Biosci. Biotechnol. Biochem., 75 (5), 1003–1005, 2011 Note Sterol Composition in Larvae of the Silkworm, Bombyx mori

y Shinji NAGATA and Hiromichi NAGASAWA

Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan

Received January 5, 2011; Accepted January 26, 2011; Online Publication, May 20, 2011 [doi:10.1271/bbb.110008]

Sterols in silkworm larvae were analyzed. in an artificial diet and in Bombyx larvae. Substantial was predominantly detected in all tissues examined. sterol components of the midgut, epidermis, silkgland, Dietary and desmosterol, a putative bio- and hemolymph of Bombyx larvae fed mulberry leaves synthetic intermediate from phytosterols to cholesterol, was reported more than 45 years ago on the basis of were also detected, indicating that imperfect intestinal preparative gas-choromatography.17) In the present conversion from phytosterols to cholesterol influences study, in order to trace dietary phytosterols in the insect the sterol composition in larval tissues. body, we analyzed the sterol compositions of major lipid-related tissues such as the midgut, hemolymph, and Key words: Bombyx mori; cholesterol; ; fat body of Bombyx larvae during the feeding period (6th desmosterol; HPLC day last instar of female and male larvae; Kinshu Showa, Ueda-Sanshu, Nagano, Japan) fed an artificial Insects require dietary sterols due to their lack of diet (Silkmate 2S; Nihon Nosanko, Yokohama, Japan) ability to synthesize sterols de novo.1–3) Sterols are by reversed-phase HPLC (RP-HPLC).18,19) crucial to insects for survival and development, since The sterol compositions were determined by RP- cholesterol is utilized not only as constituents of cell HPLC, which was carried out using a C18 column membranes, but also as precursors of molting hormones, (4:6 i.d. 250 mm, Pegasil-ODS, Senshu Kagaku, ecdysteroids.4,5) This sterol requirement of insects was Tokyo, Japan) in an isocratic mobile phase: 48.5% first demonstrated in the blow-fly, Lucilia sericata.6) To acetonitrile, 48.5% methanol, and 3% distilled water date, sterol requirements for the diet and for symbionts (flow rate 1 mL/min). Sterols were monitored by are generally observed in arthropods.7,8) Cholesterol, a measuring the absorbance at 205 nm. Sterols were main sterol in insects, supplemented by two strategies: extracted from the various lyophilized materials of the (i) direct uptake of cholesterol in the diet, and (ii) artificial diet, larval midgut, hemolymph, and fat body conversion from other sterol compounds, including with ethyl acetate 3 times. To remove contaminated phytosterols to cholesterol. General phytosterols are triacyl- and diacyl-glycerol and to collect esterified structurally different from cholesterol in an additional sterol components as free sterols, saponification was alkyl group at the 24 position in the side chain of sterols. carried out by incubation of the dried extract at 37 C Therefore, enzymatic reactions of dealkylation at posi- in 1 N KOH/ethanol for 1 h. Saponified samples were tion 24 of ingested phytosterols are crucial to their extracted with hexane 3 times. The extracts were growth and development. A number of studies have developed by a thin-layer chromatography with hex- indicated the enzymatic activity of conversion from ane:ethyl acetate (3:1) as developing solvent. Since phytosterols to cholesterol in the midgut microsomal various free sterols were developed with similar Rf fraction of phytophagous insects.9–12) values (from 0.35 to 0.5), the crude sterol fractions were The silkworm, Bombyx mori, is a monophagous easily collected from TLC plates by extraction with phytophagous lepidopteran species, feeding only on ethyl acetate 3 times. Then the crude sterol fractions mulberry leaves. Since the predominant sterol in were subjected to RP-HPLC for analyses of sterol mulberry leaves is -sitosterol,13) conversion of - composition (Fig. 1). The amount of each sterol that was sitosterol to cholesterol is crucial for Bombyx larvae. detectable in the subsequent analyses procedure was Similarly, an artificial diet specifically for Bombyx calculated by comparison with commercially available larvae14) includes sterols composed mainly of -sitos- sterols: cholesterol, campesterol, , and - terol derived from powdered mulberry leaves and lipid sitosterol. This method is much simpler than the extracts of soybean (soybean oil). In addition to nutri- conventional one by means of gas-chromatography with tional regulation of sterol requirements, -sitosterol a capillary column. These sterols were confirmed by appears to contribute to activation of biting15) and measuring the molecular weights of the picolinyl dietary selection in this species.16) Therefore, sterol derivatives of the sterols by electro-spray ionization- requirements of B. mori should provide important time of flight mass spectrometry (ESI-TOF MS; JEOL, information on the relationship between nutritional AccuTof JMS-T100LC, Tokyo, Japan)20,21) after frac- regulation and feeding behavior. tionation of the sterols by RP-HPLC. Sodium adduct ion To address the molecular conversion of -sitosterol in peaks corresponding to derivatives of cholesterol, Bombyx larvae, we analyzed the composition of sterols campesterol, stigmasterol, and -sitosterol were ob-

y To whom correspondence should be addressed. Tel: +81-3-5841-5135; Fax: +81-3-5841-8022; E-mail: [email protected] 1004 S. NAGATA and H. NAGASAWA served at m=z 514, 528, 540, and 542 respectively (data sterols were determined by comparing selected ion peak not shown), although no protonated molecular ions of areas (m=z ¼ 528 and 540 for campesterol and stigmas- them were observed. terol, respectively) on LC-TOF MS analyses after The sterols in the artificial diet consisted of - picolinyl derivatization. Campesterol, stigmasterol, and sitosterol, campesterol, and stigmasterol with a trace of -sitosterol remained in the midgut, indicating that the cholesterol (Fig. 1), as described previously.22) Since enzyme for dealkylation did not finish conversion during cholesterol is thought to be converted from phytosterols the process of passing through the midgut. In contrast, in the midgut of Bombyx larvae,23) we next measured the the compositions of the sterols in the hemolymph and fat sterols in the midgut, hemolymph, and fat body of body were similar, but considerably different from those Bombyx larvae of the 6th day last instar. RP-HPLC of the dietary sterols. On the other hand, the sterols in analyses revealed that the main sterol in the midgut and the gut contents were similar to those of the dietary hemolymph was cholesterol. By comparing peak areas, sterols, suggesting that dietary phytosterols permeated to with those of the various commercially available stand- the hemolymph across the midgut cells after imperfect ard sterol compounds, we determined the compositions enzymatic conversion to cholesterol. The data on sterol of the sterols in the Bombyx larvae (Table 1). Since composition in the present study are consistent with a campesterol and stigmasterol were eluted at the same previous report.24) retention time by RP-HPLC, the compositions of those In the analyses of sterols by RP-HPLC, an unknown peak was observed at 14.5 min in the midgut and hemolymph (Fig. 1, peak 4). To obtain sufficient peak 2 3 material, we prepared samples by RP-HPLC from the Diet 1 hemolymph and midgut of 50 individuals of last-instar Bombyx larvae. The various HPLC-purified fractions * * were analyzed after picolinyl derivatization. Since the Gut contents ion peaks of the resulting derivatives were observed at m=z 512, the unknown sterol was predicted to be desmosterol. That structure was confirmed by the fact Midgut of the same retention time as commercially available 4 desmosterol and its picolinyl derivative on RP-HPLC (data not shown). Hemolymph The results of the sterol composition analyses are 4 summarized in Table 1. As in previous studies using GC-MS analyses,9) phytosterols (cholesterol, campesterol,

Absorbance at 205 nm Fat body and stigmasterol) and cholesterol were detected in the 4 present study. Thus the present data confirm that dietary sterols are converted to cholesterol in the midgut. Feces However, using the present analytical method, desmos- * terol was detected in the midgut and hemolymph. Also, * the present finding that predominant cholesterol was present in the fat body in the composition similar to that 15 20 25 Time (min) in the hemolymph indicates that the sterol composition in the fat body might be influenced by that in Fig. 1. RP-HPLC Profiles of Sterols. hemolymph. Sterol fractions in an artificial diet, and those of Bombyx gut Previous reports postulated a biosynthetic pathway contents, larval midgut, hemolymph, fat body, and fecal pellets were analyzed. The retention times of the peaks were compared with from -sitosterol to cholesterol, as depicted in Fig. 2. In those of commercially available sterols (cholesterol, campesterol the present study desmosterol was detected, whereas no with stigmasterol and -sitosterol correspond to the peaks indicated fucosterol, 24,28-epoxyfucosterol, or other intermedi- by arrowheads 1, 2, and 3 respectively). Since campesterol and ates from phytosterol to cholesterol were detected. The stigmasterol were coincidently eluted at an elution time similar to traces of desmosterol in the examined tissues suggest peak 2, the picolinyl derivatives of the eluted sterols were analyzed by ESI-TOF MS (data not shown). An unknown peak, 4, was that the 24-reducing enzyme of desmosterol is a rate- identified as desmosterol. Asterisks indicate compounds not iden- limiting step in the conversion from phytosterols to tified in the present study. cholesterol in Bombyx larvae. Also, the presence of

Table 1. Sterol Compositions of Bombyx Larvae and an Artificial Diet (%)

Analyzed sterols Tissues Cholesterol Campesterol Stigmasterol -Sitosterol Desmosterol Diet 7:1 2:831:0 5:520:1 2:741:9 6:9 n.d. Gut content 7:7 2:025:5 3:119:4 1:947:3 3:7 n.d. Midgut 61:0 5:414:8 1:83:4 1:516:9 3:53:9 0:9 Hemolymph 59:3 18:412:1 4:66:1 3:317:3 6:45:1 2:0 Fat body 80:9 8:54:9 2:72:8 1:59:2 4:42:1 0:6 Fecal pellets 16:0 6:032:8 3:18:4 2:742:8 3:0 n.d.

Values are mean SD (n ¼ 5). n.d., not detected. Sterol Composition in the Silkworm 1005 The fact that several unidentified components were observed throughout the present study (Fig. 1) suggests that the precise phytosterol of Bombyx larvae remains to be fully elucidated. To address the molecular basis of sterol metabolism its utility in insects, further investigation is necessary. HO β-Sitosterol Acknowledgments

This study was supported partly by Grants-in-Aid for Scientific Research (nos. 18780083 and 22780099) from the Ministry of Education, Culture, Sports, Science and HO Technology of Japan, and supported partly by NAITO Fucosterol O Foundation.

References

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