Kato, Masaru

Lin, Kiryo

Yamada, Hiromi

English

Kyoto University Research Information Repository

RIGHT:URL:CITATION:AUTHOR(S):ISSUE DATE:TITLE:Free Fatty Acids Promote FeedingBehavior of the Silk-worm, Bombyxmori L.Lin, Kiryo; Yamada, Hiromi; Kato, MasaruLin, Kiryo ...[et al]. Free Fatty Acids Promote Feeding Behavior of the Silk-worm, Bombyxmori L.. Memoirs of the Faculty of Science, Kyoto University. Series of biology. New series1971, 4(2): 108-1151971-03-31http://hdl.handle.net/2433/258804      MEMOIRS OF THE FACULTY OF SCIEncE, KYOTO UixTIVERSITY, SERIES OF BIOLOGy                       Vol. IV, pp. 108-115, March 1971     Free Fatty Acids Promote Feeding Behavior of                the Silk-worm, Bombyx mori L.                                    By               Kiryo LiN, Hiromi YAMADA, and Masaru KATo,               Il)epartment of Zoology, Faculty of Science,                              Kyoto University                         (Received DeÅëember 23, 1970)     ABSTRACT It was found that adding one per cent of free fatty acids mixture  of the rnulberry leaves greatly increased feeding behavior of the newly hatched  larvae. When separately tested with commercially obtained free fatty acids in  accordance with each average value of six kinds of the confirmed ML free fatty  acids, the experimental results showed that linolenic acid caused potent activity, then followed with linoleic acid, and lauric acid. Those of myristic acid, palmitic  acid, stearic acid, and three octadecenoic acids (including elaidic acid, oleic acid,  and vaccenic acid) caused no activity. MorLeover, linoleic acid O.099oi plus linolenic  acid O.6696 N'Nere found to have nearly same effectiveness as 196 ML free fatt.y acids  mixture. And 19oi of vaccenic acid also demonstrated promotive effect on larval feeding, but its activity was fQund lower than linoleic acid plus linolenic acid. Anyhow, those ML free fatty acids, especially linoleic acid plus linoleniÅë acid, and vaccenic acSd were proved having complemental efficacities with those ML-contained larvae's  preferential substances such as sucrose and inositol. And those acids also acted synergestically with P-sitosterol, n-chlorogenic acid, and even vanillin to a fairly large extent under the existence of the biting assay diet.                                Introduct,ien    It was originally discoverecl by Hamamura (1) that the metlianolic extractof the mulberry leaves (ML) catised high activity on the promotion of larvalfeeding. The biting factors was found present in both organic solvent solublefraction (petroleum ether) and water sol'uble 'fraction of the above methanolicextract. When these two fractions were combined, and added to the meehanolextracted residue of the ML, feeding behavior of the silk-worm larvae couldbe promoted to the utmost.    Talking abottt water soluble fraction, so far, some biting factors such assucrose and inositol have been reported by Hamamuz"a (2) (3). The monogluco-side pigment, isoqttercitrin by Hayashiya (4), and n-chlorogenic acid by theauthors (5) were also proved to increase feeding behavior o'f the newly hatchedlarvae. In concerning with organic solvent soluble fraction, up to now, one                 Kiryo LiN, Hiromi YAMADA and Masaru KfxTo 109biting substance, P-sitosterol was previously described by Hamamura (3) fromthe non-saponified fraction, and linolenic acid by Wada et al. (unpublished).The free fatty acid fraction (2N-Na2C03 extract), however, inay be still con-taining naoz-e biting substances. Although llnolenic acid by Ito et al (6). andoleic acid, the existence of which is not appreciable in ML, was reported byYainada et al (7) that caused potent activities on the proinotion of the growth,yet, on feeding stimulation, the particular contributions of ehese free fattyacids fraction to the silk-worm larvae have got to be clarified in details.   Therefore, in this paper, the divulged experiments of the commercial freefatty acids from Ci2 to C22, and especially the ML-contained free fatty acidswere carried out by using our specaal siinplex biting assay diet (BAD) for thebioassay of tlieir feeding stimulating efficacies.                         Materials and Methods    Larvae Fi of the strains Gunko x Manri, Nl, N2, N3, of the Japanesestrains, and Sl, S2, S3, S4, S5 of the Chinese strains were used throughoutthe experiments. They were kindly provlded by Gunze Silk Company.                     Table 1. Components of the BADComponents g. of dry wt.       sucrose O.1       inositol O. 02       cellulose O.8       agar e.2       bnffer* 10 ml.. . .fP.•9.i.l>.fl.LS.l5P.l[i..+... .N.l.a.9.:.l.?. }tf.{.s..Rgtl}rel.1.E:g..wll.b lYlr.}{{3?.O..f..tg. .p..\...9.•.e.   Diet The BAD is shown in Table 1.   Irt4ethod for checfe offeeding behavior The newly hatched larvae were placedon the BAD in a dark incubator at 27eC constantly. And then simply counteclthe number of the defecated feces at a certain time after fetching under non-sterile condition as previously described paper (5).   Free fatly acids Except self-prepared native ML free fatty acids mixture,ten kinds of free fatty acids were obtained cornmercially. The purities of thesesamples were examined by gas chromatography.   PreParalion of free fatty acids The ML free fatty acids were prepared bythe use of J.Bonner's method (8). Those cornmercially obtained six kinds oft"ree fatty acids were combinecl according to the average values of si: ML-containecl free fatty acids from gas chromatograms as Table 2 shows. Thismixture js designated as e'6F" (8 kinds of ML components mintts Ci6:i and C}s:iacid. The foriner ls omitted for its rarity and the latter for its difficulty inidentification of i'ts isomers). Another series of 7E, 70, and 7V are the mixtureof 6F plus elaidic acid, oleic acid, and vaccenic acid of each O.92.0o! separately.These three 1<in(ls of octadecenoic acids of each was addecl because they appeared110 t t tttttt t tt ttt                      '                                     ' ' Content$. .. Ratios Average 7E .70 7V                '                            (9oi) values                     ,,,Authors .,....Ito et al.* ..                     Ist 2nd ia.ricbcid ''61'66""""2I'g6"' i.3o i.3q 2.g4 i.o7 i.2o mYristic 'acid O. Ol O. 26 O. O02 O. 09 O. 39 O. 30 O. 32 palmitic acid 23. 58 24. 00 13. 90 2e. 50 26. 46 28. 10 29. 00 C16:1 - O. 09 O. 09 O. 65 O. 28 O. 05 O. 12 O. 09 stearic acid 2. 54 O. 70 1. 4e 1. 54 O. 07 O. 85 O. 65 octadecenoic acid O. 27 1. 80 O. 70 O. 92 O. 57 1. 20 1. 63 linoleic acid 9. 60 4. 65 13. 0e 9. 08 O. 85 1. 50 2. 43 linolenic acid 63.82 66. 00 68. 95 66. 29 68. 67                                                         66. 86 64.  tti6'{e"'l"'rph5"to{al 511Iollfft'-oiM' 'il'free fatty acids mixture is 166"et'. 7E, 7o, and 7v       were combined according to the above 7 average values (Ci6:i was omitted).       * Those values were calculated by us from the gas chromatogram reported         by Ito et al.(6).sharply in the same positjon on the gas chromatogram as ML octadecenoic acid(Cis:i) appeared.    Here, we would like to point out that the commercially obtained free fattyacids were not so pure. For instance, froiTi one Shirnadzu gas chromatogram,we calculated that elaidic acid (cis-9-octadecenoic acid) was contaminated with7.4/0oi lauric acicl, 5/0oi myristic acid, l39o" palmitic acid. Oleic acid (trans-9-octadecenoic acid) was fotmd to liave contaminated with 6.99oi lauric acid, 8.29o!myristic acid, 2.49o- palmitic acid, and 7.29oi Ci6:i tmknown acid. Ancl vaccenicacid (trans-11-octadecenoic acid) was also contaminated with 1.7.0o- lauric acid,3.4!0o/ palmitic acid, and 9.59o/ stearic acid.    The samples, 7E, 70, and 7V, therefore, were analyzed to determine theircompositions. Then we found palmitic acicl great]y i-ncreased its quantity, andon the contrary, linoleic acid largely decreased. Since each industrial producedfatty acid was so impure, the true compositions of 6F were actuaily happenedto be causecl slight deflection, and hence mocre or less influenced the accuracyof our biological assays.                           Results and Discussion    According to the quantitative analysis l)y means of gas chromatograph(Shimaclzu GC-4B), eight peak$ of the free fatty acids, extracted from themulberry• leaves powder, obtainecl clearly on the gas chromatograms, from which,the proportional value of each content was precisely calculated. [l"hen we Åíoundthe quantity of each fluctuation was quite different. owing to the lots of mate-rial. For this reason, we decided to make an appropriate quantity from thecalculated average value of each free fatty acid (see Table 2) for the bioassayFree Fatty Acids Promote Feeding Behavior of the Silk-worm, Bontbyx mori L.Table.2. Ratios, of' the ML.free f•atty acids compared with 1 E;           70, and 7V as ca'lculated•from gas, chromatograms                t . ttt.ttttt . tt. ttt t.t. t.tt ttttKiryo LiN, Hiromi YAMADA and Masaru KATo 111of biting activlty. Then lt was found ln this system that linoleRic acid causedvery high feeding efficacy than those of any other free fatty acids as Wcada etal. previously discovered. Linoleic acid was,lowei?• than liiiolenic acid, whilelauric acid also showed rather liigh activity. XVhen each acld was added wit.hP-sitosterol (O.5.0o-) of the ML normal concentration reported by Hamamura (3),we found that lauric acid showed very high cooperative effect, then followedwith linolenic acid, ]inole}c acid, and vaccenic acid. Those myristic acid, pal-mitic acid, stearic acid, and two octadecenoic acids (elaidic acid and oleic acid)though showed rather poor activities, yet, were stjll activated by it. Theresults are given in Table 3.       Table 3. The comparative effects of the commercially obtained                 free fatty acids on biting promotion (used BAD)Feces no. per 4e hrs per 10 larvaeContents Addition (96)  •withoutP-sitosterol  withP-sitosterol (O.59ai)lauric acidmyristic acidpalmitic acidC16:1stearic acidoctadecenoic acids   elaidic acid   oleic acid   vaccenic acidlinoleic acidlinolenic acidcontrolO. O13O. OO09O. 20O. O15O. O09O. O09O.O09O. 09O. 66146 28 11 2 7 o 55212406 oRi3nel,i lsLTote : Each acid was dissolved in chloroform, and coated on the cellulose powder,       then evaporated chloroform with elect'ric water bath at 700C.    Furthermore, when these free fatty acids were combined, we found thecombination of linoleic acid plus linolenic acid gave 254 feces number for theft'rst 18 hours (see Table 4). It demonstrated nearly equal effect as eight 1<indsof 1.0o' ML free fatty acids mixture which was once reported by Yamada et al.(7) as a normal concentration in ML. It was also once analyzed by Ito et al.(6) as Table 2 shows. At the same experiment, 7V and 7E caused a little morehig}ier actkvities than those of 70 and 6F, but lower than linoleic acid plus lino-lenic acid or natural ML free fatty acids mixture. Here we should remind thatif linoleic acid was as pure as linolenic acicl, we stipposed that the proniotiveefficacies o'f these two fatty acids coulcl be more increased.    On the other hand, when the assaying dosage of each acid was raised to19o-, it was found that vaccenic acid (trans-H-octadecenoic acid) showed fairlyhigh specific function on feecling pyomotion. Elaidic acld was also proved to112 Free Fatty Acids Promote Feeding Behavior of the Silk-worm, B onpbyx mor'i L.Table 4. The effects of several kinds ofbiting stimulation (used BAD)free fatty acids mlxture on(a)Contents Addition (96) Feces no.    per 10per 18 larvaehrs.   6F ca.1   8F* 1   lauric + linoleic O. O13 + O. 09   lauric + linolenic O. O13 + O. 66   linoleic + linolenic O. 09 + O. 66   lauric + linoleic + linolenic O.O13 + O.09 +* Eight kinds of ML free fatty acids mixtureO. 66196220153228259  6204254240(b)linoleic acidlinolenic acidlinoleic + linolnoneenic   O. 09   O. 66O. 09 + O. 66 36121151 oTable 5. The effects of the commerciallyfatty acids on the promotion ofobtained 10 kindslarval feeding9f freeAddition (1 96)Feces no. per 18 hrs.    per 10 larvae Remarkarachidic aciderucic acidpalmitic acidstearic acidelaidic acidoleic acidvaccenic acidlinoleic acidlinolenic acidricinoleic acid8Fnoneilk,ii16These are one of six repeatedexperimental data. Each Petridish was added with ML watersoluble fraction O. 18g., cellulosee.8g., agar O.2g., and potas-sium phosphate buffer 10 ml.(pH 6.8)Kiryo LiN, I/Iiromi YAMtmA ancl ?vlasaru KATo 1113have activation next 'to vaccenic acid. Those of arachidic acid, erucic acid,linoleic acid, linolenic acjd, oleic acid, palmit'ic acid, ricinoleic acicl, and stearicaci(l were found to have caused inhibition on larval feedjng. The resultantdata are given in Table 5.    Since vaccenic acid was 'found having stimulation on feeding behavior, wewere then tried to tal<e a further step to test whether it acts synergestica}lvvwith P-sitosterol by ffamarnuura (2), vanillin and n-chlorogenic acid by theauthors (5). The experiinental results showed that xraccenic aci(l surely coop-erated witla above biting substacnces to a large extent (see Table 6). In this       Table 6. The synergestical effect of vaccenic acid on the promotion                 of larval feeding under the existence of BADPetri dishaxTo•s,t6cio,geg,no,eqgEsm8a?,ci8,50s-idVaiiiil'.".,,P'[eg;g,S6,,\:Cc?8ii'Cggi,,/,i,/1i,oPsi Al* + C**2 do.3 do.4 do.5 do.6 A2*** +C7 Al +C8 do. 9 do.10 do.11 A2+CI2 Al +C1111iill11+iil'ill ggg1Z,;;9gli2 3 gi13 Al +C14 do.t : t : + kg? NTote : "' "'        * agar O. 2g       ** cellulose O.8g      *** agarose O.2gexperiment, the compcftrative effects of the gelatinizers-agar and agarose werealso studied, because the comn)ercial agar contains considerable amounts ofprotein, oligosaccharides, natural pigments, ashes, and the inf}uential effectsof these impurities to the feecling behaviors of the sM<-worm larxrae can no't beconcluded. The results sbowed that agarose was actu,ally a litt!e more 1)etterthan agar, and hence it was confinned that these impuri'ties in agar ratherdepressed biting activity.    Niext step, in order to clarify whethei- unnatural 1ttlA ot' vaccenic acid oynatural O.09.0o! linoleic acid plus O.66.`o5 'iinolenic acid activate feeding behavjorhighly, we were then used our BAD plus "e-sitosterol plus n-chlorogenic acid, 114 Free Fatty Acids Promote Feeding Behavior of the Silk-worm, Bo,nbyx mori L.and even added vanillin to compare their, activities. The summarized resultsshowed that the promotive effects of linoleic acjd plus linolenic acid 'f'urther'                                                                    'eTXaCbeleed7agtt'oflJCg?1 lfOo' Of VaCCenic acid, but at any rate lower than young ILf.[,,L.,,g;s,.                                                        '                            t tt                The cornparison of the ynnatural dose of vaccenic acid and ,Table 7.                natural doses of linoleic acid.plus linolenic acid on feeding '                                       '                                         '                promotion (used BAD) r' . . '.,t' .v',•                                                      tt            Components Feces no.124 hrs./16 iarvae '     A*     A + 1 96 vaccenic acid     A + O. 0996 linoleic +         O. 66 96 li nole nic     A + O.0996 linoleic +         O.669o! linolenic +         1 96 vanillin     control (young ML)* BAD + P-sitosterol O.59oi + n-chlorogenic acid 196258285427448461   Now, the feeding behavior of the newly hatched hybrid larvae of the Fiof Gunlco x Manri is clearly proved activated by free fatty acids. And whatabout the other strains? It is a well-1<nown matter of fact that the Japanesestrains are more active than Chinese strains to artificial diet in generally speak-ing, therefore, we particttlarly chose its pareneal strains : Sl, S2, S3, S4, S5,and Nl, N2, N3, and then used 7V plus BAD to examine their feeding behav-     Table 8. The feeding behaviors of 8 kinds of strains cornpared to Fi of               Gunko x }lt(fanri (used BAD plus 19o/ of 7V)                                                                 ttStrains Feces no.118 hrs. 1 10 larvaeSlS2S3S4S5NTIl2NT3F, of Gunko x Manri 15 52 1721 281365812089lors.stramsstramsThe experimental data of Table 8 clear-cut proved especially Nl and N3 were found really more active, and even surpassed E hybrid that has been usingthat three Japanesethan all five Chineseas larval material for                ,KiryQ LiN, 'Hiromi YAMADA and' Masa'ru KATQ 115several years in our Kato Research Laboratory. Among Chinese strains, Sl, S3,and S4 were found pretty inactive on feeding.   Fui-ther studies on tl:e feeding. preferential substances of the silk-wormlarvae are still in progress.                            Acknowledgments   We should express hearty thanks to Dr. J. Nagai, Biochemical Laboratory,Faculty of Science, Kyoto University for the identification of free fatty acidsand gas chromatographical assistance, and to Dr. Y. Hamamura of KonanUniversity, for encouragement.                               References 1) HAMAMuRA, Y., Food selection by silkworm larvae. Nature, 183, 1746-1747, 1959 2) HAMAMuRA, Y., K. HAyAsHiyA, K. NTAiTo, K. MATsueRA, and J. NisHiDA, Food    selection by silkworm larvae, Bombyx mori L.. Bull. Kyoto Univ. Ind. & Tech.,    3(3), 567-582, 1962 3) NAMAMuRA, Y., On the feeding mechanism and prepared food of silkworm     Bomborx mori L.. J. Silkworm, 15 &16, 49-56, 1963-1964 • " 4) }{{AyAsHiyA, K., Requirements of the feeding stimulating stibstances for insect.     Shokubutsu BOeki, 23(8), 344-348, 1969 ,. .,' 5) LiN, K., ff. YAMADA and rvl. KATo, A comparative study of the effect of     chlorogenic acid and its isomers on the growth of the silkworm, Bombyx mori     L.. J. Sericult. Sci. Japan, 39(3), 153-157. 1970 ,/ 6) ITo, T. and S. NAKAsoNE, Nutrition of the silkworm, Bombyv mori L. -8.     Nutritive effects of fatty acids, BulL Sericult. Expt. Sta., (ToKyo) 20(4),375-391, 7) YAMADA, E[. and M. KATo, Oleic acid as potent growth factor for the silkworm.   • Proc. Japan Acad., 43(3), 230-233, 1967  8) BoNNER, J. Plant Biochemistry, (Academic Press inc., New York) 360-390, 1952

Free Fatty Acids Promote Feeding Behavior of the Silk-worm, Bombyx mori L.

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Free Fatty Acids Promote Feeding Behavior of the Silk-worm, Bombyx mori L.

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