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Biological and genetic differences between populations of diamondback moth from different geographic origins

A. Pichon, D. Bordat, A. Bordat, L. Arvanitakis and C. Tertois

CIRAD-ENTOTROP, CSIRO, Campus international de Baillarguet, TA 40/L, 34398 Montpellier Cedex 5, France. Corresponding author: apolline.pichon@cirad.fr

Abstract

The diamondback moth, Plutella xylostella (Lepidoptera: Yponomeutidae), is a major pest of Brassicaceae and has a worldwide distribution. Biological and genetic differences were studied between populations native to South Africa, Benin, Brazil, France, Japan, United States, Martinique, Réunion Island, Uzbekistan and five localities in Australia. To observe the variability of oviposition behaviour between populations, the eggs laid by 20 females from each population studied were counted daily. Females from Benin and South Africa laid more eggs than the females from Uzbekistan and Martinique. Oviposition duration is longest (30 days) in the Uzbekistan population and varies from 15 to 20 days in the other populations. Females from Benin, Réunion Island and Martinique laid the majority of their eggs in the first four days; those from Uzbekistan and South Africa staggered oviposition over the whole period. Concerning genetic differences, the results of isoenzyme electrophoresis reveal variability between populations for nine loci. Tests of the Hardy-Weinberg equilibrium show heterozygote deficits in many populations. Analysis of allelic frequencies gives an estimate of Fst of 0.103 for all the populations studied. The populations from Australia and Japan are the most different from other populations and from each other. Some biological and genetic differences are shown between populations. Genetic differences are not correlated with the geographic distance separating the populations.

Keywords

Plutella xylostella, polymorphism, oviposition, isoenzyme

Introduction

Plutella xylostella (L.) (Lepidoptera: Yponomeutidae) is the most important pest of cultivated brassicas in tropical areas (Talekar & Shelton 1993) and has a global distribution. Its geographic origin is generally considered to be in the eastern Mediterranean area, as are Brassica species (Tsunoda 1980). However, Kfir (1998) reports that the origin could be in South Africa. The migratory ability of diamondback moth is in part responsible for this wide distribution (Talekar & Shelton 1993). Massive migrations over long distances (>3000 km) have been reported between the south of Finland and England (Chu 1986) and populations observed in Canada probably migrate from the south of the United States of America (Harcourt 1986). Despite its ability to migrate, important differences in insecticide resistance exist between closed populations; sometimes separated by less than ten kilometres (Cheng 1981, Tabashnik et al. 1987). Gene flow between these populations may not be sufficient to overcome the differences.

The habitat of a population (temperature, humidity, environmental factors) and growing conditions (size of cultivated area, use of insecticides) differ from one area to another. Considering populations from various countries where selection pressures are variable, are they different from each other? To elucidate these differences, biological characterisation, oviposition activity and enzyme genotype polymorphism of 14 populations have been studied.

Material and methods

(a) Biological activity

The oviposition activities of populations from South Africa, Benin, Martinique, Réunion Island and Uzbekistan were studied. Each sample collected was reared at 25°C, 75% humidity and photoperiod 12L/12D. Mass rearing was maintained on cultivated brassicas: Chinese mustard (Brassica juncea), cabbage (B. oleracea var. Châteaurenard) and cauliflower (B. oleracea var. botrytis). Pupae were isolated from the first generation obtained during the rearing and 20 pairs of a female and a male were randomly chosen, soon after emergence. Each replicate for a population consisted of five females and five males placed in a plastic box. A Chinese mustard leaf received the eggs laid by females. The adults were fed with honey. Eggs were counted every day, until the death of females and the leaf was changed every day. Results were submitted to an analysis of variance (ANOVA) and to a Newman & Keuls test (α = 5%) with the software Statitcf (Gouet & Philippeau 1992).

(b) Genetic differences

Enzyme electrophoresis on starch gel (14%) was the technique used to sample populations from South Africa, Benin, Brazil, France, United States of America, Japan, Uzbekistan, Philippines, Réunion Island and five localities in Australia: Adelaide, Brisbane, Mareeba, Melbourne and Sydney. Samples collected in the field (100 individuals) were reared in the laboratory (25°C, 12L:12D, 75% HR). Final sampling was made on individuals of the field generation and of the first generation of the laboratory colony. Adults, deep-frozen in liquid nitrogen, were crushed in 20 µ1 of NADP solution. Buffers used were Tris Citrate pH 8, Histidine pH 6, Tris Maleate EDTA pH 7.4, Tris Citrate pH 8.7. Migration (150 V, 70 mA) was 6 h long. Buffers and staining solutions were prepared following Pasteur et al. (1987) and Hillis et al. (1996) protocols. Bands were analysed in terms of loci and alleles. GENEPOP version 3.1d (Raymond & Rousset 1995), Fstat version 1.0 (Goudet 1994) and DARwin version 3.6 (Perrier & Jacquemoud-Collet 2000) were used to analyse allelic frequencies.

Results

(c) Oviposition activity

Numbers of eggs laid by 20 females each day, for each population, are shown in Figure 1. Variations were observed between populations. The statistical significance of these differences is given in Table 1. The most fecund females were from South Africa and Benin, followed by those from Réunion Island, Martinique and Uzbekistan. Up to 4 days after emergence, females from Benin, Martinique and Réunion Island lay 60–70% of their eggs, while those from Uzbekistan and South Africa lay only 24 to 36%. Females from the latter populations lay more than 20% of their eggs between the 12th and the 20th day after emergence, whilst populations from Benin, Réunion Island and Martinique lay 1% to 4%. The egg laying period also varies with 13 days for populations from Benin and Martinique to 25 days for those from Uzbekistan and South Africa.

Figure 1. Number of eggs laid each day by 20 females of Plutella xylostella with different geographic origins. Black square: South Africa, black triangle: Benin, black circle: Martinique, star: Uzbekistan, diamond: Réunion Island.

Table 1. Differences observed in the oviposition activity of 20 females of Plutella xylostella with different geographic origins. a, b, c on the same row: populations significantly different. ANOVA and Newman & Keuls test α = 5%. *: period in days, mean of 4 replicates

Population

South Africa

Benin

Martinique

Uzbekistan

Réunion Island

Fisher test

P

Number of eggs

5894a

5812a

2325c

2884c

4199b

14.19

0.0001

% of eggs laid for day 1 to day 4

35.66b

76.11a

63.40a

24.44b

60.49a

15.64

0.0000

% of eggs laid for day 12 to day 20

25.95a

1.19b

1.31b

22.01a

4.51b

8.95

0.0007

Duration*

24a

14bc

13c

25a

18b

21.28

0.0000

(d) Genetic study

Over 23 enzyme systems were tested in the laboratory, 14 had diffuse bands and two had a monomorphic pattern (glyceraldehydes-3-phosphate dehydrogenase, pyruvate kinase). Seven enzymes had legible and polymorphic loci: isocitrate dehydrogenase (IDH), malate dehydrogenase NADP+ (MDHP), glucose-6-phosphate dehydrogenase (G6PDH), mannose phosphate isomerase (MPI), phosphoglucomutase (PGM), hexokinase (HK), aspartate aminotransferase (AAT). Some isoenzymes had more than one locus: IDH, with IDHs (slow) and IDHf (fast), MDHP, with MDHPs and MDHPf. Hardy-Weinberg equilibrium test revealed that populations have a deficit of heterozygotes concerning loci MDHPs, G6PDH, MPI, PGM, HK and an excess of heterozygotes concerning locus AAT (Table 2).

Table 2. Estimations of Wright’s F statistics, for each locus, for all populations of P. xylostella

Locus

Fis

Fst

Fit

IDHf

- 0.0098

0.0560

0.0467

IDHs

0.1029

0.0487

0.1466

MDHPf

- 0.0240

0.1401

0.1195

MDHPs

0.1913

0.1730

0.3312

G6PDH

0.4835

0.0199

0.4937

MPI

0.2269

0.0923

0.2983

PGM

0.0084

0.1364

0.1437

HK

0.0256

0.0872

0.1106

AAT

- 0.4165

0.1549

-0.1971

All

0.151

0.103

0.238

Locus PGM is monomorphic in the Japanese population; it is the same for IDHs in populations from Benin, France, Réunion Island and Melbourne. The Fisher test shows that allelic frequencies are significantly different for all pairs of populations, except the pair Benin/Brazil (χ2=28.029, P=0.06162). Those loci having genotypic linkage disequilibria were IDHf with IDHs, MDHPf with G6PDH and MPI. Loci IDHf and MDHPf were not maintained for further analysis. The global fixation index (Fst), which defines the level of differences among populations, has a value of 0.103, with P(Fst=0)<0.001. Populations were very different for loci MDHPs (Fst=0.1730), PGM (Fst=0.1364) and AAT (Fst=0.1549) (Table 2). Fst for pairs of populations varies from 0 to 0.2304 (Table 3). Populations from Benin and Brazil are not differentiated. Populations from Australia and Japan are the most different from other populations and from each other. The value of Fst, excluding populations from Australia and Japan is 0.047. Differentiation between populations is shown in Figure 2.

Table 3. Fixation index (Fst) for pairs of Plutella xylostella populations. SA : South Africa, BEN : Benin, BRA : Brazil, FRA : France, USA : United States of America UZB : Uzbekistan, PHI : Philippines, REU : Réunion Island, AUA : Australia (Adelaide), AUB : Brisbane, AUMa : Mareeba, AUMe : Melbourne, AUSy : Sydney

Pop

SA

BEN

BRA

FRA

JAP

USA

UZB

PHI

REU

AUA

AUB

AUMa

AUMe

BEN

0.0168

                       

BRA

0.0071

0.0002

                     

FRA

0.0238

0.0639

0.0434

                   

JAP

0.1236

0.1856

0.1751

0.1846

                 

USA

0.0317

0.0932

0.0670

0.0670

0.1659

               

UZB

0.0262

0.1019

0.0629

0.0360

0.1218

0.0350

             

PHI

0.0419

0.0186

0.0132

0.0795

0.1954

0.0809

0.1036

           

REU

0.0161

0.0404

0.0132

0.0489

0.1504

0.0676

0.0522

0.0462

         

AUA

0.0666

0.1008

0.1070

0.1390

0.0719

0.1168

0.0985

0.1164

0.1267

       

AUB

0.1364

0.1797

0.1732

0.2173

0.1664

0.1758

0.1188

0.1958

0.1972

0.1262

     

AUMa

0.0858

0.1213

0.1168

0.1689

0.1506

0.1146

0.1281

0.1127

0.1463

0.0973

0.1031

   

AUMe

0.1308

0.1756

0.1717

0.2304

0.1749

0.1352

0.1325

0.1799

0.1927

0.1241

0.0387

0.0871

 

AUSy

0.0473

0.0440

0.0426

0.0972

0.1590

0.0823

0.0851

0.0445

0.0917

0.0965

0.0878

0.0558

0.0691

Figure 2. Unrooted tree for fixation index (Fst) among populations of Plutella xylostella, calculated with the method of Unweighted Neighbour Joining.

Discussion

(e) Biological differences

In this work, the oviposition activity reveals differences among populations concerning fecundity of females, percentages of eggs laid at the start and the end of the oviposition period and duration of oviposition. Results do not reveal a correlation between these variables for the populations analysed. Females from Benin have a short oviposition period and lay a majority of eggs in the first week, whereas those from Uzbekistan show the opposite tendency. But this type of characterisation is not valid for other populations. We observed different characteristic oviposition activities. Females from Uzbekistan have the highest longevity, but the fecundity observed is not maximal. This result is in contradiction with the hypothesis of Poelking (1992) that fecundity and longevity are positively correlated.

Differences are observed among populations placed under the same laboratory conditions. It is conceivable that differences do exist in the field. Variations among females in a population are probably higher and the oviposition may vary according to environmental conditions (temperature, humidity). Whatever their geographic origin, all females do not have the same oviposition pattern. Populations of P. xylostella can adapt to their environment.

Those environmental factors affecting the reproduction capacity of females are the host plant (wild or cultivated brassicas) and temperature during biological development. Females lay fewer eggs on wild brassicas (Muhamad et al. 1994). The quality and quantity of nutrients of the host plant can influence the female’s reproductive ability (Begum et al. 1996). We used F1 adults from the first generation completely reared in the laboratory on cultivated brassicas; differences observed were not caused by rearing conditions.

Diamondback moth females collected during winter are more fecund than those collected in summer (Yamada & Umeya 1972). A generation of P. xylostella reared at 15°C will have bigger adults than a generation reared at 25°C (Shirai 1995). Moreover, fecundity is positively correlated with the size of female adults (Moller 1988, Muhamad et al. 1994). Fecundity is also correlated with the photoperiod (Harcourt et al. 1966). Numerous factors can influence oviposition and their effects have been analysed. The adaptive ability of P. xylostella is important, but still not well known.

(f) Genetic differences

Heterozygote deficits can be due to the sampling method and a Walhund effect may be observed. Moreover, some loci are completely monomorphic in several populations and these heterozygote deficits probably exist in natural populations. Fst global value (0.103) is relatively high and above values calculated in previous studies: Caprio & Tabashnik (1992), Fst=0.028–0.034; Kim et al. (1999), Fst=0.0215. For other lepidopteran species, Fst among populations are equivalent: Fst=0.109 for populations of Panolis flammea (Wainhouse & Jukes 1997) or smaller Fst=0.080 among populations of Proclossiana eunomia (Nève et al. 2000) and Fst=0.007 among populations of the migrating lepidopteran, Agrotis ipsilon (Buès et al. 1994). Analysis of data reveals no correlation between geographic distance separating populations and differentiation among them. Samples used in this work have very distant geographic origins (several thousand kilometres for the majority); migrations are reduced among them. Populations from Benin and Brazil have similar allelic frequencies. By contrast, populations from Australia are the most differentiated. Concerning the Benin and Brazil populations, results from enzyme electrophoresis are not informative enough to determine a phylogenic link among them. Further studies with DNA-based markers will elucidate this question. The effects of genetic drift are more important on small populations, as a consequence, a higher number of migrants will produce a gene flow sufficient to overcome the genetic drift (Allendorf & Phelps 1981). The small size of populations from Australia may be an explanation of the differences observed, the genetic drift is important and gene flow therefore reduced.

Concerning loci MDHPS, PGM and AAT, the level of differentiation (Fst) is very high. An explanation is a selection of genotypes of allozymes, which confers an advantage to the population. In diamondback moth, the implication of esterase in insecticide resistance (penthoate) has been demonstrated (Miyata et al. 1986). Moreover, the function of glutathione-S-transferase in resistance to organophosphorus compounds has been described in previous studies (Cheng et al. 1990). In this study, we have frequency analyses of allozymes which have a function in the central metabolic pathways in insects. For example, PGM, G6PDH and HK are implicated in glycolysis pathways or linked metabolic reactions. These enzymes are submitted to selection pressures and their genotypes may vary in function (Carter & Watt 1988). In addition, Herrero (2001) has demonstrated a correlation between the presence of an allele of the locus MPI and resistance to the toxin Cry1A of Bacillus thuringiensis in P. xylostella. A physiological process does not induce the presence of this allele and it is transmitted to the progeny. Insecticide pressure can select genotypes of enzymes that do not have a direct function in the resistance process.

Conclusions

The analyses of the oviposition activity and of the allozyme genotypes of populations of P. xylostella with different geographic origins show differences among them. Concerning the biology, the data obtained in the laboratory show that females from several populations do not lay the same quantity of eggs with the same frequency. In the field, differences probably exist among populations, but the importance of variations within a population over time is to be considered. A better knowledge of the adaptive ability of diamondback moth populations to their environment will permit improvement of the methods to control this pest.

On a genetic level, differences among populations are shown with analysis of allozyme genotypes. However, insecticide pressure and the development of resistance can select these markers. This hypothesis is to be confirmed. In this study, allozyme markers cannot give information on the phylogeny of the populations. Studies based on other molecular markers, independent of insecticide pressure, will elucidate the phylogeographic structure of P. xylostella populations.

Acknowledgements

Sincere thanks to Alan Kirk of European Biological Control Laboratory (USDA-ARS). Thanks also to Tamara Smith and Martin Villet of Rhodes University, South Africa. This work was financially supported by the CIRAD, Montpellier, France.

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