Genetic Variation of Host Populations of Liriomyza sativae Blanchard

Agricultural Sciences in China

May 2008

2008, 7(5): 585-590

Genetic Variation of Host Populations of Liriomyza sativae Blanchard WANG Li-ping1, 2, DU Yu-zhou1, HE Ya-ting1, ZHENG Fu-shan1 and LU Zi-qiang1 1 2

Institute of Applied Entomology, Yangzhou University, Yangzhou 225009, P.R.China State Key Laboratory for Biology of Plant Disease and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100094, P.R.China

Abstract In this study, partial sequences of the mitochondrial cytochrome oxidase subunit I (mtDNA-COI) gene and the ribosomal internal transcribed spacer 1 (rDNA-ITS1) gene, isolated from five artificial populations of Liriomyza sativae (Diptera: Agromyzidae), were sequenced and compared, to analyze their genetic variation. Analysis of the mtDNA-CO1 gene showed that a low genetic variation was detected among the five populations and only five variable sites were found in the nucleotide sequences. Most of the observed variations that occurred within the populations were because of nucleotide transitions, whereas, the interpopulation variation was because of the differences in haplotype frequencies occurring among the host populations. Analysis of the rDNA-ITS1 gene revealed a small diversity in the five host populations. The trend of genetic differentiation in the host populations was consistent with the preference of L. sativae to the plant hosts. Key words: Liriomyza sativae, host populations, mtDNA-COI, rDNA-ITS1, genetic differentiation

INTRODUCTION The herbivorous leafminer fly Liriomyza sativae Blanchard (Diptera: Agromyzidae) was first found in Hainan Province, China, in 1993 (Kang 1996), and since then the fly has dispersed to an additional 29 provinces within China. L. sativae has a high reproductive rate and a short life cycle. It feeds on a wide range of host plants. As a result, rapid outbreaks of this pest are common in many locations where different species of plants are grown. The outbreaks of L. sativae severely damage many plants, such as, fruits, vegetables, tobacco, and cotton, resulting in substantial economic losses and significant impacts on the agricultural products of China (Wang et al. 1999; Chen et al. 2002; Lei and Wang 2005). Thus it is important to examine different host populations of L. sativae to understand the

Received 31 October, 2007

mechanism of host population formation so that controlling strategies for this pest insect can be developed. Hosts play a vital role in speciation of polyphagous herbivorous insects. L. sativae is a typical polyphagous herbivorous insect with a wide range of hosts. Pang et al. (2005) reported that genetic differences have existed among different host populations of L. sativae. Genetic variations among different species within the genus Liriomyza can be detected using molecular markers (Morgan et al. 2000; Qiu et al. 2000; Scheffer 2000; Scheffer and Lewis 2001, 2005). Among other approaches, mitochondrial DNA and ribosomal internal transcribed spacer gene have been shown to be viable. Mitochondrial DNA is variable, has strict maternal heredity and no genetic recombination and provides a good indication of intra and interpopulation variation (Avise 1994). Sequences of ribosomal internal transcribed

Accepted 11 March, 2008

Correspondence DU Yu-zhou, Tel: +86-514-7971854, Fax: +86-514-7971584, E-mail: [email protected]

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spacer evolved rapidly with many variable and informative sites. In this study, the authors have used the mitochondrial cytochrome oxidase subunit I (mtDNA-COI) gene and the ribosomal internal transcribed spacer 1 (rDNA-ITS1) gene to analyze the genetic differentiation among different host populations of L. sativae, which were reared on different host plants for a number of generations.

MATERIALS AND METHODS Sample collection Leaves with tunnels caused by leafminers were collected from five hosts in the suburb of Yangzhou City, China, in 2004. After eclosion, the leafminers, identified to be L. sativae by using the modality method (Spencer 1973), were bred on their respective original hosts at 28°C and under a photoperiod of 14 h:10 h (L:D) for a number of generations to obtain five host populations. Tomato population was obtained by breeding L. sativae on Lycopericon esculentum for 18 generations; cowpea population on Vigna unguiculata ssp. Sesquipedalis for 18 generations; greengrocery population on Brassica campestris ssp. Chinensis (L) Makino (B. chinensis L.) for 18 generations; lettuce population on Lactuca sativa for 20 generations; cotton population on Gossypium hirsutum for 6 generations. At the end of the last generation, all samples were kept in 1.5-mL disinfected tubes filled with 100% ethanol at -70°C at the Institute of Applied Entomology, Yangzhou University, China. Nine individual leafminers were tested for each population.

DNA extraction and amplification of mtDNA-COI and rDNA-ITS1 genes Genomic DNA of L. sativae was extracted as described by Wen and He (2003) with slight modifications. Two DNA sequences were amplified and used for genetic variation analysis. One sequence was a partial sequence of mtDNA-COI. The primers for PCR amplification were C1-J-2183 (5´-CAACATTTATTTTG ATTTTTTGG-3´) and TL2-N-3014 (5´-TCCATTGC

WANG Li-ping et al.

ACTAATCTGCCATATTA-3´). The PCR reaction mixture (50 µL) contained 5 µL of 10 × buffer, 2 µL of 2.5 mM dNTPs, 2 µL of 20 µM primers, 2 U Taq DNA polymerase, and 2.0 µL DNA as template. The amplification procedure was 4 min at 95°C, 35 cycles of 1 min at 95°C, 50 s at 52.5°C, and 90 s at 72°C, followed by a final extension for 10 min at 72°C. The other target sequence the authors amplified was the complete region of rDNA-ITS1. The primers for PCR amplification were 18SF (5´-GAAGT AAAAGTCGTAACAAGG-3´) and 5.8SR (5´-GTCC TGCAGTTCACACGATG-3´). Twenty-five microliters of PCR reaction mixture contained 2.5 µL of 10×buffer, 2 µL of 2.5 mM dNTPs, 1 µL of 10 µM primers, 1.5 U Taq DNA polymerase, 1.5 µL of 2.5 mM Mg2+, and 1.0 µL DNA as a template. The amplification protocol was 3 min at 94°C, 30 cycles of 1 min at 94°C, 45 s at 55°C, and 1 min at 72°C, followed by a final extension for 7 min at 72°C. The PCR products were purified (UNIQ-10 Column PCR products purification kit) and sequenced by Shanghai Sangon Biological Engineering and Technology Service Co. Ltd (Shanghai, China).

Statistical analyses Sequences were aligned using ClustalX ver. 1.83 (Thompson et al. 1997). Genetic variation and polymorphic sites were determined using MEGA ver. 3.1 (Kumar et al. 2004). The neighbor-joining (Saitou and Nei 1987) tree with bootstrap support (1000 replicates) (Felsenstein 1985) was constructed using Kimura-2-parameter distance measures (Kimura 1980). L. trifolii (AY323210) was used as an outgroup for comparison. For the mtDNA-COI gene, a sequence network was constructed using the program TCS 1.21 (Clement et al. 2000) and analysis of molecular variance (AMOVA); pairwise Fst tests and gene flow were calculated using Arlequin ver. 3.1 (Excoffier et al. 2005).

RESULTS Analysis of the mtDNA-COI gene The authors obtained 40 partial mtDNA-CO1 sequences,

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Genetic Variation of Host Populations of Liriomyza sativae Blanchard

784 bp in length. The average A + T content (70.20%) was higher than the G + C content (29.80%). Five variable sites out of 784 bp were observed, occupying 0.638% of the sequences (Table 1). Both the tomato and cowpea populations had five variable sites, whereas, the other three host populations had four sites each. All variable sites were transitions and no insertions or deletions were found. All transition variations occurred at the first site of each codon. Four of the five transitions resulted in amino acid changes. Five haplotypes (A through E) were observed in 12.50% of all the samples. Haplotypes C, D, and E appeared in all the populations and Haplotype D occurred at rates from 30 to 60% within each of the

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populations. Haplotypes A and B were shared by at least two populations, although none of the populations had exclusive haplotypes. The NJ phylogenetic tree with bootstrap support (1 000 replicates) was constructed using L. trifolii as an outgroup (Fig.1). The five haplotypes were divided into two clusters, one of which contained Haplotype D and Haplotype E with a bootstrap percentage of 66% and the other contained Haplotypes A, B, and C with a bootstrap percentage of 66%. Haplotypes have been networked using TCS (ver. 1.21) (Fig.2). The haplotype with the highest outgroup probability is displayed as a square, whereas, other haplotypes are displayed as ovals. The size of the square

Table 1 Haplotype distribution (frequency) and sequence variable sites of L. sativae in mtDNA-CO1 Haplotype (Frequency) A (0.050) B (0.125) C (0.200) D (0.425) E (0.200)

Populations and individuals with haplotypes

Variable sites

TMP

CPP

GGP

LTP

CTP

43

130

340

376

457

GenBank accession no.

1 1 2 3 1

1 1 1 3 2

0 1 2 3 2

0 2 1 4 1

0 0 2 4 2

T C -

A G -

G A A

T C -

C T T

DQ911139 DQ911140 DQ911141 DQ911142 DQ911143

TMP, tomato population; CPP, cowpea population; GGP, greengrocery population; LTP, lettuce population; CTP, cotton population. The same as below.

Haplotype D 66 Haplotype E L. trifolii 0.01

Fig. 1 Neighbor-joining tree of the host haplotypes of L. sativae from mtDNA-CO1 sequences by MEGA 3.1 software. L. trifolii is an outgroup.

Haplotype C

A

G -340-

T-45

7-C

6-

-G

Haplotype B 37

T

43

C-

T-

Haplotype D

A

The authors obtained five complete rDNA-ITS1 sequences, each of which was 303 bp in length

Haplotype A

30-

Analysis of the rDNA-ITS1 gene

Haplotype C 66 Haplotype B

G-1

or oval corresponds to the haplotype frequency. Haplotype E is the oldest among the samples and can serve as an outgroup for the other haplotypes of the tree. The AMOVA showed that 80.0% of the overall variation occurred within populations and 20.0% occurred between populations (Table 2). The Fst value (0.29; P < 0.01) reflected very subtle differences among populations. Gene flow and Fst values are shown in Table 3. The Fst values range from 0.143 to 0.268 (P > 0.05), suggesting that variation among populations is relatively low. Gene flow among populations is expressed by Nm, where Nm > 4 suggests that gene flow is abundant and N m < 1 suggests that populations may differ because of genetic drift (Su et al. 2001). In this study, Nm values among populations range between 1 and 4, suggesting that both gene flow and genetic differences exist among the five populations.

Haplotype A

Haplotype E

Fig. 2 TCS network for the nested clade analysis of different host populations of L. sativae based on mtDNA-CO1. Haplotype with the highest outgroup probability is displayed as a square, whereas, other haplotypes are displayed as ovals. The size of the square or oval corresponds to the haplotype frequency. The sequences corresponding to each haplotype are shown in Table 1.

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WANG Li-ping et al.

Table 2 Analysis of molecular variance (AMOVA) of mtDNA-CO1 for 40 individuals in the five populations of L. sativae Source of variation

df

Among populations Within populations Total

4 35 39

Variance components 0.100 0.400 0.500

Table 3 Fst values and gene flow of mtDNA-CO1 among the five host populations of L. sativae TMP CPP GGP LTP CTP

TMP

CPP

GGP

LTP

CTP

0.143 0.161 0.196 0.214

2.997 0.161 0.196 0.214

2.606 2.606 0.214 0.268

2.051 2.051 1.836 0.232

1.836 1.836 1.366 1.655 -

The values above the diagonal are Nm and below the diagonal are Fst .

Table 4 rDNA-ITS1 diversity of the host populations of L. sativae TMP CPP LTP GGP CTP L. trifolli

TMP

CPP

LTP

GGP

CTP

L. trifolli

0.000 0.004 0.007 0.004 0.159

0.000 0.004 0.007 0.004 0.159

0.004 0.004 0.004 0.007 0.164

0.005 0.005 0.004 0.011 0.160

0.004 0.004 0.005 0.006 0.159

0.026 0.026 0.027 0.026 0.026 -

The values above the diagonal are standard error and below the diagonal are pairwise distances.

43 TMP 45 CPP LTP 53

GGP

CTP L. trifolii 0.02

Fig. 3 Neighbor-joining tree of the host populations of L. sativae based on rDNA-ITS1 by MEGA 3.1 software. L. trifolii is an outgroup.

(GenBank accession no. of EF152325-EF152329). The average A + T content (85.0%) of the five sequences was much higher than the G + C content (15.0%), which was consistent with those in other species of the genus. It was observed that three variable sites occupied 0.99% of the nucleotides in the sequences. All variable sites were transitions or transversions and no insertion or deletion was detected. The differences in rDNA-ITSI sequences of the five host populations were analyzed by the MEGA software program using the Kimura-2-parameter distance measures (Table 4). The diversity among different host populations was very small (all less than 0.011). No

Percentage of variation (%) 20.00 80.00 100

P < 0.01 < 0.0001

Fst 0.20

diversity was found between the cowpea and tomato populations. NJ phylogenetic tree with bootstrap support (1 000 replicates) was constructed using L. trifolii as an outgroup (Fig.3). Homology among the different host populations was high. The five populations were divided into two clusters. The cotton population formed one cluster even as the four other populations were grouped into another cluster, in which the cowpea and tomato populations were close to each other and the greengrocery and lettuce populations were close to each other.

DISCUSSION Analysis of the mtDNA-COI gene revealed low genetic variation among the five populations feeding on five different hosts, whereas, most of the variation occurred within populations. Interpopulation variation was on account of different haplotype frequencies occurring among different host populations. Five different haplotypes were observed in this study. All host populations contained Haplotypes C, D, and E, whereas, only two populations shared Haplotype A. This suggests that both gene flow and genetic differences existed among the populations. Among the three common haplotypes, Haplotype D occurred with the highest frequency, suggesting that it was a dominant and stable haplotype, capable of adapting to different environmental conditions. Castelloe and Templeton (1994) suggested that outgroup weights were strongly correlated with actual age and were much better indicators of haplotype age than frequency. Using outgroup weights, the authors established that Haplotype E was the oldest in the samples and could serve as an outgroup for the remainder of the haplotype tree. Cowpea and tomato populations contained all five haplotypes; greengrocery and lettuce populations had four haplotypes each, and the cotton population contained three haplotypes. Genetic variation in popula-

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Genetic Variation of Host Populations of Liriomyza sativae Blanchard

tions that had recently colonized a new habitat was expected to be lower than that of the source population, because of the smaller population of the recent dispersers (Zhu et al. 2005). Given the observed frequency of the haplotypes, the cotton population had probably dispersed from the fields of the other four host populations. The origin and mechanisms of the dispersal of L. sativae required further investigation. A single base variation in the location of a genome is referred to as a single nucleotide polymorphism (SNP). In this study of the mtDNA-COI gene, tomato and cowpea populations each had five haplotypes from only eight samples, indicating high variability. The authors could not completely rule out the possibility that the single base variation detected in each haplotype was an SNP that existed in the individuals of the population, and was not related to feeding on the host plants, given that the size of the samples verified was small in this study. It would be helpful to answer this question by increasing the sample size in further studies. Analysis of the rDNA-ITS1 gene also revealed the low genetic variation among the five populations in this study. The NJ phylogenetic tree of the five host populations showed that the greatest divergence occurred between the cotton population and any one of the other host populations. Although the cowpea and tomato populations were clustered together, the greengrocery and lettuce populations were grouped into another cluster. Giving that tomato and cowpea are the most fitting hosts, greengrocery and lettuce are the fitting hosts, and cotton is the secondary host for L. sativae (Wang et al. 2007). The authors suggest that genetic differentiation of the host populations may be associated with the preference of L. sativae to plant hosts. The results of the mtDNA-COI gene were similar to those of the rDNA-ITS1 gene for cowpea and tomato populations, each contained all five haplotypes; greengrocery and lettuce populations had four haplotypes each, and the cotton population contained only three haplotypes. Plants not only provide a food source for phytophagous insects, but also influence their characteristics of reproduction and growth. Adaptation to novel host plants may play a vital role in leading to genetic differentiation and, ultimately, speciation (Qin 1987). In this study, the authors verified that genetic differentiation

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existed among host populations, although in low degrees, at the molecular level of mtDNA-COI and rDNA-ITS1. To fully understand the relationships between population variation and adaptation of L. sativae to different hosts, one should examine the original population and increase the repeating samples in further studies.

Acknowledgements The authors are grateful to Mr. Huang Yuan for his comments and suggestions and Li Zheng-xi for statistical assistance. This study was supported by the grants from the National Natural Science Foundation of China (30370932), the National Basic Research Program of China (2006CB102002), and the Opening Foundation of the State Key Laboratory for Biology of Plant Disease and Insect Pests, China.

References Avise J C. 1994. Molecular Markers, Natural History and Evolution. Chapman & Hall Press, New York. Castelloe J, Templeton A R. 1994. Root probabilities for intraspecific gene trees under neutral coalescent theory. Molecular Phylogenetics and Evolution, 3, 102-113. Chen B, Zhao Y X, Kang L. 2002. Mechanisms of invasion and adaptation and management strategies of alien leafminers. Zoological Research, 23, 155-160. (in Chinese) Clement M, Posada D, Crandall K A. 2000. TCS: a computer program to estimate gene genealogies. Molecular Ecology, 9, 1657-1660. Excoffier L, Laval G, Schneider S. 2005. Arlequin ver. 3.0: an integrated software package for population genetics data analysis. Evolutionary Bioinformatics Online, 1, 47-50. Felsenstein J. 1985. Confidence limits on phylogenies: an approach using the bootstrap. Evolution, 39, 783-791. Kang L. 1996. Ecology and Sustainable Control of Serpentine Leafminer. Science Press, Beijing. (in Chinese) Kimura M. 1980. A simple method for estimating evolutionary rate of base substitutions though comparative studies of nucleotide sequences. Journal of Molecular Evolution, 16, 111-120. Kumar S, Tamura K, Nei M. 2004. MEGA3: Integrated software for molecular evolutionary genetics analysis and sequence alignment. Briefings in Bioinformatics, 5, 150-163. Lei Z R, Wang Y. 2005. Liriomyza sativae. In: Wan F H, Zheng X, Guo J Y, eds, Biology and Management of Invasive Alien Species in Agriculture and Forestry. Science Press, Beijing. pp. 177-205. (in Chinese) Morgan D J W, Reitz S R, Atkinson P W, Trumble J T. 2000.

© 2008, CAAS. All rights reserved. Published by Elsevier Ltd.

590

WANG Li-ping et al.

The resolution of Californian population of Liriomyza

Entomological Society of America, 98, 181-186.

huidobrensis and Liriomyza trifolii (Diptera: Agromyzidae) using PCR. Heredity, 85, 53-61.

Spencer K A. 1973. Agromyzidae (Diptera) of economic importance. Series Entomologica, 9, 1-418.

Pang B P, Cheng J A, Huang E Y, Bao Z S. 2005. Effects of different host plants on population parameters of Liriomyza

Su B, Fu Y, Wang Y, Jin L, Chakraborty R. 2001. Genetic diversity and population history of the red panda (Ailurus

sativae. Plant Protection, 31, 26-28. (in Chinese) Qin J D. 1987. Relationship of Insects and Plants. Science Press,

fulgens) as inferred from mitochondrial DNA sequence variations. Molecular Biology and Evolution, 18, 1070-1076.

Beijing. (in Chinese) Qiu Y Z, Wu W Z, Xiao X F. 2000. Application of RAPD-PCR

Thompson J D, Gibson T J, Plewniak F, Jeanmougin F, Higgins D G. 1997. The CLUSTAL X windows interface: flexible

in the quickly determining of six kind of leafminers Liriomyza spp. (Diptera: Agromyzidae). Journal of Chinese Entomology,

strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research, 25, 4876-4882.

20, 293-309. (in Chinese) Saitou N, Nei M. 1987. The neighbor-joining method: A new

Wen S Y, He X F. 2003. A method of rapid preparation of traceDNA templates of insects for PCR. Entomological

method for reconstructing phylogenetic trees. Molecular Biology and Evolution, 4, 406-425.

Knowledge, 40, 276-279. (in Chinese) Wang J, Shi B C, Gong Y J, Liao D, Lu H. 1999. Plant items of

Scheffer S J. 2000. Molecular evidence of cryptic species within the Liriomyza huidobrensis (Diptera: Agromyzidae). Journal

Liriomyza sativae. Beijing Agricultural Sciences, 17, 37-39. (in Chinese)

of Economic Entomology, 93, 1146-1151. Scheffer S J, Lewis M L. 2001. Two nuclear genes confirm

Wang L P, Du Y Z, He Y T, Zhang S Q. 2007. Population dynamics and host suitability of Liriomyza sativae

mitochondrial evidence of cryptic species within Liriomyza huidobrensis (Diptera: Agromyzidae). Annals of

(Blanchard). Scientific Research Monthly, 25, 106-108. (in Chinese)

Entomological Society of America, 94, 648-653. Scheffer S J, Lewis M L. 2005. Mitochondrial phylogeography

Zhu Z H, Ye H, Zhang Z Y. 2005. Genetic relationships among four Bact rocera cucurbitae geographic populations in Yunnan

of vegetable pest Liriomyza sativae (Diptera: Agromyzidae): divergent clades and invasive populations. Annals of the

Province. Chinese Journal of Applied Ecology, 16, 1889-1892. (in Chinese) (Edited by ZHANG Juan)

© 2008, CAAS. All rights reserved. Published by Elsevier Ltd.