Helopeltis theobromae caught off cocoa plants in Kuala Selangor and off Acalypha plants in

Serdang, Selangor, Malaysia were investigated electrophoretically for nine biochemical markers: peptidase,

a -glycerophosphate dehydrogenase, 6-phosphogluconate dehydrogenase, xanthine

dehydrogenase, aldehyde oxidase, phosphoglucose isomerase, isocitrate dehydrogenase, glutamate

transaminase and adenylate kinase. Only peptidase-2 polymorphism could be easily interpreted

Gan, Yik Yuen

Mohd Saahlan, Rusnah

Muhamad Awang, Rita

Tan, Soon Guan

Universiti Putra Malaysia Institutional Repository

Pertanika 9(1), 65 - 68 (1986)Peptidase Polymorphism in Natural Populations of the Cocoa Pest,Helopeltis theobrornae MillerRUSNAH MOHD SAAHLAN, S.G. TAN*, RITA MUHAMADland Y.Y. GANDepartment of Biology and IDepartment of Plant ProtectionUniversiti Pertanian Malaysia,43400 Serdang, Selangor, Malaysia.Key words: Helopeltis theobromae; electrophoretic analysis; biochemical markers; peptidasepolymorphism.ABSTRAKHelopeltis theobr01nae yang ditangkap danpada tanaman koko di Kuala Selangor dandanpada tanaman Acalypha di Serdang, Selangor, Malaysia telah disiasat dengan teknikelektroforesis untuk sembilan penanda biokimia iaitu: peptidase, a -gliserofosfat dehidrogenase,6josfoglukonat dehidrogenase, xanthina dehidrogenase, aldehid oksidase, fosfoglukosa isomerase,isositrat dehidrogenase, glutamat oksaloasetat transaminase dan adenilat kina~e. Hanya peptidase-2menunjukkan polimorfisma yang dapat dibincang dengan mudah.ABSTRACTHelopeltis theobromae caught off cocoa plants in Kuala Selangor and offAcalypha plants inSerdang, Selangor, Malaysia were investigated electrophoretically for nine biochemical markers: pep-tidase, a -glycerophosphate dehydrogenase, 6-phosphogluconate dehydrogenase, xanthinedehydrogenase, aldehyde oxidase, phosphoglucose isomerase, isocitrate dehydrogenase, glutamatetransaminase and adenylate kinase. Only peptidase-2 polymorphism could be easily interpreted.INTRODUCTIONHelopeltis theobromae Miller (Hemiptera:Miridae) or the mosquito bug is a serious pest ofcocoa in Malaysia. The adults and nymphsattack both the pods and shoots of cocoa. Thisleads to severe plant damage even when the in-sects are low in numbers (Ibrahim, 1983). Thepods are attacked at almost every stage ofdevelopment but young developing pods arepreferred by this pest and significant crop lossesoccur when heavy infestation is unchecked(Taylor et al., 1982). It has been observed thatH. theobramae also attacks other crops such ascashew, mango and the ornamental plant'To whom reprint requests should be addressed.Acalypha spp. in Malaysia.No previous work on enzymic polymor-phisms of this insect has been done in this coun-try. We now report our screening of this insectpest for the following biochemical markers: pep-tidase (PEP, E.C. 3.4.1.1), a-glycerophosphatedehydrogenase ( a -GPDH, E.C. 1.1.1.8), 6-phos-phogluconate dehydrogenase (6 PGD, E.C.1.1.1.4.4), xanthine dehydrogenase (XDH, E.C.1.2.1.37), aldehyde oxidase (ALDOX, E.C.1.2.3.1), phosphoglucose isamerase (PGI, E.C.5.3.1.9), isocitrate dehydrogenase (IDH, E.C.I. I. I .42), glutamate oxaloacetate transaminase(GOT, E.C. 2.6.1. I) and adenylate kinase (AK,E.C.2.7.4.3).RUSNAH MOHD SAAHLAN, S.G. TAN. RITA MUHAMAD AND Y.Y. GANPEP-2SS•-FS---A. PEPTIDASE--PEP-l-+------------_0PEP-2 FFAll the 61 KS insects caught off cocoa thatwere typed for GOT (Fig. 1B) showed one bandper sample (zone 1). However. for insects caughtoff A calypha plants in UPM. 27 samples showedonly the single band at zone 1. while 26 samplesshowed the band at zone 1 and another at zone2. When an aqueous extract of Acalypha leafwas run on the gel for GOT. a single band ofGOT activity corresponding to zone 2 wasobserved. However. no GOT activity was observ-ed when an aqueous extract of cocoa pod wassimilarly analysed. Since adult insects can feedon either cocoa pod sap or Acalypha leaf sap. itcoded for by one locus which two codaminantalleles. PEP-2 Fwhich codes for the fast band andPEP-2 S which codes for the slow band. Heter-ozygous individuals had both the fast andslow bands. Population data is presented inTable 1. This population data is a pool of bothmale and female samples since all three pheno-types were present in both sexes thus ruling outsex linkage for PEP-2. Both the UPM and KSpopulations were in Hardy-Weinberg equili-brium for PEP-2 with their allelic frequenciesclose to one another. Since the heterozygote FSshowed two bands. PEP·2 is most probably amonomeric protein. As the PEP-l banding pat-tern was unaffected by variation in the PEP-2pattern. it is likely that PEP-2 is coded for by adifferent locus. Family studies should be done inthe future to confirm the mode of inheritance ofPEP-2.RESULTS AND DISCUSSIONMATERIAL AND METHODSA seven percent polyacrylamide gel system(Tan and Teng. 1979) was used together withthe CA-7 buffer of Steiner and Joslyn (1979) forPEP. GOT. PGI. a-GPDH. IDH and AK andtogether with TEMM buffer (Spencer et al.,1964) for'6PGD. ALDOX and XDH. All elec-trophoretic runs were made in a refrigerator setat 4°C. 0.5% bromophenol blue in 1% aceticacid was used as the tracker dye and a constantcurrent of 30 rnA was applied to each gel. Therun was terminated when the tracker dye hadmigrated about 8.5 cm from the origin. Thistook about 2 to 3 hours. Staining procedures forthe various enzymes were according to Steinerand Joslyn (1979) except that 0.1 M Tris-HClpH = 8.0 was used as the staining buffer for allstains, MgCl2was used in place of MnCl 2 as a co-factor in IDH staining and that PEP was stainedaccording to Loxdale et al (1983) with L-leucyl-L-alanine as the substrate. but using the abovestaining buffer.Peptidase showed two strong regions of en-zyme activity (Fig. lAY. PEP-l which did notmigrate much anodally from the origin wasmonomorphic showing one band per sample in32 samples from UPM and 52 samples from KS.The more anodal migrating PEP-2 region waspolymorphic showing three phenotypes. FFwhich had one fast band. SS which had one slowband and FS which showed both the fast andslow bands. We propose that the PEP-2 region isSingle adult insect of both sexes was homo-genised in a drop of cold distilled water imme-diately before use. The homogenate was then ab-sorbed onto a piece of 2 X 6 mm. Whatman 3filter paper wick for insertion into the ap-propriate electrophoretic gels.Insects were collected off A calypha plants inthe campus of Universiti Pertanian Malaysia(UPM) Serdang. Selangor and off cocoa trees inKuala Selangor (KS). Selangor. Malaysia. Theywere stored in a deep freezer set at - 70°C untilused for electrophoresis.66 PERTA lKA VOL. 9 NO. I. 1986PEPTIDASE POLYMORPHISM IN HELOPELTlS THEOBROMAE MILLERTABLE 1PEP-2 phenotypic distribution and gene frequencies in Helopeltis theobromae. Within brackets are theexpected numbers assuming Hardy- Weinberg equilibriumPopulation No. TestedUPM 32K5 52Phenotypes Gene frequenciesFF F5 55 PEP-2 F PEP-2 s16 12 4 0.688 ± 0.058 0.312 ± 0.058(15.13) (13.75) (3.13) 2 P > 0.20Xl = 0.5218 30 4 0.635 ± 0.047 0.365 ± 0.047(20.94) (24.12) (6.94) 2 P > 0.05Xl = 3.10was likely that in those insects caught offA calypha plants which showed both zones 1 and2, zone 1 represented insect GOT while zone 2represented GOT from freshly sucked Acalyphaleaf sap.PGI showed a complex pattern with eachsample showing 4 to 6 bands which migratedclose to one another making interpretation dif-ficult in the 155 KS and 97 UPM samples thatwere typed.All the other enzymes that were screenedwere monomorphic and showed similiar bandsfor both the UPM and KS samples. AK showedtwo major bands per sample in 57 UPM and 107KS insects. Sometimes, the staining for the moreanodal of the two AK bands was interferred withby the appearance of a colourless band ofsuperoxide dismutase at almost the same posi-tion. 6PGD (155 UPM and 91 KS) showed oneLegend to FIgure 1.•B.GOTI•2•3I4ZONE-2ZONE-lomajor and one more anodal minor band persample. XDH (122 UPM, 75 KS) and a -GPDH(103 UPM, 98 KS) showed one band per sample.ALDOX (122 UPM, 85 KS) usually showed onemajor band sample. Some samples howevershowed, in addition, one or two more cathodalminor bands which could represent breakdownproducts of the major Aldox band.Since both alleles PEP-2 F and PEP-2 S werecommon in the two populations of H.theobromae we investigated, PEP-2 is a verypolymorphic enzymatic marker which could beuseful for future studies of this agriculturally im-portant cocoa pest. For example electrophoreticdata could provide information about thepopulation structure of intraspecific populationsfrom different geographical areas as well asreveal the existence of genetically distinct speciesin sympatric populations of morphologicallysimilar insects (Berlocher, 1984). Such informa-tion have both theoretical as well as practical ap-plications and are essential for formulating ef-fective pest control programmes. In H.theobromae it would be important to knowwhether insects that attack cashew and mangobelong to the same species as those that attackcocoa and A calypha or whether they belong togenetically distinct but morphologically-crypticspecies.ACKNOWLEDGEMENTThis work was funded by research grant no.1705-1-506, Faculty of Science and Environ-mental Studies, Universiti Pertanian Malaysia.PERTANIKA VOL. 9 NO. I, 1986 67RUS AH MOHD SAAHLA ,S.C. TAN, RITA MUHAMAD AND Y.Y. GAREFERENCESBERLOCHER, S.H. (1984): Insect molecular systema-tics. Annual Review ofEntomology 29: 403 - 433.IBRAHIM, W.A. (1983): Helopeltis: biology, ecologyand control. Proceedings of Seminar on Aduancesin Cocoa Plant Protection in Malaysia, KualaLumpur, 1983, pp. 16 -18.LOXDALE, H.D., CASTANERA, P., BROOKES, C.P.(1983): Electrophoretic study of enzymes fromcereal aphid populations. I. Electrophoretictechniques and staining systems for characteris-ing isoenzymes from six species of cereal aphids(Hemiptera: Aphididae). Bulletin of Entomolo-gical Research 73: 645 - 657.SPENCER, N., HOPKINSON, D.A., HARRIS, H. (1964).Phosphoglucomutase polymorphism III man.Science 204: 742 - 745.STEINER, W.W.M., JOSLYN, D.J. (1979). Electropho-retic techniques for the genetic study of mos-quitoes. Mosquito News 39: 35 - 54.TAYLOR, J.G., Ho, C.T. and LIM, T.M. (1982). Amechanised thermal fogging system for controll-ing Helopeltis theobromae Mill in cocoa. Pro-ceedings of Plant Protection in the Tropical,Kuala Lumpur, 1982, pp. 695 -703 ..TAN, S.G., TENG, Y.S. (1979). Human saliva as asource of biochemical genetic markers. I. Techni-ques. Human Hereditary. 29: 69 - 76.(Received 2 October, 1985)68 PERTA IKA VOL. 9 O. I, 1986

Peptidase Polymorphism in Natural Populations of the Cocoa Pest, Helopeltis theobromae Miller

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Peptidase Polymorphism in Natural Populations of the Cocoa Pest, Helopeltis theobromae Miller

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