Cocoa pod borers collected in the field from Tawau, Sabah and from Sua Betong, Negeri Sembilan

and rambutan fruit borers collected from Puchong and the campus of Universiti Pertanian Malaysia

(UPM) in Serdang, Selangor, Malaysia were analysed by polyacrylamide gel electrophoresis. Hexokinase

was found to be polymorphic in the UPM population, malate dehydrogenase in the Tawau, Sua Betong

and UPM populations and fluorescent esterase and malic enzyme were polymorphic in all four populations

Gan, Yik Yuen

Muhamad Awang, Rita

Sinha, Rita

Tan, Soon Guan

Universiti Putra Malaysia Institutional Repository

PertanQall1(l), 7-13 (1988)Hexokinase, Malate Dehydrogenase, Fluorescent Esterase andMalic Enzyme Polymorphisms in the Cocoa Pod Borer,Conopomorpha cramerella (Snellen)S.G. TA ,RITA MUHAMAD, Y.Y. GA and S. RITADepartments ofBiology, Biotechnology and Plant ProtectionUniversiti Pertanian Malaysia43400 UPM Serdang, Selangor, Malaysia.Key words: Conopomorpha cramerella; polymorphisms; hexokinase; malate dehydrogenase; fluorescentesterase; malic enzyme.ABSTRAKPengorek buah koko yang dikumpul dari ladang koko Tawau, Sabah dan Sua Betong, NegeriSembilan, dan pengorek buah rambutan yang dikumpul dari Pucung dan kampus Universiti PertanianMalaysia (UPM) di Serdang, Selangor, Malaysia telah dianalisis dengan menggunakan teknik elektroforesisgel poliakrilamid. Hexokinase didapati polimorfik pada populasi UPM, malat dehidrogenase pada populasiTawau, Sua Betong dan UPM dan esteras fluorescent dan enzim malik adalah polimorfik pada ke empat-empat populasiABSTRACTCocoa pod borers collected in the field from Tawau, Sabah and from Sua Betong, Negeri Sembilanand rambutan fruit borers collected from Puchong and the campus of Universiti Pertanian Malaysia(UPM) in Serdang, Selangor, Malaysia were analysed by polyacrylamide gel electrophoresis. Hexokinasewas found to be polymorphic in the UPM population, malate dehydrogenase in the Tawau, Sua Betongand UPM populations and fluorescent esterase and malic enzyme were polymorphic in allfour populations.INTRODUCTIONThe cocoa pod borer, Conopomorpha cramerella,is a major cocoa pest in Malaysia, Indonesia andthe Philippines. In Malaysia two biotypes are pre-sent. The biotype in Sabah attacks both cocoa(Theobromae cacao L.) pods and rambutan(Nephelium lappaceum L.) fruits whereas the onein Peninsular Malaysia attacks only rambutanfruits but an outbreak of the biotype that attackscocoa was reported for the first time in PeninsularMalaysia (in Malacca and Negeri Sembilan) to-wards the end of 1986 (Loke et al., 1986, Linget al., 1987). Fortunately this outbreak in thepeninsula has now been contained (Chin, 1987).A review of the role that biochemical poly-morphisms could play in the taxonomic studyof this species had been presented elsewhere (Ritaand Tan, 1987). We had previously reported onthe occurrence of polymorphisms for the enzymespeptidase, a-glycerophosphate dehydrogenase(Rusnah et al., 1985), phosphoglucomutase andesterase (Halmy et al., 1987). In order to obtainmeaningful data on the population genetics ofthis species, the number of polymorphic markersavailable for use to study this insect should beincreased. We have therefore continued in oureffort to find more biochemica) polymorphismsin this species. We now report our findings thatS.G. TAN, RITA MUHAMAD, Y.Y. GAN AND S. RITA.the enzymes hexokinase (HK. E.C.2.7.1.l),fluorescent esterase (FE. E.C.3.1.1.1.), malatedehydrogenase (MDH. E.C.1.1.1.37) and malicenzyme (ME. E.C.1.1.1.40) are polymorphicwhen analysed electrophoretically in this econo-mically important insect pest.and Hopkinson, 1976). Testing for inhibition bythe carbonic anhydrase inhibitor acetazolamide,was done as described by Harris and Hopkinson(1976).RESULTS AND DISCUSSIONL......- OFig. 1. DiJJgramatic representation ofHexokinase (HK)phenotypes observed in C. cramerella. 0 is theorigin.The population data for HK is presented inTable 1. Both males and females were aboutequally represented in all three phenotypes thusindicating that the HK electromorphs are codedfor by an autosomal locus with two codominantalleles, HK 100 and HK 104 . For allelic designation,A strong zone of HK activity was obser.ved within20 minutes of incubation. This zone migratedabout 5 cm anodally from the origin when thebromophenol blue tracker dye migrated 13 cmanodally. On prolonged incubation of 1-2 hours,another faint zone of enzyme activity could beseen about 2 cm from the origin. However, as theoccurrence of this faint zone was not a certaintyit was therefore not scored. Three phenotypeswere observed for the strong zone (Fig. 1), pheno-type HK 100/100 which showed a single slowband, 100/1 04 which showed a fast and a slowband and 104/1 04 which showed a single fastband.104/104-100/104---PHENOTYPES:100/100MATERIALS AND METHODSC. cramereila was collected from cocoa podsof Sua Betong Estate near Port Dickson in NegeriSembilan in November 1986 and from the TawauDistrict of Sabah, Malaysia in January 1987 basedon the collection and transportation proceduresdescribed in RuSnah et aL (1985). C. cramereilafrom rambutans was collected from Ladang 7 inthe campus of Universiti Pertanian Malaysia inSerdang in January 1987 and in Puchong, Selangor,Malaysia in September 1987 using the collectionand rearing procedures of Rusnah et ai.. 1985.The adult insects were stored at - 70°C until,they were used for electrophoresis.Sample homogenization and polycrylamidegel electrophoresis were done as in Rusnah et ai.(1985) except for the following modifications.The CA-7 buffer system that was used to typefor MDH had 0.1 % Kodak Photoflo incorporatedinto the gel. We found that the addition of 0.1 %Photoflo into our CA-7 gels improved the resolu-tion of not only MDH but also esterase, phos-phoglucomutase and a-glycerophosphate dehydro-genase bands. The concentration of Photoflo thatwe used was a reduction from the 1% used byMunstermann (1979) in his vertical gel system asthat concentration unfortunately caused bubblingproblems in the preparation of our horizontal gels.Use of 0.1 % Photoflo solved the bubbling problemas well as improved the isozyme band resolution.HK, FE and ME were typed using the buffersystem of Varvio Aho et ai. (1980). Staining forMDH, HK and ME was as described in Stainer andJoslyn (1979) except that 0.1 M Tris-HCl, pH 8.0was used as the staining buffer. Staining for FEwas done following the procedure of Harris andHopkinson (1976) for the enzyme carbonicanhydrase with fluorescein diacetate as the sub-strate. Testing for substrate specificity of FEwas done by staining for general esterases witha- and (3-napthyl acetates as substrates (Halmyet ai., 1987) and by staining for esterase D with 4methylumbelliferyl acetate as the substrate (Harris8 PERTANIKA VOL. 11 NO.1, 1988POLYMORPHISMS IN THE COCOA POD BORERTABLE 1Population data for Hexokinase (HK) and Malate Dehydrogenase (MDH) in Conopomorpha cramerella.Numbers within brackets are expected numbers assuming Hardy-Weinberg equilibrium.Enzymes & No. Phenotypes GenePopulations tested 100/100 100/104 104/104 FrequenciesHK HK100 HK 104Ladang 7 81 65 14 2 0.889 ± 0.025 0.111 ± 0.025(64.00) (16.00) (1.00) xi =1.27 P >0.20MDH MDH100 MDH 104Tawau 84 82 2 0 0.988 ± 0.008 0.012 ± 0.008(82.01 ) (1.96) (0.01) xi =0.01 P>0.90Sua Betong 103 94 9 0 0.956 ± 0.014 0.044 ± 0.014(94.20) (8.61 ) (0.20) X2 =0 22 P>0.501 .Ladang 7 63 60 3 0 0.976 ± 0.013 0.024 ± 0.013(60.04) (2.93) (0.04 ) xi= 0.04 P>0.80MDH~1------1------------0c-MDHePHENOTYPES:100/ 100/100 104Fig. 2. Diagramatic representation ofMalateDehydrogenase-l (MDH-l) phenotypes observedin G. cramerella. The Cathodal c-MDH wasmonomorphic showing one band per sample. 0is the origin.three populations are in Hardy Weinberg equili-brium for MOH-!. It is not surprising that wedid not observe the predicted phenotype 104/1 04we called the most common allele in the Ladang7 population as allele .• 100". All other alleleswere named based on the relative mobilities oftheir bands in mm to the band of allele" 100".HK was only polymorphic in the Ladang 7 popula-tion which was in Hardy-Weinberg equilibrium forthis enzyme. All 102 insects from Tawau, 112insects from Sua Betong and 29 insects fromPuchong showed phenotype 1OO/l 00 only.Two zones of MOH were observed after 30minutes of staining. The anodal MOH-l migratedabout 1 em. The cathodal c-MOH migrated 0.2em when the tracker dye migrated anodally 10em from the origin. c-MOH was monomorphicin all the four populations tested, while MOH-lwas polymorphic in the Tawau, Sua Betong andLadang 7 populations. The phenotypes observedare shown in Fig. 2. Phenotype MOH-l 100/l00showed a single slow band while phenotype100/l04 showed three bands which is the typicalpattern observed in the heterozygote for a dimericprotein such as MOH (Harris and Hopkinson,1976). Both males and females are about equallydistributed in the two phenotypes observed. Wepropose that the MDH-l phenotypes are con-trolled by an autosomal locus with two codo-minant alleles MOH_l 100 and MOH_I 104 . Thepopulation data for the three populations poly-morphic for MOH-I are presented in Table 1. AllPERTANIKA VOL. 11 NO.1, 1988 9l00025G723S.G. TAN, RITA MUHAMAO, Y.Y. GAN AND S. RITA.with only one fast band present as its expectedvalue in the all three populations was less thanone. All 30 Puchong samples showed phenotype100/100.Three anodal zones of enzyme activity wereobserved when FE was stained for by using eitherfluorescein diacetate or 4-methyl umbelliferyacetate as the substrate. FE-l migrated about 1cm, FE-2 about 2.5 cm and FE-3 about 5 cmwhen bromophenol blue migrated 13 cm from theorigin. The banding patterns observed when usingthese substrates were identical but the bands wereclearer and easier to score when fluoresceindiacetate was used as the substrate. Hence thissubstrate was routinely used to type the insectsand the gels were read after about 20 minutes ofincubation. However, none of the enzyme zoneswere inhibited by acetazolamide, the carbonicanhydrase inhibitor. Ct- and ~- napthyl acetatesstained only zone FE-3. This zone was found to besimilar to EST-2 described by Halrny etal. (1987)but on the Varvio-Aho buffer system used to typeFE-2, only alleles EST_2100 and EST-297 could beseparated from one another. Allele EST_2 102could not be typed on this buffer. Based on theirabilities to utilize both 4-methylumbelliferylacetate and fluorescein diacetate as substrates butnot Ct- and ~-napthyl acetates, and the fact thatthey are not inhibited by acetazolamide, it is likelythat FE-l and FE-2 are similar to human esteraseD (Harris and Hopkinson, 1976). FE-l was mono-morphic and showed one band per sample in allthe insects typed while FE-2 was polymorphic inall the four populations analysed. Eleven pheno-types had been observed for this enzyme (Fig. 3).The homozygous phenotypes, FE-2 100/ 100,105/105, 95/95 and 97/97 showed a single bandeach while the heterozygous phenotypes, 105/100,100/95, 105/95, 105/97, 100/97, 100/90 and95/97 showed two bands each, typical of a mono-meric protein. The population data are presentedin Table 2. Both males and females are aboutequally represented in the various phenotypes.Hence it is proposed that FE-2 is coded for by anautosomal locus with five autosomal codominantalleles FE_2 100 , FE_2 10S , FE_297, FE-29 5 andFE_2 90 . The rare· allele, FE_290 , and phenotypeFE-2, 100/90, were not taken into account when----- - - - - - --FE-3- - - -- - - -FE-2-- - - -- - - --- - - - -- - - - -FE-1-0FE - 2 PHENOTYPES95/ 105/ 105/ 100/ 100/100/ 105/ 97/ 95/ 100/ 100/100 105 97 95 90 97 97 97 95 95 105Fig. 3. Diagramatic representation of Fluorescent Esterase (FE) zones seen in C. cramerella. FE-] was monomorphicand FE-3 was not Typed. All the FE-2 phenotypes observed are presented here. 0 is the origin.10 PERTANIKAVOL.l1 NO.l,1988POL YMORPHISMS IN THE COCOA POD BORERTABLE 2Population data for Fluorescent Esterase - 2 (FE-2) in Conopomorpha cramerelw. Numbers within brackets areexpected numbers assuming Hardy-Weinberg equilibrium. N = number·tested.Phenotypes & PopulationsGene frequencies Tawau Sua Betong Ladang 7 Pucung100/100 25 (25.52) 16 (15.08) 10 (9.56) 6 (5.63)105/105 1 (0.75) 5 (3.25) 1 (0.56) 2 (1.63)97/97 o(0.02) o (0.04) 0 1 (0.30)95/95 1 (0.75) 3 (2.36) 1 (0.73) 2«(1.41)100/105 9 (8.75) 11 (13.98) 4 (4.61) 6 (6.07)100/95 9 (8.75) 12 (11.91) 5 (5.27) 5 (5.63)105/95 1 \l.50) 5 (5.54) 1 (1.27) 3 (3.03)105/97 o(0.25) 1 (0.72) 0 l( 1.40)100/97 2 (1.46) 2 (1.55) 0 3 (2.60)97/95 o(0.25) o (0.62) 0 1 (1.30)100/90 0 1 0 1N 48 56 22 30FE_2 100 0.729 ± 0.045 0.518 ± 0.047 0.659 ± 0.072 0.433 ± 0.064FE_210S 0.125 ± 0.034 0.241 ± 0.040 0.159 ± 0.055 0.233 ± 0.055FE-297 0.021 ± 0.015 0.027 ± 0.015 0 0.100 ± 0.039FE_29S 0.125 ± 0.034 0.205 ± 0.038 0.182 ± 0.058 0.217 ± 0.053FE_29O 0 0.009 ± 0.009 0 0.017 ± 0.0172 X~ = 2.75 X~ = 0.630 2 _X6 = 1.09 X6 - 2.30P> 0.95 P> 0.80 P> 0.80 P > 0.80'----------------0ME-1 PHENOTYPES100/ 102/ 98/ 100/ 100/100 102 98 98 102Fig. 4. Diagramatic represen tation ofMalic Enzyme(ME) zones seen in C. cramerella. ME-2 wasnot typed. All the ME-] phenotypes observedare presented here. 0 is the origin.- - - --_ _ ME-2- - - _.-II ME-1•---the populations were tested for Hardy-Weinbergequilibrium. All four populations were in Hardy--Weinberg equilibrium thus supporting our geneticinterpretation of this locus.Two anodal zones of ME activity were ob-served after the gels were stained for about 30minutes. The intensely stained ME-I zone mi-grated about 1 cm while the weaker ME-2 zonemigrated 2 cm when the tracker dye migrated 13cm from the origin. ME-2 showed either two orthree bands per sample but since it could not bescored with confidence it was not typed. Fivephenotypes had been observed for ME-I (Fig. 4),phenotypes ME-I 100/100, 98/98 and 102/1 02each showed a sharp single band while phenotypes100/98 and 100/102 showed a broad band each.The broad band may represent five closely migra-ting bands since according to Harris and Hopkin-son (1976), ME is a tetrameric protein. However,the molecular structure of this enzyme in Conopo-morpha can only be confirmed through furtherbiochemical studies which is beyond the scope ofour expertise. All four populations are polymor-PERTANlKA VOL. 11 NO. I, 1988 11S.G. TAN, RITA MUHAMAO, Y.Y. GAN AND S. RITA.TABLE 3Population data for Malic Enzyme-l (~E-l) in Conopomorpha cramerelkz. Numbers within brackets are expected numberassummg Hardy-Weinberg quilibrium. N = number tested.Phenotypes & PopulationsGene frequencies Tawau Sua Betong Ladang 7 Pucung100/100 21 (18.82) 26 (24.52) 15 (14.77) 15 (14.63)100/98 38 (42.35) 0 3 (1.85) 098/98 26 (23.82) 0 0(0.06) 0102/102 0 29 (27.52) 6 (4.67) 2 (1.63)102/100 0 49 (51.96) 15 (16.62) 9 (9.75)98/102 0 0 0(1.04) 0N 85 104 39 26ME_l!OO 0.471 ± 0.038 0.486 ± 0.035 0.615 ± 0.055 0.750 ± 0.060ME_1 98 0.529 ± 0.038 0 0.038 ± 0.022 0ME_l!02 0 0.514 ± 0.035 0.346 ± 0.054 0.250 ± 0.060X~ = 20.90 X = 0.34 X2 = 2.36 2! 3 X! = 0.15P > 0.20 P > 0.50 P > 0.50 P > 0.50phic and in Hardy-Weinberg equilibrium for ME-l(Table 3). Both sexes were present in about equalnumbers in the phenotypes observed. We proposethat ME-l is controlled by an autosomal lo'cuswith three codominant alleles, ME-]! 00, ME-]! 02and ME-] 98. ME_]98 is the commoner allele inthe Tawau population with a frequency of 0.529± 0.038 as against 0.471 ± 0.038 for alleleME_]!OO while in the Ladang 7 and Puchong popu-lations ME-]! 00 is the most frequent allele and. in the' Sua Betong population, ME-]! 02 is thecommoner of the two alleles present.ACKNOWLEDGEMENTSThis work was funded by Universiti PertanianMalaysia through research Grant No. 50281Genetics. We thank Dr. Tay Eong Bok, StateDirector of Agriculture, Sabah for providing uswith the Tawau samples, Ms. Sivarogini Kanasarfor collecting the Sua Betong samples, the FederalDepartment of Agriculture, Malaysia for their in-valuable cooperation and assistance during thecollection of the Sua Betong samples, and AsuanKudus and Azney Amir for collecting the UPMand Puchong samples.REFERENCESCHIN, F. (1987): Views on the eradication programmefor the cocoa pod borer in Peninsular Malaysia.Proceedings of the Symposium on Management ofthe Cocoa Pod Borer. Kuala Lumpur. 1987. pp.176-180.HALMY, A., S.G. TAN., M. RITA and Y.Y. GAN. (1987):Esterase and phosphoglucomutase polymorphismsin the rambutan fruit borer, Conopomorpha cra-merelkz (Snellen) J.P. Prot. Tropics 4:101-105.HARRIS, H. and D.A HOPIKINSON. (1976): Hand-book of 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 borer outbreakin Peninsular Malaysia. Proceedings of the Sympo-sium on Management of the Cocoa Pod Borer.Kuala Lumpur. 1987. pp.129-137.LOKE, W.H., O. MOHO. SHAMSUDIN and A.K. ZAM.(1986): Occurrence and status of Acrocercopscramerelkz Snellen in Peninsular Malaysia. In: Push-parajah, E., Chew, P.S. (Eds.) Cocoa and coconuts:Progress and Outlook. Incorporated Society ofPlanters. Kuala Lumpur. pp. 265 -267.MUNSTERMANN, L.E. (1979): Isozymes of Aedesegypti. Phenotypes, linkage and use of geneticanalysis of sympatric species populations in EastAfrica. PhD. dissertation. University of Notre Dame,U.S.A.RITA MUHAMAO and S.G. TAN (1987): Biochemicaltaxonomy of the cocoa pod borer: a prospectiveview. Proceedings of the Symposium on Manage-ment of the Cocoa Pod Borer. Kuala Lumpur. 1987.pp.1l9-126.RUSNAH MOHO. SAAHLAN., RITA MUHAMAD,12 PERTANlKA VOL. 11 NO. I, 1988POLYMORPHISMS IN THE COCOA POD BORERS.G. TAN, Y.Y. GAN and HALMY AHMAD.(1985): Biological polymorphisms of peptidase anda-GPDH in natural populations of the cocoa podborer Conopomorpha cramerella (Snellen). 1. PI.Prot. Tropics. 2:49-52.STEINER, WW.M. and DJ. JoSYLYN. (1979): Elec-trophoretic techniques for the genetic study ofmosquitoes. Mosq. News 39: 35-54.VARVIO-AHO, SIRKKA-LISA and P. PAMILO. (1980):A new buffer system with wide applicability. Iso-zyme Bulletin 13: 14.(Received 20 December 1987)PERTANlKA VOL. 11 NO.l,1988 13

Hexokinase, Malate Dehydrogenase, Fluorescent Esterase and

Malic Enzyme Polymorphisms in the Cocoa Pod Borer,

Conopomorpha cramerella (Snellen)

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Hexokinase, Malate Dehydrogenase, Fluorescent Esterase and Malic Enzyme Polymorphisms in the Cocoa Pod Borer, Conopomorpha cramerella (Snellen)

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