Cocoa pod borers from Tawau, Sabah and Sua Betong, Negeri Sembilan and rambutan fruit borers from

Serdang and Puchong, Selangor and Kuala Kangsar, Perak, Malaysia were subjected to electrophoretic analysis

in an effort to find diagnostic electromorphs between these two bio types of Conopomorpha cramerella. Thirty

enzymes and general proteins were successfully demonstrated on zymograms but none of them could serve as

diagnostic markers between cocoa pod borers and rambutan fruit borers. The allelic frequencies for 8 polymorphic

enzymes are presented

Asuan, K.

Gan, Yik Yuen

Kanasar, Sivarogini

Muhamad Awang, Rita

Sinha, Rita

Tan, Soon Guan

Universiti Putra Malaysia Institutional Repository

Pertanika 12(1), 1-6 (1989)An Electrophoretic Study of Natural Populationsof the Cocoa Pod Borer, Canopomorpha cramerella(Snellen) from Malaysia.RITA MUHAMAD, S.G. TAN, YY GAN, S. RITA,S. KANASAR and K. ASUAN.Departments of Plant Protection, Biology, and BiotechnologyUniversiti Pertanian Malaysia43400 UPM Serdang, Selangor Darul Ehsan, Malaysia.Key words: Conopomorpha cramerella; cocoa pod borers; rambutan fruit borers; electromorphs;polymorphisms.ABSTRAKPengorek buah koko dan Tawau, Sabah dan Sua Betong, Negeri Sembilan dan pengorek buah ramlmtan danSerdang dan Puchong, Selangor dan Kuala Kangsar, Perak, Malaysia telah dianalisa secara elektroforesisdalam usaha untuk mendapatkan diagnosis elektromorf antar kedua biotip Conopomorpha cramerella.30 enzim dan protein-protein umum telah dapat ditunjukkan pada zimogram-zimogram tetapi tidak ada satupun yang boleh digunakan sebagai penanda diagnosis antara pengorek buah koko dengan pengorek buahrambutan. Frekwensi alil-alil untuk 8 enzim polimorfjuga dipaparkan.ABSTRACTCocoa pod borers from Tawau, Sabah and Sua Betong, Negeri Sembilan and rambutan fruit borers fromSerdang and Puchong, Selangor and Kuala Kangsar, Perak, Malaysia were subjected to electrophoretic analysisin an effort to find diagnostic electromorphs between these two biotypes ofConopomorpha cramerella. Thirtyenzymes and general proteins were successfully demonstrated on zymograms but none of them could serve asdiagnostic markers between cocoa pod borers and rambutan fruit borers. The allelicfrequencies for 8 polymorphicenzymes are presented.INTRODUCTIONThe cocoa pod borer, Conopomorpha cramerella(Snellen) (Lepidoptera: Gracilariidae) is amajor cocoa pest in Sabah State, Malaysia butuntil late 1986 it was only present as a minorpest of rambutan in Peninsular Malaysia (Lokeet al. 1986, Ling et al. 1987). Both the cocpapod borer and the rambutan fruit borer areknown scientifically as C. cramerella. An outbreakof the cocoa biotype occurred for the first timein the states of Malacca and Negeri Sembilanin Peninsular Malaysia in September andNovember 1986 respectively. Fortunately thecocoa pod borer had not been detectedelsewhere in the Peninsula (Chin 1987).This outbreak enabled us to collect both thebiotype that attacks the pods of cocoa (Theo-bromae cocoa L.) and that which attacks rambutan(Nephelium lappaceum L.) fruits from thePeninsula within a period of three months(November 1986 to January 1987) althoughunfortunately not from the same locality. Wewere also able to obtain the cocoa biotype fromTawau, Sabah in January 1987. With thesesamples, we attempted to find diagnosticelectormorphs between the two biotypesof C. cramerella.MATERIALS AND METHODSThe following samples were available for electro-phoretic analysis: cocoa pod borers from SuaBetong Estate (SB) near Port Dickson in NegeriSembilan collected in November 1986 and fromthe Tawau district of Sabah collected inJanuaryRITA MUHAMAD, S.G. TAN, Y.Y. GAN, S. RITA, S. KANASAR AND K. ASUAN1987 (Tl); rambutan fruit borers from Ladang7 in the campus of Universiti Pertanian Malay-sia, Serdang, Selangor collected inJanuary 1987(Ll); from Kuala Kangsar, Perak State (KK)collected in January 1987 and from Puchong,Selangor state collected in January 1987 (PI)and in September 1987 (P2). The adult insectswere frozen at - 70°C until they were usedfor electrophoresis.Sample homogenization and pOlyacry-lamide gel electrophoresis were done as inRusnah et at. (1985). Various buffer systemssuch as TEB (Green 1977), CA-7 (Steiner andJoslyn 1979), TEMM (Spencer et al 1964)and that of Varvio-Aho et at. (1980) were usedin our screening for diagnostic electromorphsbetween cocoa pod borers and rambutan fruitborers. The staining procedures used were fromSteiner and Joslyn (1979), Harris andHopkinson (1976), Menken (1980), Shawand Prasad (1970) and Munstermann (1979).Isoelectric focusing was performed as in Tanet at. (1982) using LKB ampholytes with pHranges of5-7 and 4-6 after which general proteinwere stained for by using Coomassie BrilliantBlue R250.The polymorphic markers, phospho-glucomutase (PGM) , esterase (EST) (Halmyet at. 1987), a-glycerophosphate dehydro-genase (a-GPDH), peptidase (PEP) (Rusnahet al 1985) and malate dehydrogenase weretyped on CA-7 buffer with 0.1 % Kodak Photo-flo incorporated into the gel while hexokinase(HK), fluorescent esterase (FE) and malicenzyme (ME) were typed on the Varvio-Ahobuffer (Tan et al 1987). PEP was also typed onthe Varvio-Aho buffer.RESULTS AND DISCUSSIONAt least two adult insects each of cocoa podborers from Tawau (Tl) and Sua Betong (SB)and at least two adult insects each of rambutanborers from Ladang 7 (Ll) and Puchong(PI or P2) were screened for the presence ofdiagnostic electromorphs of the followingenzymes that were successfully demonstratedon zymograms:· aldehyde dehydrogenase,aldehyde oxidase, trehalase, glucose dehydro-genase, aconitase, alcohol dehydrogenase,fumarase, phosphoglucose isomerase, xanthinedehydrogenase, isocitrate dehydrogenase,glucose-6-phosphate dehydrogenase, 6-phospho-gluconate dehydrogenase, diaphorase,fluorescent acid phosphatase, fucose de-hydrogenase, L-threonine dehydrogenase,succinic dehydrogenase, uridine mono-phosphate kinase, glutamate oxaloacetate trans-minase, fructose 1, 6 diphosphatase, pyruvatekinase and acid phosphatase.However, no consistent diagnostic elec-tromorphs were observed between rambutanfruit borers and cocoa pod borer for any of theabove enzymes. No diagnostic protein bandswere observed between cocoa pod borers andrambutan fruit borers although numerous bandswere seen on the gel for each insect sampleafter isoelectric focusing runs on pH ranges4-6 and 5-7. The allelic frequencies for thepolymorphic systems are presented in Table 1.None of these polymorphic systems showed anydiagnostic electromorphs between CPB and RB.The data for populations T2 (CPB from Tawau,collected in December 1984) and P3 (RB fromPuchong, collected in January 1985) are fromRusnah et at. (1985) and that for L2 (RB fromLadang 7 UPM collected in March-April 1985)is from Halmy et at. (1987). The data for HI{,MDH, FE-2 and ME-l for populations TI, SB,P2 and Ll are obtained from Tan et at. (1987)which reported in detail the occurrence ofpolymorphisms for these enzymes in C. cramer-ella, their biochemical characteristics and theirproposed modes of genetic control. All theallelic frequencies from these papers arecompiled in Table 1 together with data reportedhere for the first time for comparative purposesand in order to calculate their heterozygosityvalues. Data for EST-l is not presented here asit could not be reliably typed in this studybecause the quality of the EST-l bands deterio-rated on storage during the time span of aboutten months from the time the samples werefirst collected to the time the electrophoreticanalysis was finally completed. Fortunately, thebands for the other polymorphic systemsremained consistent and could be typed reliablyduring the study period.The addition of Kodak Photoflo to theCA-7 gel improved the resolution of the PGMbands so that another allele, PGM98 which wasnot detected previously (Halmy et at. 1987)could now be typed. PEP was initially typed on2 PERTANlKA VOL. 12NO. I, 1989TABLE 1Gene frequency data for Esterase-2 (EST-2), phosphoglucomutase (PGM), peptidase-2 (PEP-2), a-glycerophosphate dehydrogenase (a-GPDH) ;J>zhexokinase (HK), malate dehydrogenase (MDH), fluorescent esterase -2 (FE-2) and malic enzyme (ME-I) in natural populations of tTlr'Conopomorpha cramemlla (Snellen) from five localities in Malaysia. tTlno-J;>cAllelic frequencies 0"0;r:Locus & 0~Allele 1'1 1'2 SB U L2 PI P2 P3 KK ::JnPGM eno-J105 0.231 ± 0.029 NA 0.344 ± 0.033 0.352 ± 0.038 0.302 ± 0.032 0.340 ± 0.067 0.500 ± 0.083 NA 0.304 ± 0.068 c0100 0.639 ± 0.033 0.543 ± 0.034 0.512 ± 0.039 0.476 ± 0.034 0.420 ± 0.070 0.361 ± 0.080 0.522 ± 0.074 -<98 0.038 ± 0.013 0.033 ± 0.012 0.049 ± 0.017 - 0.080 ± 0.038 0 0.044 ± 0.031 0'"0 'TJtTl 97 0.087 ± 0.020 0.080 ± 0.019 0.086 ± 0.022 0.221 ± 0.028 0.160 ± 0.052 0.140 ± 0.058 0.130 ± 0.050 z~ N 104* 106* 81* 106* 25* 18* 23* ~;J>3; H 0.538 0.591 0.593 0.642 0.640 0.611 0.565 c~ 1~S7~2 ~<: 102 0.279 ± 0.031 NA 0.361 ± 0.031 0.375 ± 0.039 0.355 ± 0.037 0.406 ± 0.087 0.375 ± 0.070 NA 0.417 ± 0.082 "00 0r 100 0.553 ± 0.035 0.574 ± 0.032 0.487 ± 0.041 0.428 ± 0.038 0.469 ± 0.088 0.479 ± 0.072 0.389 ± 0.081 "0C~ 97 0.168 ± 0.026 0.066 ± 0.016 0.138 ± 0.028 0.217 ± 0.032 0.125 ± 0.054 0.146 ± 0.051 0.] 94 ± 0.067 5~Z N 104* 122* 76* 83* 16* 24* 18'*9 H 0.567 0.525 0.592 0.614 0.563 0.708 0.444 (5:-' Zen::0 PEP-2 (CA-7 buffer) 000 F 0.947 ± 0.036 0.919 ± 0.022 1.000 1.000<.0 NA 1.000 NA 1.000 1.000 'TlS 0.053 ± 0.036 0.081 ± 0.022 0 0 0 0 0 (')N 19* 74 18* 19* ]8* 41* 14* ~H 0.105 0.162 0 0 0 0 0 S60 aS::HK a104 0 NA 0 0.111 ± 0.025 NA 0.042 ± 0.041 0 NA 0.080 ± 0.038 ~100 1.000 1.000 0.889 ± 0.025 0.958 ± 0.041 1.000 0.920 ± 0.038 :,;N 102 112 81 12* 28 25* ~H 0 0 0.173 0.083 0 0.080 ~PD~-2-(Vario-Aho's buffer) ~102 0.144 ± 0.037 NA 0 0 NA NA 0 NA NA ~()(l 100 0.800 ± 0.042 0.932 ± 0.027 0.895 ± 0.050 0.940 ± 0.034... 98 0.056 ± 0.024 0.068 ± 0.027 0.105 ± 0.050 0.060 ± 0.034N 45* 44* 19* 25*H 0.31 I . 0.136 0.211 0.12a-GPDHF = 100 0.995 ± 0.005 1.000 0.996 ± 0.004 0.956 ± 0.019 NA 1.000 0.981 ± 0.019 0.989 ± 0.005 0.917 ± 0.046S = 97 0.005 ± 0.005 0 0.004 ± 0.004 0.018 ± 0.012 0 0.019 ± 0.019 0.011 ± 0.005 0.083 ± 0.046 ~104 0 0 0 0.026 ± 0.015 0 0 0 0,..,;J>N 98* 86 118* 57* 18* 26* 184 18* 3::H 0.010 0 0.008 0.088 0 0.038 0.022 0.167 c:r:MDH;J>104 0.012 ± 0.008 NA 0.044 ± 0.014 0.024 ± 0.013 NA 0 0 NA 0 .~100 0.988 ± 0.008 0.956 ± 0.014 0.976 ± 0.013 1.000 1.000 1.000 VJN 84 103 63 5* 24 20* 0"0 H 0.024 0.087 0.048 0 0 0 52tT17-l N~'-2 .z,..,;J> l05 0.125 ± 0.034 NA 0.241 ± 0.040 0.159 ± 0.055 NA NA 0.233 ± 0.055 NA NA ~3 100 0.729 ± 0.045 0518 ± 0.047 0.659 ± 0.072 0.433 ± 0.064 ~~ (')97 0.021 ± 0.015 0.027 ± 0.015 0 0.100 ± 0.039 ;J>< Z~ 95 0.125 ± 0.034 0.205 ± 0.0380.182 ± 0.058 0.217 ± 0.053 .90 0 0.009 ± 0.009 0 0.017 ± 0.017 (/l;::; N 48 56 22 30 ~z j0 H 0.438 0.554 0.455 0.633VJ::- M/~~I'.0 102 0 NA 0.514 ± 0.035 0.346 ± 0.054 NA NA 0.250 ± 0.060 NA 0.304 ± 0.068 ~00 Z<.D 100 0.471 ± 0.038 0.486 ± 0.035 0.615 ± 0.055 0.750 ± 0.060 0.696 ± 0.068 ~98 0.529 ± 0.038 0 0.038 ± 0.022 0 0 ~N 85 104 39 26 23* ~H 0.447 0.471 0.462 0.346 0.435 I::l- ~H 0.280 0.075 0.285 0.322 0.630 0.277 0.311 0.0178 0.255 ~HI 0.240 - 0.244 0321 - 0.342 0.242 - 0.250 c::TI = Tawau 1987, T2 = Tawall 1984, SB = Slla Belong 1986, Ll = l.a5.!,ang 7 1987, 1.2 = Ladang..2. 198~, PI = Pllchong .Ian., 87, P2 = Puchong Sept., 87, P::I = Puchong ~19i15, KK = Kuala Kangsar 1987, N = sample size, H= Hell:rozygosity, H = Mean Heterozygosity, HI = Mean Heterozygosity based on PGM, E5T-2, HK, a-GPDH & MDHonly. NA = Not analysed. '" = Data presented for the first time in this paper.AN ELECTROPHORETIC STUDY OF NATURAL POPULATIONS OF CONOPOMORPHA CRAMEREUAthe CA-7 buffer system used by Rusnah et aL(1985) but it was subsequently typed on theVarvio-Aho buffer because on this bufferthree alleles could be typed for PEP-2 where-as on the CA-7 buffer, only two alleles couldbe recognised.PGM, EST-2, PEP-2 (Varvio-Aho buffer),FE-2 and ME-1 were polymorphic in all thepopulations that were analysed for theseenzymes. Slight differences were observed inthe allelic frequencies between the various geo-graphical populations and between populationsfrom the same locality but collected at differenttimes. However, there were no clear cut differ-ences between the allelic frequencies andheterozygosities for populations of rambutanfruit borers and cocoa pod borers.PEP-2 on CA-7 buffer showed poly-morphism only in the Tawau sample of cocoapod borers where alleles PEP-2 F and PEP-2 s(Rusnah et al. 1985) were present whereas in allthe other populations typed only allele PEP-'Zwas present. On the Varvio-Aho buffer, allelePEP_2 I02 was only present in the Tawau popu-lation whereas alleles PEP-2 100 and PEP_2 98 werepresent in the Tawau and Sua Betong cocoapod borer populations and in the Ladang 7 andPuchong populations of rambutan fruit borers.Hexokinase was only polymorphic in theLadang 7, Puchong and Kuala Kangsar popu-lations of rambutan fruit borers in which bothalleles HK104 and HK lOO were present but themost common allele in these populations,namely HKlOO, was fixed in the cocoa pod borerpopulations ofTawau and Sua Betong. a-GPDHwas polymorphic in the Tawau, Sua Betong,Ladang 7, Puchong and Kuala Kangsarpopulations with alleles a_GPDHIOO (a-GPDHFin the terminology of Rusnah et al. 1985) anda _GPDH97 (or -GPDHS) being present but allelea -GPDH 104 was only present at a low frequencyin the Ladang 7 population. MDH was onlypolymorphic in the cocoa pod borer popula-tions of Tawau and Sua Betong and in therambutan fruit borer population of Ladang 7where allele MDH 104 occurred at low frequen-cies. The Puchong and Kuala Kangsar popula-tions were fixed for the common MDH 100 allele.As regards ME-I, ME- 98 was the commoner allelein the cocoa pod borer populations of Tawau,ME-I 102 in the cocoa pod borer population ofSua Betong while the most frequent allele inthe rambutan fruit borer populations fromLadang 7, Puchong and Kuala Kangsar wasME-I 100.Hence, while differences do exist in allelicfrequencies between populations of C. cramerellafrom different geographical areas of Malaysia,such differences also exist between populationsfrom the same locality but collected at differenttimes. Unique alleles were present in certainpopulations but the commoner alleles werepresent in all populations. We have thereforebeen unable to find any diagnostic gene orallele or even to build up a biochemical keythat would enable us to distinguish electro-phoretically between cocoa pod borers andrambutan fruit borers in our present studyusing electrophoresis.H, the average heterozygosity value,calculated based on data for PGM, EST-2, HK,a-GPDH and MDH which are available for Tl,SB, Ll, PI, P2 and KK showed values in therange of 0.240 to 0.342 with no consistentdifferences between the two populations ofcocoa pod borers and the four populations oframbutan fruit borers. (PI & P2 differed intheir times of collection from the same locality).Rusnah et al. (1985) had sugge~ted thepossibility that a large scale electrophoreticsurvey of C. cramerella from Sabah and PeninsularMalaysia may reveal the existence of two species.However, in this study we were unable to findany evidence to support this suggestion. Fur-ther work using more powerful biochemicalgenetic tools like restriction fragment lengthpolymorphisms (RFLP) should be done todetermine whether diagnostic biochemicalmarkers really do not exist between cocoa podborers and rambutan fruit borers. However,financial constraints have prevented us fromusing these techniques.ACKNOWLEDGEMENTThis work was supported by research grant no.50281: Genetics from Universiti PertanianMalaysia. We thank Dr Tay Eong Bok, StateDirector of Agriculture, Sabah for providingthe Tawau Samples, Dr. Loke Wei Hong(MARDI) for providing the Kuala Kangsarsamples, the Federal Department of Agri-culture, Malaysia and Mr. Liau Siau Suan fromPERTANIKA VOL. 12 NO.1, 1989 5RITA MUHAMAD, S.C. TAN, Y.Y. CAN, S. RITA, S. KANASAR AND K. ASUANChemara Research Centre for their invaluableco-operation and assistance during the col-lection of the Sua Betong samples.REFERENCESCHIN, F. 1987. Views on the Eradication Pro-gramme for the Cocoa Pod Borer in PeninsularMalaysia. In Proceedings of the Symposium onManagement of the Cocoa Pod BOT/ff, pp. 176-180.Kuala Lumpur.GREEN, C.A. 1977. A Sex-limited Esterase in theAccessory Glands of Males of Anaphel£s funestus.Mosquito News 37: 46-48.HALMY, A., S.G. TAN, M. RITA, and Y.Y. GAN. 1987.Esterase and Phosphoglucomutase Polymorphismin the Rambutan Fruit Borer, Conapomorphacramerella (Snellen). Journal of Plant Protection inthe Trapics. 4: 101-105.HARRIs, H., and D.A. HOPKINSON. 1976. Handbookof Enzyme Electrophoresis in Human Genetics.North - Holland. Amsterdam.LING, A.H., C.C. YEw, and T.S. KOH. 1987. Ex-perience in Handling the Cocoa Pod BorerOutbreak in Peninsular Malaysia. In Proceedings ofthe Symposium on Management ofthe Cocoa Pod Borer,pp. 129-137. Kuala Lumpur.LOKE, W.H. O. MOHO. SHAMSUDIN, and A.K.ZAM. 1986. Occurrence and Status of Acrocer-caps cramerella Snellen in Peninsular Malaysia. InCocoa and Coconuts: Progress and Outlook. KualaLumpur: ed E. Pushparajah and P.S. Chew, pp265-267. Incorporated Society of Planters.MENKEN, S.BJ. 1980. Allozyme Polymorphism and theSpeciation Process in Small Ermine Moths (Lepidap-tera, Yponomeutidae) Speciation. (Studies in Yponomeu-tidae). PhD. dissertation. University ofLeiden, TheNetherlands.MUNSTERMANN, L.E. 1979. Isozymes ofAedes aegypti.Phenotypes Linkage and Use of Genetic Analysis ofSympatric Species papulation in East Africa. PhD.dissertation. University of Notre Dame, U,S.A.RUSNAH MOHO. SAAHLAN, RITA MUHAMMAD, S.G.TAN, Y.Y. GAN, HALMY AHMAD. 1985. BiologicalPolymorphisms of Peptidase and ex - GPDH inNatural Populations of the Cocoa Pod BorerConapomorpha cramerella (Snellen). Journal ofPlantProtection in the Tropics. 2: 49-52.SHAW, C.R. and R. PRAsAD. 1970. Starch GelElectrophoresis of Enzymes. A Compilation ofRecipes. Biochemical Genetics 4: 297-320.SPENCER, N., D.A. HOPKINSON and H.PARRISH. 1964. Phosphoglucomutase Poly-morphism in Man. Nature (Lond.) 204: 742-745.STEINER, W.W.M. and DJ. JOSLYN. 1979. Elec-trophoretic Techniques for the Genetic Study ofMosquitoes. Mosquito News. 39: 35-54.TAN, S.G., Y.Y. GAN, and K. AsUAN. 1982. Transfer-rin Subtyping in Malaysians and Indonesians fromNorth Sumatra. Human Genetics 60: 369-370.TAN, S.G., M. RITA, Y.Y. GAN, and S. RITA. 1988.Hexokinase, Malate Dehydrogenase, FluorescentEsterase and Malic Enzyme in the Cocoa PodBorer, Conapomorpha cramerella Snellen. Pertanika11(1): 7-13.VARVIo-AHO, SIRKKA-UISA, and P. PAMlLO. 1980. ANew Buffer System with Wide Applicability. IsozymeBulletin. 13: 14.(Received 9 November, 1988)6 PERTANIKA VOL. 12 NO.1, 1989

An Electrophoretic Study of Natural Populations of the Cocoa Pod Borer, Canopomorpha cramerella (Snellen) from Malaysia

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An Electrophoretic Study of Natural Populations of the Cocoa Pod Borer, Canopomorpha cramerella (Snellen) from Malaysia

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