Production,Characterization, and Antioxidant Activity of Fucoxanthin from the Marine Diatom Odontella aurita

The production, characterization, and antioxidant capacity of the carotenoid fucoxanthin from the marine diatom Odontella aurita were investigated. The results showed that low light and nitrogen-replete culture medium enhanced the biosynthesis of fucoxanthin. The maximum biomass concentration of 6.36 g L⁻¹ and maximum fucoxanthin concentration of 18.47 mg g⁻¹ were obtained in cultures grown in a bubble column photobioreactor (Ø 3.0 cm inner diameter), resulting in a fucoxanthin volumetric productivity of 7.96 mg L⁻¹ day⁻¹. A slight reduction in biomass production was observed in the scaling up of O. aurita culture in a flat plate photobioreactor, yet yielded a comparable fucoxanthin volumetric productivity. A rapid method was developed for extraction and purification of fucoxanthin. The purified fucoxanthin was identified as all-trans-fucoxanthin, which exhibited strong antioxidant properties, with the effective concentration for 50% scavenging (EC₅₀) of 1,1-dihpenyl-2-picrylhydrazyl (DPPH) radical and 2,2′-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid (ABTS) radical being 0.14 and 0.03 mg mL⁻¹, respectively. Our results suggested that O. aurita can be a natural source of fucoxanthin for human health and nutrition.     

Extraction of Fucoxanthin from Raw Macroalgae excluding Drying and Cell Wall Disruption by Liquefied Dimethyl Ether

Macroalgae are one of potential sources for carotenoids, such as fucoxanthin, which are consumed by humans and animals. This carotenoid has been applied in both the pharmaceutical and food industries. In this study, extraction of fucoxanthin from wet brown seaweed Undaria pinnatifida (water content was 93.2%) was carried out with a simple method using liquefied dimethyl ether (DME) as an extractant in semi-continuous flow-type system. The extraction temperature and absolute pressure were 25 °C and 0.59 MPa, respectively. The liquefied DME was passed through the extractor that filled by U. pinnatifida at different time intervals. The time of experiment was only 43 min. The amount of fucoxanthin could approach to 390 μg/g dry of wet U. pinnatifida when the amount of DME used was 286 g. Compared with ethanol Soxhlet and supercritical CO₂ extraction, which includes drying and cell disruption, the result was quite high. Thus, DME extraction process appears to be a good method for fucoxanthin recovery from U. pinnatifida with improved yields.     

Biological Properties of Fucoxanthin in Oil Recovered from Two Brown Seaweeds Using Supercritical CO2 Extraction

The bioactive materials in brown seaweeds hold great interest for developing new drugs and healthy foods. The oil content in brown seaweeds (Saccharina japonica and Sargassum horneri) was extracted by using environmentally friendly supercritical CO₂ (SC-CO₂) with ethanol as a co-solvent in a semi-batch flow extraction process and compared the results with a conventional extraction process using hexane, ethanol, and acetone mixed with methanol (1:1, v/v). The SC-CO₂ method was used at a temperature of 45 °C and pressure of 250 bar. The flow rate of CO₂ (27 g/min) was constant for the entire extraction period of 2 h. The obtained oil from the brown seaweeds was analyzed to determine their valuable compounds such as fatty acids, phenolic compounds, fucoxanthin and biological properties including antioxidant, antimicrobial, and antihypertension effects. The amounts of fucoxanthin extracted from the SC-CO₂ oils of S. japonica and S. horneri were 0.41 ± 0.05 and 0.77 ± 0.07 mg/g, respectively. High antihypertensive activity was detected when using mixed acetone and methanol, whereas the phenolic content and antioxidant property were higher in the oil extracted by SC-CO₂. The acetone–methanol mix extracts exhibited better antimicrobial activities than those obtained by other means. Thus, the SC-CO₂ extraction process appears to be a good method for obtaining valuable compounds from both brown seaweeds, and showed stronger biological activity than that obtained by the conventional extraction process.     

In Vivo Induction of Apoptosis by Fucoxanthin, a Marine Carotenoid, Associated with Down-Regulating STAT3/EGFR Signaling in Sarcoma 180 (S180) Xenografts-Bearing Mice

Previous in vitro researches have showed that fucoxanthin, a natural carotenoid isolated from sargassum, can inhibit proliferation or induce apoptosis in human neuroblastoma, hepatoma, leukemia, colon carcinoma, prostate cancer or urinary bladder cancer cells. But the precise mechanism by which fucoxanthin exerts anticarcinogenic effects is not yet fully understood. In this study, we performed an in vivo study to investigate the anti-tumor effect and mechanisms of fucoxanthin on xenografted sarcoma 180 (S180) in mice. Results revealed that fucoxanthin significantly inhibited the growth of sarcoma at the dose of 50 or 100 mg/kg. TUNEL analysis showed that the number of positive cells in the fucoxanthin-treated group was higher than that in the control group. Western blotting analysis also revealed the suppressed expression of bcl-2 and enhanced expression of cleaved caspase-3 by fucoxanthin. In addition, immunohistochemistry analysis and Western blotting analysis showed that fucoxanthin significantly decreased the expressions of survivin and vascular endothelial growth factor (VEGF). Most importantly, fucoxanthin inhibited the expressions of the epidermal growth factor receptor (EGFR) and STAT3 and phosphorylated STAT3 proteins. These results indicated that in vivo induction of apoptosis by fucoxanthin is associated with down-regulating STAT3/EGFR signaling in S180 xenografts-bearing mice.     

Ecklonia cava Attenuates PM2.5-Induced Cognitive Decline through Mitochondrial Activation and Anti-Inflammatory Effect

To evaluate the effects of Ecklonia cava (E. cava) on ambient-pollution-induced neurotoxicity, we used a mouse model exposed to particulate matter smaller than 2.5 µm in aerodynamic diameter (PM_2.5). The intake of water extract from E. cava (WEE) effectively prevented the learning and memory decline. After a behavioral test, the toll-like receptor (TLR)-4-initiated inflammatory response was confirmed by PM_2.5 exposure in the lung and brain tissues, and the WEE was regulated through the inhibition of nuclear factor-kappa B (NF-κB)/inflammasome formation signaling pathway and pro-inflammatory cytokines (IL-6 and IFN-γ). The WEE also effectively improved the PM_2.5-induced oxidative damage of the lungs and brain through the inhibition of malondialdehyde (MDA) production and the activation of mitochondrial activity (mitochondrial ROS content, mitochondria membrane potential (MMP), adenosine triphosphate (ATP) content, and mitochondria-mediated apoptotic molecules). In particular, the WEE regulated the cognition-related proteins (a decreased amyloid precursor protein (APP) and p-Tau, and an increased brain-derived neurotrophic factor (BDNF)) associated with PM_2.5-induced cognitive dysfunction. Additionally, the WEE prevented the inactivation of acetylcholine (ACh) synthesis and release as a neurotransmitter by regulating the acetylcholinesterase (AChE) activity, choline acetyltransferase (ChAT), and ACh receptor (AChR)-α3 in the brain tissue. The bioactive compounds of the WEE were detected as the polysaccharide (average Mw; 160.13 kDa) and phenolic compounds including 2′-phloroeckol.     

Supercritical Fluid Extraction of Fucoxanthin from the Diatom Phaeodactylum tricornutum and Biogas Production through Anaerobic Digestion

Phaeodactylum tricornutum is the marine diatom best known for high-value compounds that are useful in aquaculture and food area. In this study, fucoxanthin was first extracted from the diatom using supercritical fluid extraction (SFE) and then using the extracted diatom-like substrate to produce bioenergy through anaerobic digestion (AD) processes. Factors such as temperature (30 °C and 50 °C), pressure (20, 30, and 40 MPa), and ethanol (co-solvent concentration from 10% to 50% v/v) were optimized for improving the yield, purity, and recovery of fucoxanthin extracted using SFE. The highest yield (24.41% w/w) was obtained at 30 MPa, 30 °C, and 30% ethanol but the highest fucoxanthin purity and recovery (85.03mg/g extract and 66.60% w/w, respectively) were obtained at 30 MPa, 30 °C, and 40%ethanol. Furthermore, ethanol as a factor had the most significant effect on the overall process of SFE. Subsequently, P.tricornutum biomass and SFE-extracted diatom were used as substrates for biogas production through AD. The effect of fucoxanthin was studied on the yield of AD, which resulted in 77.15 ± 3.85 LSTP CH₄/kg volatile solids (VS) and 56.66 ± 1.90 LSTP CH₄/kg VS for the whole diatom and the extracted P.tricornutum, respectively. Therefore, P.tricornutuman can be considered a potential source of fucoxanthin and methane and both productions will contribute to the sustainability of the algae-biorefinery processes.     

Effect of an Introduced Phytoene Synthase Gene Expression on Carotenoid Biosynthesis in the Marine Diatom Phaeodactylum tricornutum

Carotenoids exert beneficial effects on human health through their excellent antioxidant activity. To increase carotenoid productivity in the marine Pennales Phaeodactylum tricornutum, we genetically engineered the phytoene synthase gene (psy) to improve expression because RNA-sequencing analysis has suggested that the expression level of psy is lower than other enzyme-encoding genes that are involved in the carotenoid biosynthetic pathway. We isolated psy from P. tricornutum, and this gene was fused with the enhanced green fluorescent protein gene to detect psy expression. After transformation using the microparticle bombardment technique, we obtained several P. tricornutum transformants and confirmed psy expression in their plastids. We investigated the amounts of PSY mRNA and carotenoids, such as fucoxanthin and β-carotene, at different growth phases. The introduction of psy increased the fucoxanthin content of a transformants by approximately 1.45-fold relative to the levels in the wild-type diatom. However, some transformants failed to show a significant increase in the carotenoid content relative to that of the wild-type diatom. We also found that the amount of PSY mRNA at log phase might contribute to the increase in carotenoids in the transformants at stationary phase.     

Vibrio vulnificus MO6-24/O Lipopolysaccharide Stimulates Superoxide Anion,Thromboxane B2, Matrix Metalloproteinase-9, Cytokine and Chemokine Release by Rat Brain Microglia in Vitro

Although human exposure to Gram-negative Vibrio vulnificus (V. vulnificus) lipopolysaccharide (LPS) has been reported to result in septic shock, its impact on the central nervous system’s innate immunity remains undetermined. The purpose of this study was to determine whether V. vulnificus MO6-24/O LPS might activate rat microglia in vitro and stimulate the release of superoxide anion (O₂⁻), a reactive oxygen species known to cause oxidative stress and neuronal injury in vivo. Brain microglia were isolated from neonatal rats, and then treated with either V. vulnificus MO6-24/O LPS or Escherichia coli O26:B6 LPS for 17 hours in vitro. O₂⁻ was determined by cytochrome C reduction, and matrix metalloproteinase-2 (MMP-2) and MMP-9 by gelatinase zymography. Generation of cytokines tumor necrosis factor alpha (TNF-α), interleukin-1 alpha (IL-1α), IL-6, and transforming growth factor-beta 1 (TGF-β1), chemokines macrophage inflammatory protein (MIP-1α)/chemokine (C-C motif) ligand 3 (CCL3), MIP-2/chemokine (C-X-C motif) ligand 2 (CXCL2), monocyte chemotactic protein-1 (MCP-1)/CCL2, and cytokine-induced neutrophil chemoattractant-2alpha/beta (CINC-2α/β)/CXCL3, and brain-derived neurotrophic factor (BDNF), were determined by specific immunoassays. Priming of rat microglia by V. vulnificus MO6-24/O LPS in vitro yielded a bell-shaped dose-response curve for PMA (phorbol 12-myristate 13-acetate)-stimulated O₂⁻ generation: (1) 0.1–1 ng/mL V. vulnificus LPS enhanced O₂⁻ generation significantly but with limited inflammatory mediator generation; (2) 10–100 ng/mL V. vulnificus LPS maximized O₂⁻ generation with concomitant release of thromboxane B₂ (TXB₂), matrix metalloproteinase-9 (MMP-9), and several cytokines and chemokines; (3) 1000–100,000 ng/mL V. vulnificus LPS, with the exception of TXB₂, yielded both attenuated O₂⁻ production, and a progressive decrease in MMP-9, cytokines and chemokines investigated. Thus concentration-dependent treatment of neonatal brain microglia with V. vulnificus MO6-24/O LPS resulted in a significant rise in O₂⁻ production, followed by a progressive decrease in O₂⁻ release, with concomitant release of lactic dehydrogenase (LDH), and generation of TXB₂, MMP-9, cytokines and chemokines. We hypothesize that the inflammatory mediators investigated may be cytotoxic to microglia in vitro, by an as yet undetermined autocrine mechanism. Although V. vulnificus LPS was less potent than E. coli LPS in vitro, inflammatory mediator release by the former was clearly more efficacious. Finally, we hypothesize that should V. vulnificus LPS gain entry into the CNS, it would be possible that microglia might become activated, resulting in high levels of O₂⁻ as well as neuroinflammatory TXB₂, MMP-9, cytokines and chemokines.     

Evaluation of Sensitivity to Phoxim and Cypermethrin in an Endoparasitoid,Meteorus pulchricornis (Wesmael) (Hymenoptera: Braconidae), and Its Parasitization Efficiency Under Insecticide Stress

Insecticides can have consequences for beneficial arthropods. Insect parasitoids can contact insecticides through direct exposure spray droplets or residues on crop foliage. Here, we focus on better understand the response of Meteorus pulchricornis (Wesmael), a parasitoid wasp of lepidopteran pests, and its detoxification mechanisms on stress caused by phoxim and cypermethrin. Hence, we determined the dose–mortality curves and estimating the sublethal concentrations (LC₃₀ and LC₅₀). Then, we applied the sublethal concentrations against adult parasitoids to assess its survival, parasitism efficacy, and also developmental and morphometric parameters of their offspring. Simultaneously, we check the activities of glutathione S-transferase (GST), acetylcholinesterase (AChE), and peroxidase (POD) after sublethal exposure of both insecticides, which has measured until 48 h after treatment. Overall, phoxim and cypermethrin exhibited acute lethal activity toward the parasitoid with LC₅₀ values 4.608 and 8.570 mg/liter, respectively. Also, we detect that LC₃₀ was able to trigger the enzymatic activity of GST, AChE, and POD, suggesting a potential detoxification mechanism. However, even when subjected to sublethal exposure, our results indicate strong negatives effects, in particular for phoxim, which has affected the parasitism efficacy and also the developmental and morphometric parameters of M. pulchricornis offspring. Therefore, it can be concluded that both phoxim and cypermethrin have negative impacts on M. pulchricornis and we suggest cautioning their use and the need for semifield and field assessments to confirm such an impact.     

Isolation,Characterization, Kinetics,and Enzymatic and Nonenzymatic Microbicidal Activities of a Novel c-Type Lysozyme from Plasma of Schistocerca gregaria (Orthoptera: Acrididae)

A protein, designated as Sgl, showing a muramidase lytic activity to the cell wall of the Gram-positive bacterium Micrococcus lysodeikticus was isolated for the first time from plasma of Escherichia coli-immunized fifth instar Schistocerca gregaria. The isolated Sgl was detected as a single protein band, on both native- and SDS-PAGE, has a molecular weight of ∼15.7 kDa and an isoelectric point (pI) of ca 9.3 and its antiserum has specifically recognized its isolated form. Fifty-nine percentage of Sgl lytic activity was recovered in the isolated fractions and yielded ca 126-fold increase in specific activity than that of the crude. The partial N-terminal amino acid sequence of the Sgl has 55 and 40% maximum identity with Bombyx mori and Gallus gallus c-type lysozymes, respectively. The antibacterial activity against the Gram-positive and the Gram-negative bacteria were comparatively stronger than that of the hen egg white lysozyme (HEWL). The detected Sgl poration to the inner membrane that reach a maximum ability after 3 h was suggested to operate as a nonenzymatic mechanism for Gram-negative bacterial cell lysis, as tested in a permease-deficient E. coli, ML-35 strain. Sgl showed a maximal muramidase activity at pH 6.2, 30–50°C, and 0.05 M Ca²⁺ or Mg²⁺; and has a K_m of 0.5 μg/ml and a V_max of 0.518 with M. lysodeikticus as a substrate. The Sgl displayed a chitinase activity against chitin with a K_m of 0.93 mg/ml and a V_max of 1.63.     

Fucoidan Suppresses Hypoxia-Induced Lymphangiogenesis and Lymphatic Metastasis in Mouse Hepatocarcinoma

Metastasis, the greatest clinical challenge associated with cancer, is closely connected to multiple biological processes, including invasion and adhesion. The hypoxic environment in tumors is an important factor that causes tumor metastasis by activating HIF-1α. Fucoidan, extracted from brown algae, is a sulfated polysaccharide and, as a novel marine biological material, has been used to treat various disorders in China, Korea, Japan and other countries. In the present study, we demonstrated that fucoidan derived from Undaria pinnatifida sporophylls significantly inhibits the hypoxia-induced expression, nuclear translocation and activity of HIF-1α, the synthesis and secretion of VEGF-C and HGF, cell invasion and lymphatic metastasis in a mouse hepatocarcinoma Hca-F cell line. Fucoidan also suppressed lymphangiogenesis in vitro and in vivo. In addition, accompanied by a reduction in the HIF-1α nuclear translocation and activity, fucoidan significantly reduced the levels of p-PI3K, p-Akt, p-mTOR, p-ERK, NF-κB, MMP-2 and MMP-9, but increased TIMP-1 levels. These results indicate strongly that the anti-metastasis and anti-lymphangiogenesis activities of fucoidan are mediated by suppressing HIF-1α/VEGF-C, which attenuates the PI3K/Akt/mTOR signaling pathways.     

Simultaneous Determination of Fucoxanthin and Its Deacetylated Metabolite Fucoxanthinol in Rat Plasma by Liquid Chromatography-Tandem Mass Spectrometry

Fucoxanthin and its deacetylated metabolite fucoxanthinol are two major carotenoids that have been confirmed to possess various pharmacological properties. In the present study, fucoxanthinol was identified as the deacetylated metabolite of fucoxanthin, after intravenous (i.v.) and intragastric gavage (i.g.) administration to rats at doses of 2 and 65 mg/kg, respectively, by liquid chromatography-tandem mass spectrometric (LC-MS/MS) analysis. Next, an accurate and precise LC-MS/MS method was developed to quantitatively determine fucoxanthin and fucoxanthinol in rat plasma. Plasma samples were resolved by LC-MS/MS on a reverse-phase SB-C18 column that was equilibrated and eluted with acetonitrile (A)/aqueous 0.1% formic acid (B; 92/8, v/v) at a flow rate of 0.5 mL/min. Analytes were monitored by multiple-reaction monitoring (MRM) under positive electrospray ionization mode. The precursor/product transitions (m/z) were 659.3→109.0 for fucoxanthin, 617.2→109.0 for fucoxanthinol, and 429.4→313.2 for the internal standard (IS). Calibration curves for fucoxanthin and fucoxanthinol were linear over concentrations ranging from 1.53 to 720 and 1.17 to 600 ng/mL, respectively. The inter- and intraday accuracy and precision were within ±15%. The method was applied successfully in a pharmacokinetic study and the resulting oral fucoxanthin bioavailability calculated.     

Antitumor and Antimicrobial Potential of Bromoditerpenes Isolated from the Red Alga,Sphaerococcus coronopifolius

Cancer and infectious diseases continue to be a major public health problem, and new drugs are necessary. As marine organisms are well known to provide a wide range of original compounds, the aim of this study was to investigate the bioactivity of the main constituents of the cosmopolitan red alga, Sphaerococcus coronopifolius. The structure of several bromoditerpenes was determined by extensive spectroscopic analysis and comparison with literature data. Five molecules were isolated and characterized which include a new brominated diterpene belonging to the rare dactylomelane family and named sphaerodactylomelol (1), along with four already known sphaerane bromoditerpenes (2–5). Antitumor activity was assessed by cytotoxicity and anti-proliferative assays on an in vitro model of human hepatocellular carcinoma (HepG-2 cells). Antimicrobial activity was evaluated against four pathogenic microorganisms: Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Candida albicans. Compound 4 exhibited the highest antimicrobial activity against S. aureus (IC₅₀ 6.35 µM) and compound 5 the highest anti-proliferative activity on HepG-2 cells (IC₅₀ 42.9 µM). The new diterpene, sphaerodactylomelol (1), induced inhibition of cell proliferation (IC₅₀ 280 µM) and cytotoxicity (IC₅₀ 720 µM) on HepG-2 cells and showed antimicrobial activity against S. aureus (IC₅₀ 96.3 µM).     

A Comparative In Vitro Evaluation of the Anti-Inflammatory Effects of a Tisochrysis lutea Extract and Fucoxanthin

In this study, we compared the effects of a Tisochrysis lutea (T. lutea) F&M-M36 methanolic extract with those of fucoxanthin (FX) at equivalent concentration, on lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. The T. lutea F&M-M36 methanolic extract contained 4.7 mg of FX and 6.22 mg of gallic acid equivalents of phenols per gram. HPLC analysis revealed the presence of simple phenolic acid derivatives. The T. lutea F&M-M36 extract exhibited a potent and concentration-dependent inhibitory activity against COX-2 dependent PGE2 production compared to FX alone. Compared to LPS, T. lutea F&M-M36 extract and FX reduced the expression of IL-6 and of Arg1 and enhanced that of IL-10 and of HO-1; T. lutea F&M-M36 extract also significantly abated the expression of NLRP3, enhanced mir-223 expression and reduced that of mir-146b, compared to LPS (p < 0.05). These findings indicate that T. lutea F&M-M36 methanolic extract has a peculiar anti-inflammatory activity against COX-2/PGE2 and NLRP3/mir-223 that might be attributable to the known anti-inflammatory effects of simple phenolic compounds found in the extract that may synergize with FX. Our data suggest that T. lutea F&M-M36 may serve as a source of anti-inflammatory compounds to be further evaluated in in vivo models of inflammation.     

Peridinin from the Marine Symbiotic Dinoflagellate,Symbiodinium sp., Regulates Eosinophilia in Mice

Peridinin and fucoxanthin, which are natural carotenoids isolated from a symbiotic dinoflagellate, Symbiodinium sp., and a brown alga, Petalonia fascia, respectively, were compared for inhibitory effects on delayed-type hypersensitivity in mice. The number of eosinophils at the site of inflammation and in peripheral blood was compared for the administration of peridinin and fucoxanthin applied by painting and intraperitoneally. Peridinin, but not the structurally-related fucoxanthin, significantly suppressed the number of eosinophils in both the ear lobe and peripheral blood. Furthermore, peridinin applied topically, but not administered intraperitoneally, suppressed the level of eotaxin in the ears of sensitized mice. Fucoxanthin weakly suppressed the concentration of eotaxin in ears only by intraperitoneal administration. Although both carotenoids inhibited the migration of eosinophils toward eotaxin, the inhibitory effect of peridinin was higher than that of fucoxanthin. Peridinin may be a potential agent for suppressing allergic inflammatory responses, such as atopic dermatitis, in which eosinophils play a major role in the increase of inflammation.     

Biochemical characterization of acetylcholinesterase, cytochrome P450 and cross-resistance in an omethoate-resistant strain of Aphis gossypii Glover

The degree of insecticide resistance, synergism effects, acetylcholinesterase (AChE) activity kinetics, specific activities of detoxification enzymes and cross-resistance were investigated in omethoate resistant and relatively susceptible strains of Aphis gossypii Glover. The resistant cotton aphid strain (ORR) exhibited 231.3-fold resistance to omethoate compared to the susceptible strain (OSS). Synergist piperonyl butoxide (PBO) dramatically increased the toxicity of omethoate in the resistant strain, while triphenyl phosphate (TPP) and diethyl maleate (DEM) did not exhibit synergism effects. The calculated AChE activity, V_max and K_m ratios of ORR to OSS were 0.1, 0.2 and 0.4, respectively. Based on analysis of IC₅₀ indices, enzyme inhibition experiments showed that AChE from the ORR strain was 10.6-, 3.2-, 6.2-, 10.5- and 4.4-fold more insensitive to inhibition by eserine, omethoate, paraoxon, paraoxon-methyl and malaoxon, respectively, than that from the OSS strain. The cytochrome P450-mediated O-demethylation activity (3.7-fold) and ethoxycoumarin-O-deethylase activity (2.6-fold) in the ORR strain were significantly higher than in the OSS strain. Specific activity of carboxylesterase (CarE) and glutathione S-transferase (GST) were not significantly different in both the ORR and OSS strains. Bioassay results indicated the ORR strain had developed high levels of cross-resistance to chlorpyrifos (24.2-fold), malathion (21.1-fold), acephate (10.2-fold), esfenvalerate (30.6-fold), methomyl (22.4-fold), carbofuran (33.2-fold), but had negative cross-resistance to bifenthrin (0.4-fold). Overall, these results demonstrate that reduced AChE sensitivity, combined with increased cytochrome P450 monooxygenase detoxification, plays an important role in the high levels of omethoate resistance and can cause cross-resistance to other insecticides in the ORR strain.     

Identification and Characterization of Three Chitinases with Potential in Direct Conversion of Crystalline Chitin into N,N′-diacetylchitobiose

Chitooligosaccharides (COSs) have been widely used in agriculture, medicine, cosmetics, and foods, which are commonly prepared from chitin with chitinases. So far, while most COSs are prepared from colloidal chitin, chitinases used in preparing COSs directly from natural crystalline chitin are less reported. Here, we characterize three chitinases, which were identified from the marine bacterium Pseudoalteromonas flavipulchra DSM 14401^T, with an ability to degrade crystalline chitin into (GlcNAc)₂ (N,N’-diacetylchitobiose). Strain DSM 14401 can degrade the crystalline α-chitin in the medium to provide nutrients for growth. Genome and secretome analyses indicate that this strain secretes six chitinolytic enzymes, among which chitinases Chia4287, Chib0431, and Chib0434 have higher abundance than the others, suggesting their importance in crystalline α-chitin degradation. These three chitinases were heterologously expressed, purified, and characterized. They are all active on crystalline α-chitin, with temperature optima of 45–50 °C and pH optima of 7.0–7.5. They are all stable at 40 °C and in the pH range of 5.0–11.0. Moreover, they all have excellent salt tolerance, retaining more than 92% activity after incubation in 5 M NaCl for 10 h at 4 °C. When acting on crystalline α-chitin, the main products of the three chitinases are all (GlcNAc)₂, which suggests that chitinases Chia4287, Chib0431, and Chib0434 likely have potential in direct conversion of crystalline chitin into (GlcNAc)₂.