitten crab, Eriocheir sinensis.

In crustaceans, the steroid hormone 20-hydroxyecdysone (20E) initiates molting, and the molting process is also regulated by energy metabolism. AMPK is an energy sensor and plays a critical role in systemic energy balance. Here, the regulatory mechanism in the interaction between 20E and AMPK was investigated in Chinese mitten crab, Eriocheir sinensis. The results showed that the 20E concentration and the mRNA expression levels of 20E receptors in hepatopancreas were down-regulated post AMPK activator (AICAR) treatment, and were up-regulated after AMPK inhibitor (Compound C) injection in crabs. Besides, the molt-inhibiting hormone (MIH) gene expression in eyestalk showed the opposite patterns in response to the AICAR and Compound C treatment, respectively. Further investigation found that there was a significant reduction in 20E concentration post PI3K inhibitor (LY294002) treatment, and the phosphorylation level of PI3K was increased in hepatopancreas after AMPK inhibitor injection. On the other hand, the positive regulation of PI3K-mediated activation of AMPK was also observed, the phosphorylation levels of AMPKα, AMPKß and PI3K in hepatopancreas were significantly increased post 20E injection. In addition, the phosphorylation levels of AMPKα and AMPKß induced by 20E were decreased after the injection of PI3K inhibitor. Taken together, these results suggest that the regulatory cross-talk between 20E and AMPK is likely to act through PI3K pathway in E. sinensis, which appeared to be helpful for a better understanding in molting regulation.

Identification of a novel CYP4V gene in the polychaete Perinereis aibuhitensis: transcriptional comparison with a CYP4B gene exposed to PAHs.

Polychaete worms can biotransform polycyclic aromatic hydrocarbons (PAHs) in environments, and the cytochrome P450 (CYP) enzyme plays an important role in this process. Herein, a novel cytochrome P450 gene was identified and characterized from the polychaete worm Perinereis aibuhitensis. The full-length cDNA, which is named CYP4V82, is 1709 bp encoding a protein of 509 amino acids and has high similarity to CYP4V. The expression levels of CYP4V82 and CYP4BB4 (a CYP gene identified from P. aibuhitensis in a previous study, Chen et al. Mar Pollut Bull 64:1782-1788, 2012) exposure to various concentrations of benzo[a]pyrene (B[a]P) (0.5, 2, 4, and 8 µg/L) and same mass concentrations of fluoranthene (Flu, 3.2 µg/L), phenanthrene (Phe, 2.9 µg/L), B[a]P (4.0 µg/L) were detected to identify the function of the CYP4 family in P. aibuhitensis. Compared with CYP4BB4, CYP4V82 mRNA was minimally expressed on day 7 but highly sensitive on day 14. Notably, the expression levels of CYP4V82 and CYP4BB4 were relatively different in short-term responses to PAHs with different benzene rings of the same concentration. The expression of CYP4V82 in the B[a]P group was the highest, while that of CYP4BB4 in the Phe group was relatively higher than the two other groups. These findings suggest that PAHs are associated with the induction of CYP4V82 and CYP4BB4 expressions in P. aibuhitensis, which may have different efficiencies in the detoxification of PAHs.

Expression Profile of Hydroxysteroid Dehydrogenase-like 2 in Polychaete Perinereis aibuhitensis in Response to BPA.

Hydroxysteroid dehydrogenases (HSDs) play an important role in the metabolism of steroids and xenobiotics. However, the function of HSDs in invertebrates is unclear. In this study, we cloned the hydroxysteroid dehydrogenase-like 2 (HSDL2) gene in Perinereis aibuhitensis, which is 1652 bp in length, encoding 400 amino acids. This sequence contains conserved short-chain dehydrogenase and sterol carrier protein-2 domain, and the alignment analysis showed its close relationship with other invertebrate HSDL2. Further, the tissue distribution analysis of the HSDL2 gene showed it is expressed strongly in the intestine. The expression level of HSDL2 after inducement with bisphenol A (BPA) was also detected both at transcriptional and translational levels. The results inferred that BPA exposure can induce HSDL2 expression, and the inductive effect was obvious in the high-concentration BPA group (100 µg/L). In summary, our results showed the detoxification function of HSDL2 in polychaetes.

Expression profile of a novel glutathione S-transferase gene in the marine polychaete Perinereis aibuhitensis in short-term responses to phenanthrene, fluoranthene, and benzo[α]pyrene.

Polychaete worms can eliminate polycyclic aromatic hydrocarbons (PAHs) in environments through a mechanism that increases their water solubility. This detoxification starts with cytochrome P450 enzymes (CYPs) and then with glutathione S-transferases (GSTs). Here, a novel GST gene was identified and characterized from the widespread polychaete Perinereis aibuhitensis. The full-length cDNA of GST is 1544 bp and encodes 256 amino acids, belonging to the omega class. Gene expression patterns in P. aibuhitensis showed that its transcriptional level was positively correlated with the concentration of benzo[α]pyrene (0.5, 2, 4, and 8 µg/L) exposure but was negatively correlated with a PAH benzene ring after it was exposed to the same mass concentrations of fluoranthene (3.2 µg/L), phenanthrene (2.9 µg/L), and benzo[α]pyrene (4.0 µg/L) during the 14-day experimentation. These findings indicate that omega GST may play an important role in the phase II detoxification of PAHs in polychaete worms, and the persistence and bioavailability of PAHs may depend on benzene rings.

Quantitative immunodetection of metalloprotein II in Perinereis aibuhitensis after heavy metal exposure.

The metal detoxification mechanism is essential for the accumulation activity of some polychaetes. In order to investigate the detoxification function of metalloprotein II in polychaete Perinereis aibuhitensis, a recombinant fusion protein of MP II was successfully expressed in vitro and an anti-MP II polyclonal antibody was got. The tissue distribution of MP II in P. aibuhitensis and the protein expression under Cd, Pb and Zn exposure were detected by ELISA with this antibody. The results showed that MP II was higher in the intestine of P. aibuhitensis, followed by the body wall and parapodium. Cd, Zn and Pb exposure can induced MP II expression, but the change trend of MP II under various heavy metal exposure was different. The amount of MP II induced in P. aibuhitensis increased with the rises of Cd concentration, but there is no significant relationship between Zn and Pb concentration and MP II level.

Molecular characterization of G-protein-coupled receptor (GPCR) and protein kinase A (PKA) cDNA in Perinereis aibuhitensis and expression during benzo(a)pyrene exposure.

BACKGROUND: G-protein-coupled receptors (GPCRs) are one of the most important molecules that transfer signals across the plasma membrane, and play central roles in physiological systems. The molecular architecture of GPCRs allows them to bind to diverse chemicals, including environmental contaminants. METHODS: To investigate the effects of benzo(a)pyrene (B(a)P) on GPCR signaling, GPCR and the protein kinase A (PKA) catalytic subunit of Perinereis aibuhitensis were cloned. The expression patterns of these two genes during B(a)P exposure were determined with real-time fluorescence quantitative PCR. The PKA content in P. aibuhitensis under B(a)P exposure was examined. RESULTS: The full-length cDNAs of PaGPCR and the PaPKA catalytic subunit were 1,514 and 2,662 nucleotides, respectively, encoding 338 and 350 amino acids, respectively. Multiple sequence alignments indicated that the deduced amino acid sequence of PaGPCR shared a low level of similarity with the orphan GPCRs of polychaetes and echinoderms, whereas PaPKA shared a high level of identify with the PKA catalytic subunits of other invertebrates. B(a)P exposure time-dependently elevated the expression of PaGPCR and PaPKA. The expression of both PaGPCR and PaPKA was also dose-dependent, except at a dose of 10 µg/L B(a)P. The PKA content in concentration group was elevated on day 4, with time prolonging the PKA content was down-regulated to control level. DISCUSSION: These results suggested that GPCR signaling in P. aibuhitensis was involved in the polychaete's response to environmental contaminants.

Characterization and phylogenetic analysis of the complete mitochondrial genome from Rock Scallop (Crassadoma gigantea) using next-generation sequencing.

In this study, the complete 18,495 bp mitochondrial genome was determined from Rock scallop (Crassadoma gigantea) using next-generation sequencing technology. The complete mitochondrial genome contained 12 protein-coding genes (PCGs), 2 ribosomal RNA genes, 23 transfer RNA genes, without ATP8 and D-loop, which was similar with most mitochondrial genomes of marine bivalve molluscs. Gene annotations, including gene order, genetic code, start and stop codons and codons bias, were identified. Phylogenetic tree was constructed using Neighbor-Joining (NJ) method based on the PCGs showed the present species clustered within the Pteriomorphia clade. This work should be of importance not only for the better understanding of the relationships within Pectinidae, but also for the development of useful genetic markers in Rock scallop aquaculture and restoration efforts.

Cu/Zn superoxide dismutase (SOD) and catalase (CAT) response to crude oil exposure in the polychaete Perinereis aibuhitensis.

Cu/Zn superoxide dismutase (SOD) and catalase (CAT) cDNAs from the polychaete Perinereis aibuhitensis were cloned and characterized in order to investigate the relationship between crude oil exposure and stress response in this worm. The full length of PaSOD was 870 bp and PaCAT was 1967 bp encoding 150 and 506 amino acids, respectively. Gene expression and enzyme activity of Cu/Zn SOD and CAT in response to crude oil contaminated soil (500, 1500, and 3000 mg/kg) were measured. The results showed that expression of the CAT gene and enzyme activity in P. aibuhitensis was positively correlated to the concentration of crude oil and reached a maximum at 15 days of exposure to 3000 mg/kg crude oil. The expression of the SOD gene and enzyme activity of SOD in P. aibuhitensis also increased during exposure to crude oil and reached a maximum at 10 days of exposure to 3000 mg/kg crude oil. These results indicated that SOD and CAT are important for maintaining the balance of cellular metabolism and protecting P. aibuhitensis from crude oil toxicity.

Bioenergetic trade-offs in the sea cucumber Apostichopus japonicus (Echinodermata: Holothuroidea) in response to CO2-driven ocean acidification.

Ocean acidification (OA) caused by excessive CO2 is a potential ecological threat to marine organisms. The impacts of OA on echinoderms are well-documented, but there has been a strong bias towards sea urchins, and limited information is available on sea cucumbers. This work examined the effect of medium-term (60 days) exposure to three pH levels (pH 8.06, 7.72, and 7.41, covering present and future pH variability) on the bioenergetic responses of the sea cucumber, Apostichopus japonicus, an ecologically and economically important holothurian in Asian coasts. Results showed that the measured specific growth rate linearly decreased with decreased pH, leading to a 0.42 %·day(-1) decrease at pH 7.41 compared with that at pH 8.06. The impacts of pH on physiological energetics were variable: measured energy consumption and defecation rates linearly decreased with decreased pH, whereas maintenance energy in calculated respiration and excretion were not significantly affected. No shift in energy allocation pattern was observed in A. japonicus upon exposure to pH 7.72 compared with pH 8.06. However, a significant shift in energy budget occurred upon exposure to pH 7.41, leading to decreased energy intake and increased percentage of energy that was lost in feces, thereby resulting in a significantly lowered allocation into somatic growth. These findings indicate that adult A. japonicus is resilient to the OA scenario at the end of the twenty-first century, but further acidification may negatively influence the grazing capability and growth, thereby influencing its ecological functioning as an "ecosystem engineer" and potentially harming its culture output.

User-friendly optimization approach of fed-batch fermentation conditions for the production of iturin A using artificial neural networks and support vector machine

Background In the field of microbial fermentation technology, how to optimize the fermentation conditions is of great crucial for practical applications. Here, we use artificial neural networks (ANNs) and support vector machine (SVM) to offer a series of effective optimization methods for the production of iturin A. The concentration levels of asparagine (Asn), glutamic acid (Glu) and proline (Pro) (mg/L) were set as independent variables, while the iturin A titer (U/mL) was set as dependent variable. General regression neural network (GRNN), multilayer feed-forward neural networks (MLFNs) and the SVM were developed. Comparisons were made among different ANNs and the SVM. Results The GRNN has the lowest RMS error (457.88) and the shortest training time (1 s), with a steady fluctuation during repeated experiments, whereas the MLFNs have comparatively higher RMS errors and longer training times, which have a significant fluctuation with the change of nodes. In terms of the SVM, it also has a relatively low RMS error (466.13), with a short training time (1 s). Conclusion According to the modeling results, the GRNN is considered as the most suitable ANN model for the design of the fed-batch fermentation conditions for the production of iturin A because of its high robustness and precision, and the SVM is also considered as a very suitable alternative model. Under the tolerance of 30%, the prediction accuracies of the GRNN and SVM are both 100% respectively in repeated experiments.

Evaluation models for soil nutrient based on support vector machine and artificial neural networks.

Soil nutrient is an important aspect that contributes to the soil fertility and environmental effects. Traditional evaluation approaches of soil nutrient are quite hard to operate, making great difficulties in practical applications. In this paper, we present a series of comprehensive evaluation models for soil nutrient by using support vector machine (SVM), multiple linear regression (MLR), and artificial neural networks (ANNs), respectively. We took the content of organic matter, total nitrogen, alkali-hydrolysable nitrogen, rapidly available phosphorus, and rapidly available potassium as independent variables, while the evaluation level of soil nutrient content was taken as dependent variable. Results show that the average prediction accuracies of SVM models are 77.87% and 83.00%, respectively, while the general regression neural network (GRNN) model's average prediction accuracy is 92.86%, indicating that SVM and GRNN models can be used effectively to assess the levels of soil nutrient with suitable dependent variables. In practical applications, both SVM and GRNN models can be used for determining the levels of soil nutrient.

Molecular cloning and expression of the gene for G protein alpha subunit induced by bisphenol A in marine polychaete Perinereis aibuhitensis.

A G protein alpha subunit gene named Pa Gα was isolated from the marine polychaete Perinereis aibuhitensis. The full-length cDNA of Pa Gα was 1832 bp and contained a 205 bp 5' untranslated region (5'UTR), a 565 bp 3' UTR and a 1062 bp open reading frame encoding 353 amino acid residues. The deduced protein sequence of Pa Gα showed 73% homology with the Gα protein of Dipolydora quadrilobata. Tissue-specific expression induced by exposure to bisphenol A (BPA) in P. aibuhitensis was detected by real-time PCR, which showed BPA induced expression of the Pa Gα gene, and the level of transcription was related positively to the concentration of BPA and the length of exposure time. With increasing concentration of BPA and length of exposure time, the level of mRNA transcription was raised gradually, but the level of increasing expression of Pa Gα mRNA induced by exposure to BPA varied significantly among different tissues.

Molecular cloning, sequencing, and expression analysis of cDNA encoding metalloprotein II (MP II) induced by single and combined metals (Cu(II), Cd(II)) in polychaeta Perinereis aibuhitensis.

We amplified and analyzed the complete cDNA of metalloprotein II (MP II) from the somatic muscle of the polychaete Perinereis aibuhitensis, the full length cDNA is 904 bp encoding 119 amino acids. The MP II cDNA sequence was subjected to BLAST searching in NCBI and was found to share high homology with hemerythrin of other worms. MP II expression of P. aibuhitensis exposed to single and combined metals (Cu(II), Cd(II)) was analyzed using real time-PCR. MP II mRNA expression increased at the start of Cu(II) exposure, then decreased and finally return to the normal level. Expression pattern of MP II under Cd(II) exposure was time- and dose-dependent. MP II expression induced by a combination of Cd(II) and Cu(II) was similar to that induced by Cd(II) alone.

CYP4 mRNA expression in marine polychaete Perinereis aibuhitensis in response to petroleum hydrocarbon and deltamethrin.

A CYP4 cDNA was cloned and characterized to identify the relationship between persistent organic pollutants and stress response in marine polychaete Perinereis aibuhitensis. The full length of PaCYP4 cDNA is 1857bp and encodes 481 amino acids. The deduced amino acid sequence showed 73% identity with CYP4BB1 from polychaete Nereis virens and shared high homology to other known CYP4 sequences. The expression level of PaCYP4 under petroleum hydrocarbon (PH) and deltamethrin (DM) exposure was detected using Real-time PCR. PH and combined toxicity treatments elevated the mRNA level of PaCYP4 in a dose- and time-dependent manner. The mRNA transcripts of PaCYP4 increased at the beginning of DM exposure and then eventually decreased, and the expression level of PaCYP4 down-regulated with increasing concentration of DM. CYP4 in P. aibuhitensis plays an important role in the metabolism of petroleum hydrocarbon and organochlorine pesticide.