Pertanika 12(1), 1-6(1989)

An Electrophoretic Study of Natural Populationsof the Cocoa Pod Borer, Conopomorpha cramerella

(Snellen) from Malaysia.

RITA MUHAMAD, S.G. TAN, Y.Y GAN, S. RITA,S. KANASAR and *L ASUAN.

Departments of Plant Protection, Biology, and BiotechnologyUniversiti Pertanian Malaysia

43400 UPM Serdang, Selangor Darul Ehsan, Malaysia.

Key words: Conopomorpha cramerella; cocoa pod borers; rambutan fruit borers; electromorphs;polymorphisms.

ARSTRAKPengorek buah koko dari Tawau, Sabah dan Sua Betong, Negeri Sembilan dan pengorek buah rambutan dariSerdang 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 polimorf juga dipaparkan.

ABSTRACTCocoa pod borers from Tawau, Sabah and Sua Belong, 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 o/Conopomorpha 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 allelic frequencies for 8 polymorphicenzymes are presented.

INTRODUCTION bromae cocoaL.) and that which attacks rambutanThe cocoa pod borer, Conopomorpha cramerella (Nephelium lappaceum L.) fruits from the(Snellen) (Lepidoptera: Gracilariidae) is a Peninsula within a period of three monthsmajor cocoa pest in Sabah State, Malaysia but (November 1986 to January 1987) althoughuntil late 1986 it was only present as a minor unfortunately not from the same locality. Wepest of rambutan in Peninsular Malaysia (Loke were also able to obtain the cocoa biotype frometal 1986, Ling et at 1987). Both the cocpa Tawau, Sabah in January 1987. With thesepod borer and the rambutan fruit borer are samples, we attempted to find diagnosticknown scientifically as C. cramerella. An outbreak electormorphs between the two biotypesof the cocoa biotype occurred for the first time of C.cramerella.in the states of Malacca and Negeri Sembilanin Peninsular Malaysia in September and MATERIALS AND METHODSNovember 1986 respectively. Fortunately the The following samples were available for electro-cocoa pod borer had not been detected phoretic analysis: cocoa pod borers from Suaelsewhere in the Peninsula (Chin 1987). Betong Estate (SB) near Port Dickson in NegeriThis outbreak enabled us to collect both the Sembilan collected in November 1986 and frombiotype that attacks the pods of cocoa (Theo- the Tawau district of Sabah collected in January

RITA MUHAMAD, S.G. TAN, Y.Y. GAN, S. RITA, S. KANASAR AND K. ASUAN

1987 (Tl); rambutan fruit borers from Ladang7 in the campus of Universiti Pertanian Malay-sia, Serdang, Selangor collected in January 1987(LI); 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 al (1985). Various buffer systemssuch as TEB (Green 1977), CA-7 (Steiner andJoslyn 1979), TEMM (Spencer et al 1964)and that of Varvio-Aho et al (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 al (1982) using LKB ampholytes with pHranges of 5-7 and 4-6 after which general proteinwere stained for by using Coomassie BrilliantBlue R250.

The polymorphic markers, phospho-glucomutase (PGM), esterase (EST) (Halmyet al. 1987), a-glycerophosphate dehydro-genase (ct-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 (LI) 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 al (1985) and that for L2 (RB fromLadang 7 UPM collected in March-April 1985)is from Halmy et al (1987). The data for HK,MDH, FE-2 and ME-1 for populations Tl, SB,P2 and LI are obtained from Tan et al (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-1 is not presented here asit could not be reliably typed in this studybecause the quality of the EST-1 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, PGM9g which wasnot detected previously (Halmy et al 1987)could now be typed. PEP was initially typed on

PERTANIKA VOL. 12 NO. 1, 1989

TABLE 1Gene frequency data for Esterase-2 (EST-2), phosphoglucomutase (PGM), peptidase-2 (PEP-2), a-glycerophosphate dehydrogenase (a-GPDH)

hexokinase (HK), malate dehydrogenase (MDH), fluorescent esterase -2 (FE-2) and malic enzyme (ME-1) in natural populations ofConopomorpha cramerella (Snellen) from five localities in Malaysia.

z

or

p

iss

LocusAllele

PGM1051009897NHEST-210210097NH

PEP-2FSNH

HK1041.00NH

&

0.2310.639

;• 0.0380.087

0.279: 0.553• 0.168

77

± 0.029± 0.033±0.013± 0.020

104*0.538

± 0.031± 0.035± 0.026

104*0.567

(CA-7 buffer)0.9470.053

PEP'2-(Varia-Aho's102100

0.1440.800

± 0.036± 0.036

19*0.105

0

01.000

1020

buffer)± 0.037± 0.042

12

NA

. ^ •

r• i . ' '• •

NA

- , • r •v ' ; " •-• K

, " - • ' .

0.919 ± 0.0220.081 ± 0.022

740.162

. NA ;^

.-. ' r . ' • •

*NA ^

0.3440.5430.0330.080

0.3610.5740.066

! • * & • • • •

».932

±±±±

±±±

±

0.0330.0340.0120.019106*

0.591

0.0310.0320.016122*

0.525

1.0000

18*0

01.000

1120

00.027

0.3520.5120.0490.086

0.3750.4870.138

0.1110.889

0.895

LI

±±±±

±±±

±±

±

Allelic frequencies

0.0380.0390.0170.022

81*0.593

0.0390.0410.028

76*0.592

1.0000

0

0.0250.025

810.173

00.050

L2

0.302 ±0.476 ±

_0.221 ±

0.355 ±0.428 ±0.217 ±

r NA

2 NA,

NA

0.0320.034

0.028106*

0.642

0.0370.0380.032

83*0.614

19*

PI

0.340 ± 0.0670.420 ± 0.0700.080 ± 0.0380.160 ±0.052

25*0.640

0.406 ± 0.0870.469 ± 0.0880,125 ± 0.054

16*- 0.563

:• ^ 1.000:. ' 0. ; •• 1 8 *: - . °

0.042 ± 0.0410.958 ±0.041

12*••,•: 0 . 0 8 3

^ N A !i

0.5000.361

0.140

0.3750.4790.146

P2

±±

±

±±

NA

0.940 ±

0.0830.080

00.058

18*0.611

0.0700.0720.051

24*0.708

01.000

280

00.034

P3

NA

NA

^ 1.000

r °u 41*0

NA

NA

0.3040.5220.0440.130

0.4170.3890.194

0.0800.920

•••

• - '

KK

± 0.068± 0.074± 0.031± 0.050

23*0.565

± 0.082± 0.081± 0.067

18*0.444

1.0000

14*0

± 0.038± 0.038

25*0.080

NA

RO

PH

OR

ET

H

c;

w

0*nZ

£

Sc

1c

11i1

—

PI

IKA

\

0

0

19*

98NH

a-cmnF= 100S = 97104NH

MDH104100NH

FE-2105100979590NH

ME-110210098NII

HIII

0.056

0.9950.005

0.0120.988

0.1250.7290.0210.125

0.4710.529

t

±±

±±

±±±±

±±

0,02445*

0.311 .

0.0050.005

098*

0.010

0.0080.008

840.024

0.0340.0450.0150.034

048

0.438

00.0380.038

850.447

0.2800.240

1.00000

860

NA

NA

NA

0.075

0.068 ± 0.02744*

0.136

0.996 ± 0.0040.004 ± 0.004

0118*

0.008

0.044 ±0.0140.956 ± 0.014

1030.087

0.241 ± 0.0400.518 ± 0.0470.027 ±0.0150.205 ± 0.0380.009 ± 0.009

560.554

0.514 ±0.0350.486 ± 0.035

0104

0.471

0.2850.244

0.105 ± 0.05019*

0.211

0.956 ± 0.0190.018 ±0.0120.026 ±0.015

57*0.088

0.024 ± 0.0130.976 ± 0.013

630.048

0.159 ± 0.0550.659 ± 0.072

00.182 ± 0.058

022

0.455

0.346 ± 0.0540.615 ± 0.0550.038 ± 0.022

390.462

0.3220.321

NA

NA

NA

1.00000

18*0

01.000

5*0

NA

NA NA

0.630 0.2770.342

0.060 ± 0.03425*

0.12

0.981 ± 0.0190.019 ±0.019

026*

0.038

01.000

240

0.233 ± 0.0550.433 ± 0.0640.100 ± 0.0390.217 ± 0.0530.017 ± 0.017

300.633

0.250 ± 0.0600.750 ± 0.060

026

0.346

0.3110.242

0.989 ± 0.0050.011 ±0.005

0184

0.022

NA

NA

0.917 ±0.0460.083 ± 0.046

018*

0.167

01.000

20*0

NA

NA

0.0178

Tl - Tawau 1987, T2 • Tawau 1984, SB = Sua Betong 1986, LI = Ladang 7 1987, L2 = Ladang_7 1985, PI = Puchong Jan., 87, P2 = Puchong Sept., 87, P3 = Puchong1985, KK = Kuala Kangsar 1987, N = sample size, H= Heterozygosity, H - Mean Heterozygosity, HI * Mean Heterozygosity based on PGM, EST-2, HK, ct-GPDH & MDHonly. NA = Not analysed. * • Data presented for the first time in this paper.

2X

CO

p

O

2

0.3040.696

±+

0.0680.068

023*

0.435

0.2550.250

2:

>

a

G

AN ELECTROPHORETIC STUDY OF NATURAL POPULATIONS OF CONOPOMORPHA CRAMERELLA

the 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* and PEP-2S

(Rusnah et al. 1985) were present whereas in allthe other populations typed only allele PEP?was present. On the Varvio-Aho buffer, allelePEP-2102 was only present in the Tawau popu-lation whereas alleles PEP-2 10° 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 HKm4 and HK100 were present but themost common allele in these populations,namely HKm, was fixed in the cocoa pod borerpopulations of Tawau and Sua Betong. a-GPDHwas polymorphic in the Tawau, Sua Betong,Ladang 7, Puchong and Kuala Kangsarpopulations with alleles a-GPDH 10° (a-GPDH¥

in the terminology of Rusnah et al. 1985) anda-GPDH97 (or -GPDHS) being present but allelea -GPDH104 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 MDH104 occurred at low frequen-cies. The Puchong and Kuala Kangsar popula-tions were fixed for the common MDH 10° allele.As regards ME-1, ME-m was the commoner allelein the cocoa pod borer populations of Tawau,ME-1 102 in the cocoa pod borer population of

Sua Betong while the most frequent allele inthe rambutan fruit borer populations fromLadang 7, Puchong and Kuala Kangsar wasME-1 10°.

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, LI, 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 8c P2 differed intheir times of collection from the same locality).

Rusnah et al. (1985) had suggested 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 from

PERTANIKA VOL. 12 NO. 1, 1989

RITA MUHAMAD, S.G. TAN, Y.Y. GAN, S. RITA, S. KANASAR AND K. ASUAN

Chemara Research Centre for their invaluablecooperation and assistance during the col-lection of the Sua Betong samples.

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6 PERTANIKA VOL. 12 NO. 1, 1989