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Insect Biochemistry and Molecular Biology 43 (2013) 991e996

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Insect Biochemistry and Molecular Biology

journal homepage: www.elsevier .com/locate/ ibmb

Biosynthesis of linoleic acid in Tyrophagus mites (Acarina: Acaridae)

Takako Aboshi a, Nobuhiro Shimizu b, Yuji Nakajima a, Yoshiyuki Honda d,Yasumasa Kuwahara b, Hiroshi Amano c, Naoki Mori a,*aDivision of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, JapanbDepartment of Bioenvironmental Science, Kyoto Gakuen University, Kameoka City, Kyoto 621-8555, JapancDivision of Environmental Science & Technology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japand Yamaguchi Prefectural Agriculture & Forestry, General Technology Center, OouchiMihori, Yamaguchi, Yamaguchi 753-0214, Japan

a r t i c l e i n f o

Article history:Received 25 June 2013Received in revised form30 July 2013Accepted 7 August 2013

Keywords:Tyrophagus mitesBiosynthesis of linoleic acidD12-DesaturaseEssential fatty acids

Abbreviations: CDL, curved desolvation line; DMelectron spray ionization; GC/MS, gas chromatographliquid chromatography mass spectrometry; LPC, lysopphospatidylethanolamine; PC, phosphatidylcholineamine; PI, phosphatidylinositol.* Corresponding author. Tel.: þ81 75 753 6307; fax

E-mail address: mokurin@kais.kyoto-u.ac.jp (N. M

0965-1748/$ e see front matter � 2013 Elsevier Ltd.http://dx.doi.org/10.1016/j.ibmb.2013.08.002

a b s t r a c t

We report here that Tyrophagus similis and Tyrophagus putrescentiae (Astigmata: Acaridae) have theability to biosynthesize linoleic acid [(9Z, 12Z)-9, 12-octadecadienoic acid] via a D12-desaturation step,although animals in general and vertebrates in particular appear to lack this ability. When the mites werefed on dried yeast enriched with d31-hexadecanoic acid (16:0), d27-octadecadienoic acid (18:2), producedfrom d31-hexadecanoic acid through elongation and desaturation reactions, was identified as a majorfatty acid component of phosphatidylcholines (PCs) and phosphatidylethanolamines (PEs) in the mites.The double bond position of d27-octadecadienoic acid (18:2) of PCs and PEs was determined to be 9 and12, respectively by dimethyldisulfide (DMDS) derivatization. Furthermore, the GC/MS retention time ofmethyl 9, 12-octadecadienoate obtained from mite extracts agreed well with those of authentic linoleicacid methyl ester. It is still unclear whether the mites themselves or symbiotic microorganisms areresponsible for inserting a double bond into the D12 position of octadecanoic acid. However, we presenthere the unique metabolism of fatty acids in the mites.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

The Tyrophagus genus comprises a group of primarily fungivo-rous mites, including the mould mites, commonly found indecaying organic matter and stored food products. Some Tyropha-gus mites are also phytophagous and can cause economic damageto plants. Tyrophagus similis Volgin, 1949 and Tyrophagus putres-centiae Schrank, 1781 are commonwith a wide distribution and areeconomically important pests attacking growing plants, as well asinfesting stored foodstuffs, and domestic dwellings (Griffiths,1979). T. similis is the mite species that causes the most damageto spinach in Japan (Kasuga and Amano, 2000). Damage by T. similisis mostly observed in greenhouses in early spring and late autumnwhen greenhouse temperatures are generally moderate.

DS, dimethyldisulfide; ESI,y mass spectrometry; LC/MS,hosphatidylcholine; LPE, lyso; PE, phosphatidylethanol-

: þ81 75 753 6312.ori).

All rights reserved.

T. putrescentiae is one of the most common and intensively studiedgroups of mites.

Linoleic acid [(9Z, 12Z)-9,12-octadecadienoic acid] is an essentialfatty acid for most animals, creating proper fluidity and perme-ability in cell membranes, and required as a precursor of prosta-glandins, thromboxanes, and leukotrienes. The biosynthesis oflinoleic acid is restricted to organisms capable of inserting a doublebond into the D12 position of oleic acid. Plants and fungi have theability to synthesize linoleic acid from oleic acid bound to phos-pholipids through the actions of D12-desaturase (Sperling et al.,2003). By contrast, animals in general and vertebrates in partic-ular appear to lack this ability. However, lower animals such asnematodes (Peyou-Ndi et al., 2000), protozoa (Sayanova et al.,2006), and insects (de Renobales et al., 1987; Zhou et al., 2008),have the ability to synthesize linoleic acid. This ability has beenreported in 16 insect species representing five orders, Orthoptera,Homoptera, Isoptera, Neuroptera and Coleoptera. The de novobiosynthesis of linoleic acid was also demonstrated in the landsnail, Bulimulis alternates mariae, and in the garden slug Arion cir-cumscriptus (Weinert et al., 1993).

In this study, we examined fatty acid metabolites of T. similisand T. putrescentiae fed on dried yeast enriched with d31-hex-adecanoic acid (16:0). We identified phospholipids containing

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Fig. 1. Comparison of the phospholipid composition of dried yeast, T. similis, and T. putrescentiae (mean � SEM, n ¼ 5). 1, LPC 16:0; 2, LPC 16:1; 3, LPC 18:0; 4, LPC 18:1; 5, LPC 18:2; 6,PC 26:0; 7, PC 26:1; 8, PC 28; 9, PC 28:1; 10, PC 28:4; 11, PC 30:1; 12, PC 16:0e16:1; 13, PC 16:1e16:1; 14, PC 34:1; 15, PC 34:2; 16, PC 34:3; 17, PC 18:0e18:2; 18, PC 18:1e18:2; 19, PC18:2e18:2; 20, LPE 16:1; 21, LPE 18:0; 22, LPE 18:1; 23, PE 16:0e16:1; 24, PE 16:1e16:1; 25, PE 16:0e18:1; 26, PE 16:1e18:1; 27, PE 34:3; 28, PE 18:0e18:2; 29, PE 18:1e18:2; 30, PE18:2e18:2; 31, PI 16:0e16:1; 32, PI 16:0e18:1/16:1e18:0; 33, PI 18:0e18:1; 34, PI 18:0e18:2; 35, PI 18:1e18:2.

T. Aboshi et al. / Insect Biochemistry and Molecular Biology 43 (2013) 991e996992

d27-octadecadienoic acid (18:2) as well as those containing d-labeled hexadecanoic acid (16:0), octadecanoic acid (18:0), andoctadecenoic acid (18:1). Furthermore, the d27-octadecadienoicacid was identified as linoleic acid by GC/MS. Thus, it wasconcluded that Tyrophagus mites have the ability to convert hex-adecanoic acid into a dienoic fatty acid, which was indeed linoleicacid.

2. Materials and methods

2.1. Mites

T. similis (Astigmata: Acaridae) is a strain derived from a spinachfield in Yamaguchi Prefecture, Japan. T. putrescentiae is maintainedin our laboratory. The culture lines have been established in thelaboratory and maintained for generations on dried yeast at 70%relative humidity and 25 �C. To know the metabolic pathway offatty acid, mites were fed on dried yeast enriched with d31-hex-adecanoic acid (16:0) (5%, w/w, Isotec, Tokyo) for 3 days.

2.2. Analysis of phospholipids

Lipids from each sample were extracted by the Bligh and Dyerprocedure (Bligh and Dyer, 1959). Fifteen male or female miteswere transferred with a needle from the stock culture into a ho-mogenizer and homogenized with 380 ml of a chloroform/meth-anol/water (2/1/0.8) mixture. After chloroform and water wereadded to the extract to give final proportions of 1/1/1 (v/v/v), thechloroform layer was separated and the solvent was evaporated

dry. The dry residue was dissolved with 1 ml of methanol andapplied to a Sep-Pak C8 cartridge (400 mg/cartridge, Waters, MA).Phospholipids were extracted with methanol (5 ml), concentratedin vaccuo, and dissolved in methanol (1 ml). One portion of thesamples (80 ml) was diluted with 20 ml of dipentadecanoylPC(500 ng/ml, methanol), as an internal standard, for quantitativeanalysis and then prepared samples (5 ml) were analyzed by LCMS-IT-TOF equipped with a Prominence HPLC system (Shimadzu,Kyoto). The CDL temperature was 250 �C, block heater temperaturewas 200 �C, voltage was 1.8 kV, nebulizer gas flow was 1.5 l/min,probe voltagewas 4.50 kV, ion accumulation timewas 30msec, andionization was ESI. A Capcell Pak C8 column (UG120, 50 � 2.0 mmI.D., Shiseido, Tokyo) was eluted (0.2 ml/min) with a gradient of70e99% acetonitrile in water containing 0.05% ammonium formateover 5 min and the final concentration was maintained for 15 min.The column temperature was kept at 40 �C.

2.3. Determination of 18:2 fatty acid double bond positions

The lipid extract of T. putrescentiae, as described above, wasdissolved in 1.7 ml of diethyl ether/ethanol (95/5), and then 42 ml ofCaCl2 (100 mM), 84 ml of TriseHCl (25 mM, pH 7.5), and 5 ml ofphospholipase A2 solution (50 units, SigmaeAldrich Japan K.K.,Tokyo) were added to the solution. After incubation at 40 �C for 2 h,the organic layer was separated, placed in a screw-capped vial, andevaporated dry. Residues were dissolved in 1 ml of solution con-taining 20% methanol in benzene and 0.5 ml of trimethylsilyldia-zomethane solution (2 M in hexane, SigmaeAldrich Japan K.K.).Trimethylsilyldiazomethane is a reagent which methylates only

Fig. 2. MS spectrum of PE (18:0e18:2) obtained from T. similis and T. putrescentiae mites fed on dried yeast and MS, MS2 spectra of PE (18:0e18:2) containing d-labeled acyl groupsfrom the mites fed on dried yeast enriched with d31-hexadecanoic acid. The incorporation of d31-hexadecanoic acid into PE (18:0e18:2) was observed in the mites.

T. Aboshi et al. / Insect Biochemistry and Molecular Biology 43 (2013) 991e996 993

free fatty acids in the sample. The solution was kept at room tem-perature for 30 min and then evaporated dry. Residues wereincubated with 0.3 ml of dimethyldidulfide (DMDS) containing4mg of I2 at 35 �C for 30min. After 1 ml of hexanewas added, a 10%aqueous Na2S2O3 solution was added with vigorous vortexing untilthe color of I2 disappeared. The mixture was allowed to stand for afew minutes, and an aliquot of the upper phase containing theresultant DMDS adducts was withdrawn and injected directly intothe GC/MS system (Vincenti et al., 1987).

GC/MS was performed with an Agilent 6890N GC linked to anAglient 5975B (Agilent Technologies, Santa Clara, CA) operated at69.9 eV with an HP-5ms capillary column (0.25 mm � 30 m,0.25 mm in film thickness) with helium carrier gas at 1.0 ml/min inthe splitless mode. The oven temperature was programmed to in-crease from 120 �C (2 min holding) to 290 �C at 10 �C/min and thenheld at 290 �C for 5 min. The injector temperature was maintainedat 220 �C, the ion source temperature at 300 �C, and the quadrupoletemperature at 150 �C. The software ChemStation (Agilent Tech-nologies) was used for data acquisition.

Methyl ester derivatives of Z/E-linoleic acid isomers (Supelco,Bellefonte, PA) were analyzed using the same GC/MS equipment ona CP Sil-88 capillary column (0.25 mm � 100 m, 0.20 mm in filmthickness, Chrompack, Middleburg) with helium carrier gas at0.6 ml/min in the splitless mode (Haas et al., 1999). The oventemperature was held at 120 �C (1 min), then raised from 120 to160 �C at 20 �C/min, from 160 to 180 �C at 0.5 �C/min, and from 180to 225 �C at 3 �C/min, and finally kept at 225 �C for 2 min. Theinjector temperature was maintained at 220 �C.

2.4. Analysis of fatty acid moieties of total lipids

Total lipids of 50 mites were extracted with a chloroform/methanol/water mixture as described above. The extracts werehydrolyzed with a 20% potassium hydroxide/ethanol-water (6/4, v/v) solution at 85 �C for 2 h. After some water was added into thesolution and the mixture was adjusted to pH 1 with 6 N hydro-chloric acid, free fatty acids were extracted three times with 300 mlof diethyl ether and then washed three times with 200 ml of satu-rated sodium carbonate solution and water. After evaporation ofthe organic layer to dryness, residues were dissolved in methanol(100 ml), containing 16-hydroxyhexadecanoic acid (250 ng) as an

internal standard, and one portion (1 ml) of the samples wasanalyzed by LC/MS (LCMS-2020 equipped with a Prominence UFLC,Shimadzu, Kyoto). A Mightysil RP-18 GP column (50 � 2.0 mm,Kanto Chemical, Tokyo) was eluted (0.2 ml/min) with a gradient of50e99% acetonitrile in water containing 0.05% acetic acid over15 min with the column temperature maintained at 40 �C.

3. Results

3.1. Phospholipid composition of mites and dried yeast

Fig. 1 shows the phospholipid composition of the dried yeast,T. similis, and T. putrescentiae. Phospholipids were identified usingaccurate mass MS and MSn data described before (Aboshi et al.,2010). The phospholipid composition of the mites is differentfrom that of the dry yeast diet. Octadecadienoic acid (18:2) wasidentified as a major component of PCs and PEs in T. putrescentiaeand T. similis, whereas hexadecanoic acid (16:0) is a main phos-pholipid fatty acid in dry yeast.

To know whether the octadecadienoic acid (18:2) moiety ofphospholipids was synthesized de novo, we also analyzed phos-pholipids of the mites fed on the dry yeast enriched with d31-hexadecanoic acid (16:0). d31-Hexadecanoic acid (16:0) was incor-porated into acyl moieties of phospholipids, such as octadecadienyl(18:2), hexadecanoyl (16:0), octadecanoyl (18:0), and octadecenoyl(18:1) groups. MS spectra of PE containing nonlabeled/d-labeledoctadecanoyl-octadecadienoyl (18:0e18:2) moieties and MS2 dataof the PE containing the d-labeled acyl groups are shown in Fig. 2.The [M�H]� ion at m/z 742.54 represented PE containing non-labeled (18:0e18:2) (exact mass, 742.53), whereas the[M�Hþ31]� ion at m/z 773.72 and the [M�Hþ31 þ 27]� ion at m/z800.90 corresponded to PE containing d31-labeled octadecanoicacid (18:0) with unlabeled octadecadienoic acid (18:2) and PEcontaining the d27- and d31-labeled fatty acids (18:0e18:2),respectively. For identification of d-labeled PE (18:0e18:2), LCMS-IT-TOF analysis was conducted to obtain additional information.When the [M�Hþ31 þ 27]� ion at m/z 800.90 was used as theprecursor, characteristic product ions were observed at m/z 306.39(C15D27C2H4COO�) and at m/z 310.42 (C15D31C2H4COO�). Theseresults suggest that the d27-octadecadienoic acid (18:2) moiety of

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(A) Methyl linoleate isomer mixture

Fig. 4. GC/MS chromatograms of (A) authentic (9E, 12E)-, (9Z, 12E)-, (9E, 12Z)-, and (9Z,12Z)-isomers of methyl linoleate and (B) methyl 9, 12-octadecadienoate obtained fromphospholipids of the mites.

T. Aboshi et al. / Insect Biochemistry and Molecular Biology 43 (2013) 991e996994

phospholipids of mites was synthesized from d31-hexadecanoicacid (16:0).

3.2. Determination of the position of double bonds inoctadecadienoic acid (18:2) obtained from T. putrescentiae

T. putrescentiae was used to determine the position of doublebonds in octadecadienoic acid (18:2). The lipid extracts from themites were subjected to hydrolysis by phospholipase A2, methyl-ated to form fatty acid methyl esters, and then converted to DMDSadducts. Six major peaks of unlabeled/labeled octadienoate methylester adducts were resolved by capillary GC/MS between tR 22.3and 23.0 min (Carballeira and Cruz, 1996). Each peak was charac-terized as 2-(7-methoxycarbonylheptan-1-yl)-3,5-dimethylthio-6-(pentan-1-yl)tetrahydrothiopyran (A, tR 22.35 min) [m/z 420 (Mþ,C21H40S3O2

þ, 20%), 373 (92%), 339 (38%), 325 (100%), 293 (32%)],2-(8-methoxycarbonyl-1-methylthiooctan-1-yl)-4-(1- methyl-thiohexan-1-yl)thietane (B, tR 22.75 min) [m/z 420 (Mþ, 57%), 373(29%), 325 (40%), 241 (54%), 209 (79%), 155 (100%)], 2-(8-methoxycarbonyl-1-methylthiooctan- 1-yl)- 4-methylthio-5-(pen-tan-1-yl)tetrahydrothiophene (C, tR 22.85 min) [m/z 420 (Mþ, 4%),372 (Mþ�CH3SH, 29%), 325 (C19H33SO2, 48%), 217 (C11H21SO2

þ, 35%),155 (C9H15Sþ, 100%)], and 2-(7-methoxycarbonylheptan-1-yl)-3-methylthio-5- (1-methylthiohexan-1-yl)tetrahydrothiophene (D,tR 22.95 min) [m/z 420 (Mþ, 4%), 372 (80%), 325 (80%), 241 (49%),209 (100%), 131 (43%)], respectively by reference to mass spectralfragmentation patterns as described (Carballeira et al., 1994;Kuwahara et al., 1995; Pepe et al., 1998). The peak of d27-labeled2-(8-methoxycarbonyl-1- methylthiooctan-1-yl)-4-methylthio-5-(pentan-1-yl)tetrahydrothiophene (C0) was detected at 22.53 min.

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In themass spectrumofpeakC0, cleavagebetweenC9andC10affordedthe key fragments at m/z 170 [C9D15Sþ, 100%] and m/z 228[C11H10D11SO2

þ, 48%] with diagnostic ions at m/z 447 (Mþ,C21H13D27S3O2,10%), 398 (Mþ�CH3SD, 33%), 351 (C19H7D26SO2

þ, 50%),as shown in Fig. 3. The peak of d27-labeled 2-(7-methoxycarbonyl-heptan-1-yl)-3-methylthio-5-(1-methylthiohexan-1-yl) tetrahy-drothiophene (D0) was also detected at 22.64min. Cleavage betweenC12 and C13 afforded the key fragments at m/z 222 [C12H4D13SOþ,100%], m/z 255 [C13H7D14SO2

þ, 82%] and m/z 143 [C7H3D12Sþ, 45%]with diagnostic ions at m/z 447 (Mþ, C21H13D27S3O2, 8%), 398(Mþ�CH3SD, 84%), 351 (C19H7D26SO2

þ, 94%)(Supplemental data).These results clearly indicated that the double bonds of

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Fig. 5. Chromatograms of free fatty acids hydrolyzed from lipid extracts of mites feddried yeast with or without d31-hexadecanoic acid. d-Labeled fatty acids (18:2, octa-decadienoic acid; 16:0, hexadecanoic acid; 18:1, octadecenoic acid; 18:0, octadecanoicacid) were detected in the mite extracts.

T. Aboshi et al. / Insect Biochemistry and Molecular Biology 43 (2013) 991e996 995

octadecadienoic acid (18:2) were located at C9eC10 and C12eC13(Carballeira et al., 1994; Kuwahara et al., 1995; Pepe et al., 1998).

As Kramer et al. described previously (2002), GC/MS analysis ofauthentic geometric isomers of methyl linoleate with a CP Sil-88capillary column showed that the (9E, 12E)-, (9Z, 12E)-, (9E, 12Z)-,and (9Z, 12Z)-isomers were detected at tRs 41.3, 42.6, 43.1, and43.8 min (Fig. 4A), respectively. The peak of methyl 9, 12-octadecadienoate obtained from the mites appeared at 43.8 min,and corresponded to that of the (9Z, 12Z)-isomer (Fig. 4B). Theseresults clearly showed that d31-hexadecanoic acid was incorporatedinto the linoleic acid moiety of phospholipids.

3.3. D-labeled fatty acids of total lipids of T. putrescentiae

The lipid extracts of the mites fed on the dry yeast enriched withd31-hexadecanoic acid (16:0) were hydrolyzed to determine thefatty acid profile. Fatty acids were identified by a comparison ofpeak retention times and m/z with those of commercial fatty acids.D-labeled fatty acids elute slightly ahead of the unlabeled fattyacids. d31-Hexadecanoic acid (16:0), d31-octadecanoic acid (18:0),d29-octadecenoic acid (18:1), and d27-octadecadienoic acid (18:2)were detected (Fig. 5), whereas d-labeled hexadecenoic acid (16:1)and hexadecadienoic acid (16:2) were not.

4. Discussion

Linoleic acid is an essential fatty acid in a number of insects(Chippendale et al., 1965; Dadd, 1960, 1964; Gordon, 1959; House,1974; Sivapalan and Gnanapragasam, 1979). Certain insects, how-ever, have the ability to synthesize linoleic acid de novo (Blomquistet al., 1982; Cripps et al., 1986; de Renobales et al., 1987; Dwyer andBlomquist, 1981; Mauldin et al., 1972; Louloudes et al., 1961;Stanley-Samuelson and Loher, 1986; Strong, 1963). Early radio-tracer studies with the cockroach Perplaneta americana (Louloudeset al., 1961), the peach aphid Myzus persicae (Strong, 1963), and thesubterranean termite Coptotermes formosanus (Mauldin et al., 1972)showed the incorporation of radiolabeled acetate into the fractionthat co-eluted with linoleic acid. The synthesis of linoleic acid wasalso reported in the house cricket Acheta domestica, the pacificdampwood termite Zootermopsis angusticollis (Blomquist et al.,1982), and the black field cricket Teleogryllus commodus (Stanley-Samuelson and Loher, 1986). In most of the species which synthe-sized linoleic acid, there was a time-dependent increase in the

incorporation of radiolabeled acetate into linoleic acid. Interest-ingly, labeled linoleic acid was accumulated primarily in thephospholipid fraction in all insects except the cockroaches (2 spe-cies) (Cripps et al., 1986). Labeled linoleic acid from the cockroachesaccumulated equally in both triacylglycerol and phospholipidfractions.

In this study, we demonstrated that the linoleic acid moieties ofphospholipids of mites were synthesized from hexadecanoic acid(16:0). The double bond positions of octadecadienoic acid (18:2)were determined to be C9eC10 and C12eC13 in T. putrescentiae withall Z-geometry. As far as we know, this is a first report convincinglydetermining the two double bond positions and geometrical con-figurations of biosynthesized linoleic acid in arthropods. Althoughanimals in general are thought to lack the ability to insert a doublebond at the 12 position of 9-octadecenoic acid and the symbioticbacteria were implicated in linoleic acid synthesis, de Renobleset al. demonstrated that aposymbiotic aphid (Acyrthosphom pisum)incorporate radioactivity from 14C-acetate into linoleic acid (1986).With respect to mites, it is still unclear whether the mites them-selves or symbiotic microorganisms are responsible for inserting adouble bond into the D12 position of 9-octadecenoic acid. However,the production of linoleic acid by some insects, land slugs, gardensnails (Weinert et al., 1993), and mites suggests that the capacity toproduce linoleic acid is much more widespread among in-vertebrates than previously thought.

Recently, Zhou et al. (2008) reported the isolation of D12-desaturase genes, AdD12Des from house cricket and TcD12Desfrom the red flour beetle, responsible for the production of linoleicacid from oleic acid. Interestingly, the cricket and flour beetle D12-desaturases acted on acyl-CoA substrates rather than phospholipids(Cripps et al., 1990; Zhou et al., 2008), although all D12-desaturasesin plants and fungi are thought to act on phospholipid-boundsubstrates (Sperling et al., 2003). Consequently, the insect D12-desaturase genes have been suggested to have evolved indepen-dently from plant-like D12-desaturases (Sayanova et al., 2006) anddifferent insect species may have acquired the genes by indepen-dent evolution (Zhou et al., 2008). More investigation is necessaryto know whether the D12-desaturase of the mites is more closelyassociated with insects than plants.

Mite metabolic systems have not been extensively studied andremain to be fully elucidated. We identified crinosterol in astig-matid mites (Murakami et al., 2007). Crinosterol has been found inmarine invertebrates, marine algae, and the seeds of orientalmustard, but not in terrestrial animals. Furthermore, pumiliotoxin251D, precoccinelline 193C, and another coccinelline-type alkaloid,known as dendrobatid alkaloids, have been identified in two spe-cies of oribatid mites (Takada et al., 2005). The discovery of linoleicacid, crinosterol, and dendrobatic alkaloids in mites implies theexistence of interesting pathways of metabolism in Acari.

Acknowledgments

This study was supported partly by Grants-in-aid for ScientificResearch (nos. 22380068 and 24120006) from the Ministry of Ed-ucation, Culture, Sports, Science and Technology of Japan and byDevelopmentWorks for Practical Technology Driving Novel Policiesfor Agriculture, Forestry and Fisheries (no. 22005) from the Min-istry of Agriculture, Forestry and Fisheries.

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.ibmb.2013.08.002.

T. Aboshi et al. / Insect Biochemistry and Molecular Biology 43 (2013) 991e996996

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