Pancreatic lipase inhibitory activity of monogalactosyldiacylglycerols isolated from the freshwater microalga <Emphasis Type="Italic">Chlorella sorokiniana</Emphasis>

Chemical investigation of the freshwater microalga Chlorella sorokiniana led to the isolation of a monogalactosyldiacylglycerol (MGDG)-rich fraction possessing dose-dependent inhibitory activity against pancreatic lipase activity. The MGDG-rich fraction contains 12 MGDGs identified by LC/HRMS analysis. Among them, three MGDGs were new compounds, namely, (2S)-1-O-(7Z,10Z-hexadecadienoyl)-2-O-(7Z,10Z,13Z-hexadecatrienoyl)-3-O-β-D-galactopyranosylglycerol (1), (2S)-1-O-linoleoyl-2-O-(7Z,10Z-hexadecadienoyl)-3-O-β-D-galactopyranosylglycerol (6), and (2S)-1-O-oleoyl-2-O-(7Z,10Z-hexadecadienoyl)-3-O-β-D-galactopyranosylglycerol (8). The major galactolipids were isolated by semipreparative HPLC and tested for their effect toward pancreatic lipase inhibitory activity. All the tested MGDGs showed significant reduction of pancreatic lipase activity indicating possible beneficial use for management of lipase-related disorders such as obesity.     

An efficient method for the sequential production of lipid and carotenoids from the Chlorella Growth Factor-extracted biomass of <Emphasis Type="Italic">Chlorella vulgaris</Emphasis>

Efficient methodology for simultaneous extraction of multiple bioactive compounds from microalgae still remains a major challenge. The present study provides a method for the sequential production of three major products: Chlorella Growth Factor (CGF, a nucleotide-peptide complex enriched with vitamins, minerals, and carbohydrates), lipid, and carotenoids from Chlorella vulgaris biomass in an economically feasible manner. After protein-rich CGF was extracted, the spent biomass was found to contain 12% lipid and 3% carotenoids when extracted individually, compared to that of the un-utilized (fresh) biomass (lipid, 14%; carotenoids, 4%). When extracted simultaneously using conventional methods, the yield of lipid from “CGF and carotenoids-extracted biomass,” and carotenoids from “CGF and lipid-extracted biomass” were significantly reduced (50%). However, simultaneous extraction using different solvent mixtures such as hexane:methanol:water and pentane:methanol:water mixture-augmented lipid yield by 38.5% and carotenoids by 14%, and additionally retained chlorophyll and its derivatives. Column chromatographic approach yielded sequential production of lipid (18%), lutein (9%) with better yields as well as without chlorophyll interference. Different geometric isomers of lutein all-E-(trans)-(3R,3′R,6′R)-β,ε-carotene-3,3′diol, 9Z(cis)-(3R,3′R,6′R)-β,ε-carotene-3,3′diol, and 13Z(cis)-(3R,3′R,6′R)-β,ε-carotene-3,3′diol were purified by HPLC and elucidated by CD, UV, NMR, FT-IR, and Mass spectra. In conclusion, the study provides an efficient and economically viable methodology for sequential production of lipid and lutein along with its geometrical isomers without chlorophyll influence and yield loss from the protein-rich CGF-extracted spent biomass of marine microalga, Chlorella vulgaris.     

One pot simultaneous preparation of both enantiomer of β-amino alcohol and vicinal diol via cascade biocatalysis

Objectives

To investigate the efficiency of a new cascade biocatalysis system for the conversion of R, S-β-amino alcohols to enantiopure vicinal diol and β-amino alcohol.

Results

An efficient cascade biocatalysis was achieved by combination of a transaminase, a carbonyl reductase and a cofactor regeneration system. An ee value of > 99% for 2-amino-2-phenylethanol and 1-phenyl-1, 2-ethanediol were simultaneously obtained with 50% conversion from R, S-2-amino-2-phenylethanol. The generality of the cascade biocatalysis was further demonstrated with the whole-cell approaches to convert 10–60 mM R, S-β-amino alcohol to (R)- and (S)-diol and (R)- and (S)-β-amino alcohol in 90–99% ee with 50–52% conversion. Preparative biotransformation was demonstrated at a 50 ml scale with mixed recombinant cells to give both (R)- and (S)-2-amino-2-phenylethanol and (R)- and (S)-1-phenyl-1, 2-ethanediol in > 99% ee and 40–42% isolated yield from racemic 2-amino-2-phenylethanol.

Conclusions

This cascade biocatalysis system provides a new practical method for the simultaneous synthesis of optically pure vicinal diol and an β-amino alcohol.

     

Organic Solvent-Tolerant Marine Microorganisms as Catalysts for Kinetic Resolution of Cyclic β-Hydroxy Ketones

Chiral cyclic β-hydroxy ketones represent key motifs in the production of natural products of biological interest. Although the molecules are structurally simple, they require cumbersome synthetic steps to get access to them and their synthesis remains a challenge in organic chemistry. In this report, we describe a straightforward approach to enantiomerically enriched (R)- and (S)-3-hydroxycyclopentanone 2a, (R)- and (S)-3-hydroxycyclohexanone 2b, and (R)- and (S)-3-hydroxycycloheptanone 2c involving a transesterification resolution of the racemates using whole cells of marine microorganisms as catalysts and vinyl acetate the acyl donor and solvent. Twenty-six strains from a wide collection of isolates from marine sediments were screened, and seven strains were found to markedly catalyze the resolution in an asymmetric fashion. Using the strain Serratia sp., (R)-2a was isolated in 27% yield with 92% ee and (S)-2a in 65% yield with 43% ee, corresponding to an E-value of 37; (R)-2b was isolated in 25% yield with 91% ee and (S)-2b in 67% yield with 39% ee, corresponding to an E-value of 40; and (R)-2c was isolated in 30% yield with 96% ee and (S)-2c in 63% yield with 63% ee, corresponding to an E-value of 75.     

Effect of oxylipins on <Emphasis Type="Italic">Neurospora crassa</Emphasis> growth and differentiation

The effect of the natural oxylipins 3(R)-hydroxy-(5Z,8Z,11Z,14Z)-eicosatetraenoic acid (3-HETE) and 18-hydroxy-(9Z,12Z)-octadecadienoic acids (18-HODE) on the growth and hypha aggregation, as well as on some light-depending processes, such as carotenoid biosynthesis, protoperithecia formation (sexual cycle), and conidiation (asexual cycle), of the ascomycete Neurospora crassa was studied. Hypha aggregation and growth slowdown were induced by 3-HETE, 18-HODE, and linoleic acid. At concentrations from 5 to 50 μM, these compounds had no significant effect on the light-induced carotenogenesis. At the same time, these 3-HETE and 18-HODE concentrations, unlike linoleic acid, induced the formation of protoperithecia in the dark. At the concentration of 5 μM, an additive effect of oxylipins and light was revealed. The studied oxylipins had different effects on the asexual reproduction of N. crassa: 3-HETE induced conidiation in the dark, whereas 18-HODE induced conidiation in the light. The possible involvement of oxylipins in the regulation of the processes of sexual and asexual reproduction of N. crassa is discussed.     

Highly enantioselective production of a chiral intermediate of sitagliptin by a novel isolate of <Emphasis Type="Italic">Pseudomonas pseudoalcaligenes</Emphasis>

Objective

To produce (S)-3-hydroxy-1-(3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-4-(2,4,5-trifluorophenyl)butan-1-one (S)-1 from 4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro [1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)-1-(2,4,5-trifluorophenyl)butan-2-one (2) by microbial bioreduction.

Results

A new isolate of Pseudomonas pseudoalcaligenes reduced enantioselectively prochiral ketone 2 to chiral alcohol (S)-1. Whole cells of the bacterium were tolerant towards 20 % (v/v) DMSO and 10 g 2/l. Under the optimal conditions, the preparative-scale bioreduction yielded (S)-1 at 90 % yield and >99 % ee. Cells could be re-used with the yield and ee of product being 45 % and >99 %, respectively, after five cycles.

Conclusion

Bioreduction using whole cells of P. pseudoalcaligenes is an attractive approach to produce (S)-1, as a chiral intermediate of the anti-diabetic drug, sitagliptin.

     

Steroid Compounds from Far Eastern Starfishes <Emphasis Type="Italic">Henricia aspera</Emphasis> and <Emphasis Type="Italic">H. tumida</Emphasis>

Six new natural compounds were isolated from two Far Eastern starfish species, Henricia aspera and H. tumida, collected in the Sea of Okhotsk. Two new glycosylated steroid polyols were obtained from H. aspera: asperoside A and asperoside B, which were shown to be (20R,24R, 25S)-3-O-(2,3-di-O-methyl-β -D-xylopyranosyl)-24-methyl-5α-cholest-4-ene-3β, 6β,8,15α,16β,26-hexaol and (20R, 24R,25S,22E)-3-O-(2,4-di-O-methyl-β-D-xylopyranosyl)-24-methyl-5α-cholest-22-ene-3β,4β,6β,8,15α,26-hexaol, respectively. Two other glycosylated polyols, tumidoside A, with the structure elucidated as (20R, 22E)-3-O-(2,4-di-O-methyl-β -D-xylopyranosyl)-26,27-dinor-24-methyl-5α-cholest-22-ene-3β,4β,6β,8,15α,25-hexaol, and tumidoside B, whose structure was elucidated as (20R,24S)-3-O-(2,3-di-O-methyl-β-D-xylopyranosyl)-5α-cholestan-3β,4β,6β,8,15α,24-hexaol, were isolated from the two starfish species. (20R, 24S)-5α-Cholestan-3β,6β,15α,24-tetraol and (20R, 24S)-5α-cholestan-3β,6β,8,15α,24-pentaol were identified only in H. tumida. The known monoglycosides henricioside H1 and laeviuscolosides H and G were also identified in both species.     

Behavioral responses of <Emphasis Type="Italic">Hyalesthes obsoletus</Emphasis> to host-plant volatiles cues

The polyphagous planthopper Hyalesthes obsoletus Signoret is considered to be the principal vector of stolbur phytoplasma, which is associated with yellow diseases of grapevine. To explore the possibility of developing novel control strategies, the behavioral responses to six synthetic mixtures and nine single compounds, previously identified from the headspace of Vitex agnus-castus L. (chaste tree) and Urtica dioica L. (nettle), were investigated in Y-tube bioassays. Choice tests revealed differences in the behavioral responses of males and females to the volatiles that they were exposed to. Males were attracted to a mixture containing (E)-β-caryophyllene, 1,8-cineole, (E,E)-α-farnesene, (E)-β-farnesene, and methyl salicylate (mixture 2). The addition of methyl benzoate to this five-compound mixture (mixture 3) did not attract males but elicited positive responses in females. Furthermore, females were attracted to a mixture containing (E)-β-caryophyllene, (E,E)-α-farnesene, (Z)-3-hexenyl acetate, (Z)-3-hexen-1-ol, and benzothiazole (mixture 4), but here addition of methyl salicylate (mixture 5) did not attract females. Neither males nor females showed attractivity or repellency toward the singly tested compounds. This study enhances knowledge on the interaction of insect behaviorally effective constituents in complex plant volatile mixtures. The attractive mixtures of plant volatiles identified suggest the possibility of using them in monitoring and management of H. obsoletus.     

Karyotypic study of five Lutjanid species using conventional and Ag-NORs banding techniques

The first cytogenetic comparisons of five snapper species from Thailand were presented here. Renal cell samples were taken from blacktail snapper (Lutjanus fulvus), five lined snapper (L. quinquelineatus), dory snapper (L. fulviflamma), brownstripe red snapper (L. vitta), and mangrove red snapper (L. argentimaculatus). The mitotic chromosome preparation was prepared directly from kidney cells. Conventional staining and Ag-NOR banding techniques were applied to stain the chromosomes. The results exhibited that all five snapper species have the diploid chromosome numbers of 2n = 48 and the fundamental numbers (NF) of 48. The presences of large, medium, and small telocentric chromosomes were 22-24-2, 24-20-4, 36-10-2, 28-16-4 and 36-10-2, respectively. The Ag- NORs banding technique provides the pair of nucleolar organizer regions (NORs) at subcentromeric region of the long arm of the respective telocentric chromosome pairs 9, 1, 3, 4 and 9. Their karyotype formulas is as follows: L. fulvus (2n = 48): L ₂₂ ^t + M ₂₄ ^t + S ₂ ^t , L. quinquelineatus (2n = 48): L ₂₄ ^t + M ₂₀ ^t + S ₄ ^t , L. fulviflamma (2n = 48): Lt36 + Mt10 + St2, L. vitta (2n = 48): L ₂₈ ^t + M ₁₆ ^t + S ₄ ^t , and L. argentimaculatus (2n = 48): L ₃₆ ^t + M ₁₀ ^t + S ₂ ^t .     

Polyhydroxylated steroid compounds from the Far Eastern starfish <Emphasis Type="Italic">Distolasterias nipon</Emphasis>

Two new steroid glycosides: distolasteroside D₆, (24S)-24-O-(β-D-xylopyranosyl)-5α-cholestane-3β,6α,8,15β,16β,24-hexaol, and distolasteroside D₇, (22E,24R)-24-O-(β-D-xylopyranosyl)-5α-cholest-22-ene-3β,6α,8,15β,24-pentaol were isolated along with the previously known distolasterosides D₁, D₂, and D₃, echinasteroside C, and (25S)-5α-cholestane-3β4β,6α,7α,8,15α,16β,26-octaol from the Far Eastern starfish Distolasterias nipon. The structures of new compounds were elucidated by NMR spectroscopy and MALDI TOF mass spectrometry. Like neurotrophins, distolasterosides D₁, D₂, and D₃ were shown to induce neuroblast differentiation in a mouse neuroblastoma C1300 cell culture.     

5-Lipoxygenase-mediated Endogenous DNA Damage

Lipoxygenases (LOs) convert polyunsaturated fatty acids into lipid hydroperoxides. Homolytic decomposition of lipid hydroperoxides gives rise to endogenous genotoxins such as 4-oxo-2(E)-nonenal, which cause the formation of mutagenic DNA adducts. Chiral lipidomics analysis was employed to show that a 5-LO-derived lipid hydroperoxide was responsible for endogenous DNA-adduct formation. The study employed human lymphoblastoid CESS cells, which expressed both 5-LO and the required 5-LO-activating protein (FLAP). The major lipid peroxidation product was 5(S)-hydroperoxy-6,8,11,14-(E,Z,Z,Z)-eicosatetraenoic acid, which was analyzed as its reduction product, 5(S)-hydroxy-6,8,11,14-(E,Z,Z,Z)-eicosatetraenoic acid (5(S)-HETE)). Concentrations of 5(S)-HETE increased from 0.07 ± 0.01 to 45.50 ± 4.05 pmol/10⁷ cells upon stimulation of the CESS cells with calcium ionophore A23187. There was a concomitant increase in the 4-oxo-2(E)-nonenal-derived DNA-adduct, heptanone-etheno-2′-deoxyguanosine (HϵdGuo) from 2.41 ± 0.35 to 6.31 ± 0.73 adducts/10⁷ normal bases. Biosynthesis of prostaglandins, 11(R)-hydroxy-5,8,12,14-(Z,Z,E,Z)-eicosatetraenoic acid, and 15(R,S)-hydroxy-5,8,11,13-(Z,Z,Z,E)-eicosatetraenoic acid revealed that there was cyclooxygenase (COX) activity in the CESS cells. Western blot analysis revealed that COX-1 was expressed by the cells, but there was no COX-2 or 15-LO-1. FLAP inhibitor reduced HϵdGuo-adducts and 5(S)-HETE to basal levels. In contrast, aspirin, which had no effect on 5(S)-HETE, blocked the formation of prostaglandins, 15-HETE, and 11-HETE but did not inhibit HϵdGuo-adduct formation. These data showed that 5-LO was the enzyme responsible for the generation of the HϵdGuo DNA-adduct in CESS cells.PUFAs² can be converted into lipid hydroperoxides enzymatically by the action of LOs (1) and COXs (2) or nonenzymatically by reactive oxygen species (ROS) (3). Arachidonic acid, one of the essential PUFAs present in cell membranes, is released from phospholipids by different phospholipase A₂ isoforms upon diverse physical, chemical, inflammatory, and mitogenic stimuli (4). The free arachidonic acid then serves as a substrate for LOs, COXs, or ROS to produce a variety of lipid hydroperoxides (5, 6). ROS, 12-LO, and 15-LO can also act directly upon arachidonic acid esterified in phospholipids to produce lipid hydroperoxides (7), which are reduced (8), hydrolyzed by phospholipase A₂ (4), and then secreted as the corresponding free HETEs (9). COX-2-mediated (10) and 15-LO-1-mediated (11) metabolism of linoleic acid results in the formation of 13(S)-hydroperoxy-9,11-(Z,E)-octadecadienoic acid, which is rapidly reduced to 13(S)-hydroxy-9,11-(Z,E)-octadecadienoic acid (HODE) and secreted from cells. Arachidonic acid is specifically metabolized by 5-LO into 5(S)-HpETE, which is either reduced to 5(S)-HETE or serves as precursor to the formation of leukotrienes (LTs) (Scheme 1) (12). In contrast, ROS-mediated reactions produce racemic mixtures of all possible regioisomers of HpETEs and 13(S)-hydroperoxy-9,11-(Z,E)-octadecadienoic acids (3) that are subsequently secreted from cells as complex mixtures of racemic HETEs and HODEs. Therefore, the ability to analyze different HETE and HODE enantiomers and regioisomers is important for elucidating specific cellular lipid peroxidation pathways (13).Open in a separate windowSCHEME 1.5-LO-mediated formation of arachidonic acid metabolites and dGuo-adducts. HPNE, 4-hydroperoxy-2(E)-nonenal; DOOE, dioxo-6-octenoic acid.The conversion of arachidonic acid to 5(S)-HpETE by 5-LO is critically dependent upon the presence of FLAP (14). 5-LO and FLAP are expressed primarily in inflammatory cells such as polymorphonuclear leukocytes, monocytes, macrophages, and mast cells (12, 15–17). Therefore, 5-LO-mediated LT formation is thought to play a critical role in inflammation and allergic disorders (18–21). In addition, a number of studies have implicated 5-LO-derived arachidonic acid metabolites as mediators of atherogenesis and heart disease (12, 22, 23). The 5-LO pathway of arachidonic acid metabolism has also been proposed to play a role in prostate and pancreatic cancer (24–26).Lipid hydroperoxides undergo homolytic decomposition into bifunctional electrophiles such as 4-hydroxy-2(E)-nonenal, ONE, 4,5-epoxy-2(E)-decenal, and 4-hydroperoxy-2(E)-nonenal (27). These bifunctional electrophiles are highly reactive and can readily modify intracellular molecules including glutathione (GSH) (28, 29), DNA, (5, 6), and proteins (30, 31). Our previous in vitro studies characterized the bifunctional electrophiles ONE and 4-hydroperoxy-2(E)-nonenal as major products arising from the homolytic decomposition of 5-LO-derived 5(S)-HpETE (32). Reactions with DNA resulted in the formation of etheno-2′-deoxyguanosine (ϵdGuo) from 4-hydroperoxy-2(E)-nonenal and heptanone-ϵdGuo (HϵdGuo) from ONE (Scheme 1). 5,8-Dioxo-6-octenoic acid, a bifunctional electrophile from the carboxyl terminus of 5(S)-HpETE, gave rise to the novel DNA-adduct carboxypentanone-ϵdGuo (CPϵdGuo)-adduct as shown in Scheme 1.Previous studies have demonstrated that lipid hydroperoxides generated by COX-2 could lead to the formation of endogenous DNA adducts in epithelial cells (6). Cellular 5-LO, like COX-2, synthesizes lipid hydroperoxides on the nuclear membrane. Therefore, it is highly possible that 5-LO could also mediate the formation of lipid hydroperoxide-derived endogenous DNA adducts in cells. CESS is a human lymphoblastic cell line that expresses both 5-LO and FLAP, and they have been used as a model for inflammatory cells to examine the role of 5-LO metabolites in signal transduction (33, 34). In the present study, CESS cells provided an ideal model to elucidate the relationship of 5-LO mediated-lipid peroxidation and DNA-adduct formation in a cellular setting. Stable isotope dilution chiral LC-electron capture (EC) APCI/MRM/MS (13) was used to monitor the concomitant formation of lipid hydroperoxides in the presence of different enzyme stimulator or inhibitors. DNA-adduct formation in the same cells was measured by a stable isotope dilution APCI/MRM/MS method. The powerful tool of chiral lipid analysis enabled us to dissect the complicated lipid peroxidation pathways and to correlate them with endogenous DNA-adduct formation. The results demonstrated that 5-LO-mediated lipid peroxidation could cause HϵdGuo formation in cells. This novel finding provided additional explanation for the previous observation that increased 5-LO activity was associated with cancers and cardiovascular diseases (24–26).     

Mechanisms and reactivity differences for the cobalt-catalyzed enantioselective intramolecular hydroacylation of ketones and alkenes: insights from density functional calculations

Density functional theory (DFT) was used to study the cobalt(I)-catalyzed enantioselective intramolecular hydroacylation of ketones and alkenes. All intermediates and transition states were fully optimized at the M06/6-31G(d,p) level (LANL2DZ(f) for Co). The results demonstrated that the ketone and alkene present different reactivities in the enantioselective hydroacylation. In ketone hydroacylation catalyzed by the cobalt(I)–(R,R)-Ph-BPE complex, reaction channel “a” to (R)-phthalide was more favorable than channel “b” to (S)-phthalide. Hydrogen migration was both the rate-determining and chirality-limiting step, and this step was endothermic. In alkene hydroacylation catalyzed by the cobalt(I)–(R,R)-BDPP complex, reaction channel “c” leading to the formation of (S)-indanone was the most favorable, both thermodynamically and kinetically. Reductive elimination was the rate-determining step, but the chirality-limiting step was hydrogen migration, which occurred easily. The results also indicated that the alkene hydroacylation leading to (S)-indanone formation was more energetically favorable than the ketone hydroacylation that gave (R)-phthalide, both thermodynamically and kinetically.

Open image in new window

Graphical abstract A DFT study demonstrated that the ketone and alkene in the cobalt(I)-catalyzed enantioselective intramolecular hydroacylation showed different reactivities

     

Comparing the methods for quantifying the culm surface area of bamboo, <Emphasis Type="Italic">Phyllostachys pubescens</Emphasis>

We compared several methods for quantifying the culm surface area (S) of one of the most common bamboos in Japan, Phyllostachys pubescens Mazel ex Houz. Nine sample culms of P. pubescens were felled, and the true S was determined by the fine resolution analysis of the culm form (S _FRA). The S was then calculated independently with the sectional measurement method from the successive diameters measured at equal intervals of one-twentieth (S ₂₀), one-tenth (S ₁₀), one-fifth (S ₅) and one-half (S ₂) of the total culm length. The S was also quantified geometrically from the total culm length and the diameter at breast height or at base by assuming that the culm form could be approximated by a cone (S _DBH and S _DAB). The S _FRA was compared with each of the computed S values. For S ₁₀, S ₅ and S ₂, both the mean relative bias (%BIAS) and relative root mean square error (%RMSE) decreased with an increase in the number of measured diameters. The %BIAS and %RMSE of the S ₁₀ were, respectively, comparable and smaller compared to those of S ₂₀. The bias of the S _DBH and S _DAB suggested that the cone assumption of the culm form was violated. In conclusion, we recommend that the S should be quantified from the successive diameters measured at equal intervals of one-tenth of the total culm length using the sectional measurement method.     

Changes in defense allomone compositions of <Emphasis Type="Italic">Eutrichodesmus elegans</Emphasis> and <Emphasis Type="Italic">Eutrichodesmus</Emphasis><Emphasis Type="Italic">armatus</Emphasis> (Polydesmida: Haplodesmidae) during different stages of their life cycles

A mixture of E- and Z-(2-nitrovinyl)benzenes is a known allomone of two adult haplodesmid millipedes, Eutrichodesmus elegans (Miyosi) (Polydesmida: Haplodesmidae) and Eutrichodesmus armatus (Miyosi), as is (2-nitroethyl)benzene in E. armatus. However, the proportions of these compounds have not yet been studied in detail at the nymph stage. In the present study, the ratios of these three nitro compounds were shown to change during ontogenetic development. (2-Nitroethyl)benzene was newly detected as the second major component of the mixture in both species at stage I, just after eggs hatched (mean 43.0% in E. armatus and 7.8% in E. elegans), decreasing rapidly to less than 0.1% during growth. These changes occurred in a species-specific manner; field-collected E. armatus maintained a characteristic mixture of E- and Z-(2-nitrovinyl)benzenes (59.9–98.2 and 40.0–1.4%, respectively) during all stages including the adult stage. On the other hand, E. elegans contained E-(2-nitrovinyl)benzene as the major component (98.7–99.7%) with Z-(2-nitrovinyl)benzene as a trace component (less than 1.2%), while a minute amount of (2-nitroethyl)benzene was always retained during all nymph and adult stages. No volatiles were detected in eggs before hatching, and sequential changes of composition were observed among the three compounds after emergence in both species.     

Species- and density-dependent induction of volatile organic compounds by three mite species in cassava and their role in the attraction of a natural enemy

Upon damage by herbivores, plants induce an array of volatile organic compounds (VOCs) that mediate ecological interactions involving communication with organisms of the second and third trophic levels. VOC-mediated tritrophic interactions have largely been studied in various systems, including cassava (Manihot esculenta), but little is known about the chemical nature of herbivore-induced VOCs in this crop and the response they evoke in natural enemies. Several tetranychid and predatory mites are associated with cassava. Here, VOC emissions from uninfested plants and plants infested with 200 or 400 Mononychellus tanajoa, a specialist herbivore on cassava, and the generalists Tetranychus urticae and T. gloveri were measured. Dual-choice experiments were also conducted to assess the preference of inexperienced (reared on prey-infested bean plants) and experienced (adapted on prey-infested cassava plants) predatory mites, Neoseiulus idaeus (Phytoseiidae), between odors of uninfested plants versus odors of plants infested with M. tanajoa, T. urticae or T. gloveri. Two hundred individuals significantly increased the emissions of (Z)-3-hexen-1-ol, (E)-β-ocimene, β-caryophyllene, alloaromadendrene and (E)-geranyl acetone in T. urticae-infested plants, and (E)-β-ocimene and methyl salicylate (MeSA) in T. gloveri-infested plants. Four hundred individuals significantly increased the emissions of (Z)-3-hexen-1-ol, MeSA, α-pinene and D-limonene in M. tanajoa-infested plants. In addition, T. urticae at this density induced (E)-β-ocimene, D-limonene, (E)-geranyl acetone and six compounds that were not detected in other treatments. Tetranychus gloveri-infested plants induced the emissions of (E)-2-hexenal and D-limonene. Regardless of the infesting species, inexperienced N. idaeus did not discriminate between uninfested or infested plants. Upon experience, they discriminated between the odors of uninfested and T. urticae-damaged plants. Our findings reveal that mite infestations in cassava result in density-dependent and species-specific emission of VOCs, and that N. idaeus relies on associative learning to forage for its prey.     

Steroid compounds from two pacific starfish of the genus <Emphasis Type="Italic">Evasterias</Emphasis>

Three new steroid glycosides (evasteriosides C, D, and E) along with six known compounds were isolated from two Pacific starfish of the genus Evasterias. Evasterioside C from E. retiferacollected from the Sea of Japan was identified as (20R, 22E)-3-O-(β-D-xylopyranosyl)-24-nor-5α-cholest-22-ene-3β,6β,15α,26-pentaol 26-sulfate sodium salt. The structures of evasteriosides D and E from E. echinosoma (collected from the Gulf of Shelichov, the Sea of Okhotsk) were established as (20R, 24S)-24-O-(β-D-glucopyranosyl)-5α-cholestane-3β,6α,8,15β,24-pentaol and (20R,24S)-3,24-di-O-(β-D-xylopyranosyl)-cholest-4-ene-3β,6β,8,15α,24-pentaol, respectively. In addition, the known compounds pycnopodiosides A and C, luridoside A, 5α-cholestane-3β,6α,8,15β,16β,26-hexaol. 5α-Cholestane-3β,6α,8,15β,24-pentaol 24-sulfate sodium saltand marthasterone sulfate sodium salt were identified in E. echinosoma. The structures of the isolated compounds were established on the basis of spectroscopic analyses, using 1D and 2D NMR techniques, mass spectrometry, and some chemical transformations.     

Identification of 21 novel <Emphasis Type="Italic">S-RNase</Emphasis> alleles and determination of <Emphasis Type="Italic">S</Emphasis>-genotypes in 66 loquat (<Emphasis Type="Italic">Eriobotrya</Emphasis>) accessions

As observed in other self-incompatible species in the Pyrinae subtribe, loquat (Eriobotrya japonica) demonstrates gametophytic self-incompatibility that is controlled by the S-locus, which encodes a polymorphic stylar ribonuclease (S-RNase). This allows the female reproductive organ (style) to recognize and reject the pollen from individuals with the same S-alleles, but allows the pollen from individuals with different S-alleles to effect fertilization. The S-genotype is therefore an important consideration in breeding strategies and orchard management. In an attempt to optimize the selection of parental lines in loquat production, the S-RNase alleles of 35 loquat cultivars and their 26 progeny, as well as five wild loquat species, were identified and characterized in this study. The best pollinizer cultivar combinations were also explored. A total of 28 S-alleles were detected, 21 of which constituted novel S-RNase alleles. The S-haplotypes S₂ and S₆ were the most frequent, followed by S ₂₉ , S ₃₁ , S ₅ , S ₂₄ , S ₂₈ , S ₃₃ , S ₃₄ , S ₃₂ , and S ₁₅ , while the rare alleles S ₁ , S ₉ , S ₁₄ , S ₁₆ , S ₁₇ , S ₁₈ , S ₁₉ , S ₂₀ , S ₂₁ , S ₂₂ , S ₂₃ , S ₂₇ , and S ₃₅ were only observed in one of the accessions tested. Moreover, the S-genotypes of five wild loquat species (E. prinoides, E. bengalensis, E. prinoides var. dadunensis, E. deflexa, and E. japonica) are reported here for the first time. The results will not only facilitate the selection of suitable pollinators for optimal orchard management, but could also encourage the crossbreeding of wild loquat species to enhance the genetic diversity of loquat cultivars.     

Acaricidal and repellent effects of <Emphasis Type="Italic">Cnidium officinale</Emphasis>-derived material against <Emphasis Type="Italic">Dermanyssus gallinae</Emphasis> (Acari: Dermanyssidae)

The acaricidal activity of a methanolic extract and fractions from the rhizome of Cnidium officinale against Dermanyssus gallinae adults was investigated. The C. officinale methanolic extract exhibited 100% acaricidal activity after 48 h of treatment at a dose of 4000 ppm. The acaricidal constituents of the plant were sequentially partitioned with several solvents and then purified using silica gel column chromatography and high-performance liquid chromatography. Gas chromatography–mass spectrometry and nuclear magnetic resonance spectroscopy revealed (Z)-ligustilide as a constituent of C. officinale. Acaricidal activity was examined in three experimental tests (spray, fumigation and contact), with the spraying method being the most effective. The methanolic extract of C. officinale showed both contact and fumigant activities, though only fumigant activity was observed with (Z)-ligustilide. The fumigant effects of the methanolic extract and (Z)-ligustilide caused 86.5 and 62.6% mortality, respectively, of D. gallinae adults at 48 h. Among (Z)-ligustilide, acaricides (bifenthrin, cypermethrin and spinosad) and butylidenephthalide, bifenthrin displayed the highest acaricidal activity, and the activity of butylidenephthalide was 2.3-fold higher than that of (Z)-ligustilide. These results suggest that C. officinale-derived material can be used for the development of a control agent for D. gallinae.