Two putative bridgehead populations of Aphelinus mali (Hymenoptera: Aphelinidae) introduced in China as revealed by mitochondrial DNA marker.
During the 1940s-1950s, A. mali was introduced as an endoparasitoid of E. lanigerum in China, i.e., A. mali was introduced into Dalian, Liaoning from Japan in 1942, and into Qingdao, Shandong from the former Soviet Union during 1953-1955 (Long et al. 1960). Since then, A. mali has spread into most regions of China including Shandong, Liaoning, Shanxi, Yunnan, Hebei, Henan, and Xinjiang provinces (Zhou et al. 2010; the present study). However, we did not know which introduction of this endoparasitoid played an important role in the control of E. lanigerum in China. The information might be helpful in the management of the E. lanigerum, which is a severely damaging invasive species in China. For instance, the A. mali populations with strong adaptability may be used or re-introduced for the control of the E. lanigerum.
To determine the genetic structures of various A. mali populations and their status as a biological control agent in China, we collected 16 populations from 6 provinces (Shandong, Liaoning, Hebei, Shanxi, Xinjiang, and Yunnan) and analyzed the mtCOI gene of specimens from each of these samples.
MATERIALS AND METHODS
Sample Collection and Species Identification
Adults of the woolly apple aphid, E. lanigerum, were collected from apple trees in 6 provinces in China during 2007, 2008 and 2012. The sampled localities cover a great part of the invasion areas in China, ranging from Huludao, Liaoning (HLD) in the northeast to Zhaotong, Yunnan (ZT) in the southwest. Samples from the initial areas of introduction in Dalian, Liaon ing and Qingdao, Shandong (DL and QD) were also collected. The collected specimens of E. lanigerum were placed in Petri dishes and kept at room temperature until A. mali eclosion. The emerging adult A. mali were collected and stored in 95% ethanol at -20 [degrees]C until DNA extraction.
Before storage in alcohol, each individual was examined and identified unambiguously by a microscope.
DNA Extraction, PCR Amplification, and Sequencing
Genomic DNA was extracted from individual female adults of A. mali by the procedure described by Frohlich et al. (1999). The lysate was stored at -20 [degrees]C and was used as DNA template in PCR amplification. All 948 individual DNA samples were amplified using the newly designed primers F (5'-TCTCATATAATTTGTAATGAAAG-3') and R (5'-TGATAACTAGGAGGAAAATTTAT-3') to yield a 648-bp fragment of the mtCOI gene. PCRs were performed on a TP600 machine (TaKaRa) in a 25-[micro]L reaction volume containing 0.25 [micro]L of EasyTaq polymerase, 2.5 [micro]L of 10 x EasyTaq Buffer (+[Mg.sup.2+]), 0.5 [micro]L of dNTPs (Sangon), 3 [micro]L of DNA (concentration not estimated), and 0.5 [micro]L of each oligonucleotide primer. The thermal profile included an initial denaturation step at 94 [degrees]C for 4 min; followed by 35 cycles of denaturing at 94 [degrees]C for 30 s, annealing at 54 [degrees]C for 45 s, and extension at 72 [degrees]C for 1 min; and a final extension at 72 [degrees]C for 7 min. PCR products were run on a 1.5% agarose gel stained with ethidium bromide and then were sequenced in both directions using the same primer pairs. Sequences were aligned with Clustal W (Thompson 1994) and were then checked for indels and nuclear copies.
Haplotype Determination and Phylogenetic Analyses
The mtCOI sequences of all individuals were manually edited using DNASTAR and were aligned using MEGA 5. Only 1 sequence was selected from the same sequences from each location to conduct a phylogenetic analysis. We constructed a phylogenetic tree using the Maximum Likelihood (ML) method in MEGA5 with the Aphelinus varipes mtCOI sequence (GenBank No. HQ599571) as an outgroup. Also we downloaded the entire sequence of A. mali from GenBank and only 1 sequence was obtained (GenBank No. DQ350507). We determined the type of the clades based on the phylogenetic tree.
Geographical Distribution of Aphelinus mali
We genotyped 948 A. mali adult females from 16 populations (6 populations from Shandong; 3 each from Shanxi and Hebei, 2 from Liaoning; and 1 each from Xinjiang and Yunnan), yielding an average sample size of 59 individuals per population (Table 1).
Haplotype Composition of Aphelinus mali Based on the Mitochondrial Gene
Within the mtCOI sequences, 31 positions were polymorphic representing 1 singleton variable site and 30 parsimony informative sites. These polymorphic sites defined 3 haplotypes (coded as Hap1-Hap3; GenBank numbers: KF039708KF039710) within the 948 individuals from 16 localities across China. Hap1 was found in 679 individuals from 12 locations, and Hap2 was found in 268 individuals from 10 locations across China. However, Hap3 was only found in 1 individual from Yantai, Shandong (YT). All of the COI haplotypes defined 2 main clades: SD and LN (Fig. 1). The SD (Shandong) clade was mainly distributed in Shandong and neighboring regions and characterized by Hap1 while the LN(Liaoning) clade was mainly distributed in Liaoning and neighboring regions by Hap2 (Fig. 2).
Our analyses show that the A. mali populations in Shandong Province and neighboring regions mainly belong to the same clade (SD clade) and that the A. mali population in Liaoning Province and neighboring regions mainly belong to another clade (LN clade). The distribution of mtCOI haplotypes and clades of A. mali (Fig. 1) is consistent with previous reports concerning the intentional introduction of A. mali into China (see Long et al. 1960).
Combined with the historical records of the initial introductions of A. mali and the haplotype data in this study, our results strongly suggest that each of the 2 populations that had been introduced into Shandong (QD) and Liaoning (DL) can establish in many regions of China and play an important role in the control of E. lanigerum in China. Indeed both original introductions appear to have served either as bridgeheads to establish A. mali in adjacent areas, or as source populations to establish A. mali in distant areas in China. This should be further explored using nuclear markers. The role of initially introduced population as a bridgehead has also been shown for several invasive insect pest species (Miller et al. 2005; Ciosi et al. 2008; Ascunce et al. 2011; Lombaert et al. 2010; Lombaert et al. 2011; Kajita et al. 2012; Yang et al. 2012). Moreover, based on genetic variation of the adventive western flower thrips, Frankliniella occidentalis (Pergande), in China, Yang et al. (2012) revealed that the introduced population in Kunming probably served as a bridgehead to other populations in China.
Within the 16 populations, 6 were mixed populations that contained more than 1 haplotype. Hybridization or gene flow may have occurred between the 2 mitochondrial clades, which should be further explored using nuclear DNA markers. We suggest that the genetic introgression of A. mali clades may have facilitated the adaptation of the species to conditions in China. Prior studies demonstrated that such genetic introgressions may have facilitated adaptation by allowing the appearance of new gene combinations in Harmonia axyridis Pallis (Coccinellidae) (Lombaert et al. 2011; Facon et al. 2011). The biological and ecological effects of genetic admixture should also be further explored for A. mali in the future research.
Caption: Fig. 1. A Maximum Likelihood tree of Aphelinus mali with the Aphelinus varipes mtCOI sequence as an outgroup. Bootstrap values were computed over 1000 replications. The sequence code consists of the location code (shown in Table 1) plus the haplotype. The data shown that the A. mali populations in Shandong Province and in neighboring regions mainly belong to the same clade (SD clade) and the A. mali population in Liaoning Province and in neighboring regions mainly belong to another clade (LN clade).
Caption: Fig. 2. Sampling sites and haplotype frequencies in the 16 populations of Aphelinus mali in China. Three haplotypes were identified in China: Hap1 (dark red), Hap2 (orange), and Hap3 (dark yellow). The circular symbol for each population indicates the proportion of each haplotype in that population.
ASCUNCE, M. S., YANG, C. C., OAKEY, J., CALCATERRA, L., WU, W. J. ET AL. 2011. Global invasion history of the fire ant Solenopsis invicta. Science 331: 1066-1068.
BEERS, E. H. 2012. Effect of trap color and orientation on the capture of Aphelinus mali (Hymenoptera: Aphelinidae), a parasitoid of woolly apple aphid (Hemiptera: Aphididae). J. Econ. Entomol. 105: 1342-1349.
CIOSI, M., MILLER, N. J., KIM, K. S., GIORDANO, R., ESTOUP, A. ET AL. 2008. Invasion of Europe by the western corn rootworm, Diabrotica virgifera virgifera: multiple transatlantic introductions with various reductions of genetic diversity. Mol. Ecol. 17: 3614-3627.
FACON, B., CRESPIN, L., LOISEAU, A., LOMBAERT, E., MAGRO, A. ET AL. 2011. Can things get worse when an invasive species hybridizes? The harlequin ladybird Harmonia axyridis in France as a case study. Evol. Appl. 4: 71-88.
FROHLICH, D. R., TORRES-JEREZ, I., BEDFORD, I. D. ET AL. 1999. A phylogeographical analysis of the Bemisia tabaci species complex based on mitochondrial DNA markers. Mol. Ecol. 8: 1683-1691.
HOWARD, L. O. 1929. Aphelinus mali and its travels. Ann. Entomol. Soc. America 22: 311-368. Kajita, Y., O'Neill, E. M., Zheng, y. B., Obrycki, J. J., and Weisrock, D. W. 2012. A population genetic signature of human releases in an invasive ladybeetle. Mol. Ecol. 21: 5473-5483.
LAVANDERO, B., AND TYLIANAKIS, J. M. 2013. Genotype matching in a parasitoid-host genotypic food web: an approach for measuring effects of environmental change. Mol. Ecol. 22: 229-238.
LESSANDO, M. G., STEPHEN, D. C., AND ELIZABETH, H. B. 2012. Natural enemies of woolly apple aphid (Hemiptera: Aphididae) in Washington state. Environ. Entomol. 41: 1364-1371.
LOMBAERT, E., GUILLEMAUD, T., CORNUET, J. M., MA LAUSA, T., FACON, B. ET AL. 2010. Bridgehead effect in the worldwide invasion of the biocontrol harlequin ladybird. PLoS ONE 5: e9743.
LOMBAERT, E., GUILLEMAUD, T., THOMAS, C. E., LAWSON HANDLEY, L. J., LI, J. ET AL. 2011. Inferring the origin of populations introduced from a genetically structured native range by approximate Bayesian computation: case study of the invasive ladybird Harmonia axyridis. Mol. Ecol. 20: 4654-4670.
LONG, C. D, WANG, Y. P, AND TANG, P. Z. 1960. Investigations on the biology and utilization of Aphelinus mali Hald., the specific parasite of the woolly apple aphis, Eriosoma lanigerum Hausm. Acta Entomol. Sinica 10: 1-39.
MILLER, N., ESTOUP, A., TOEPFER, S., BOURGUET, D., LAPCHIN, L. ET AL. 2005. Multiple transatlantic introductions of the western corn rootworm. Science 310: 992.
MUELLER, T. F, BLOMMERS, L. H. M., AND MOLS, P. J. M. 1992. Woolly apple aphid (Eriosoma lanigerum Hausm., Hom., Aphididae) parasitism by Aphelinus mali Hal. (Hym., Aphelinidae) in relation to host stage and host colony size, shape and location. J. Appl. Entomol. 114: 143-154.
THOMPSON, J. D., HIGGINS, D. G., AND GIBSON, T. J. 1994. CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position specific gap penalties, and weight matrix choice. Nucleic Acids Res. 22: 4673-4680.
YANG, X. M., SUN, J. T., XUE, X. F., LI, J. B., AND HONG, X. Y. 2012. Invasion genetics of the western flower thrips in China: evidence for genetic bottleneck, hybridization and bridgehead effect. PLoS ONE 7: e34567.
ZHOU, H. X., GUO, J. Y., WAN, F. H., AND YU, Y. 2010. Natural control of Aphelinus mali on Eriosoma lanigerum and the parasitoid's over wintering protection measures. Acta Phytophylacica Sinica 37: 153-158.
Rui-Ming Zhang and Hong-Xu Zhou contributed equally to this work. We are grateful to Dr. Gabor Lovei (Department of Agroecology, Aarhus University) and Blas Lavandero (Instituto de Biologia Vegetal y Biotecnologia, Laboratorio de Interacciones Insecto-Planta, Universidad de Talca, Chile) for his detailed comments on this manuscript. This work was supported by the National Key Basic Research Development Plan Project (2013CB127600), the National Natural Science Foundation (31371994), the Special Fund for Agro-scientific Research in the Public Interest (201103026-5-2), the Science and Technology Development Planning Program of Qingdao (13-1-3-108-nsh), and the Taishan Mountain Scholar Constructive Engineering Foundation of Shandong, China.
RUI-MINO ZHANG (1), HONG-XU ZHOU (1), DONG GUO (1), YUN-LI TAO (1), FANG-HAO WAN (2), QIANG WU (2) AND DONG CHU (1) *
(1) Key Lab of Integrated Crop Pest Management of Shandong Province, College of Agronomy and Plant Protection, Qingdao Agricultural University, Qingdao 266109, China
(2) State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100081, China
* Corresponding author; E-mail: firstname.lastname@example.org
TABLE 1. CODES, NAMES, GEOGRAPHICAL COORDINATES, NUMBER OF INDIVIDUALS, AND SAMPLING DATES FOR 16 CHINESE POPULATIONS OF APHELINUS MALI COLLECTED FOR THIS STUDY. Code Location Longitude Latitude DL Dalian, Liaoning 39[degrees]01' 121[degrees]44' HLD Huludao, Liaoning 40[degrees]36' 120[degrees]22' CZ Changzhi, Shanxi 36[degrees]11' 113[degrees]6' JZ Jinzhong, Shanxi 37[degrees]41' 112[degrees]44' YC Yuncheng, Shanxi 35[degrees]1' 111[degrees]0' QHD Qinhuangdao, Hebei 39[degrees]56' 119[degrees]35' SJZ Shijiazhuang, Hebei 38[degrees]2' 114[degrees]30' BD Baoding, Hebei 38[degrees]52' 115[degrees]27' YL Yili, Xinjiang 44[degrees]24' 84[degrees]48' ZT Zhaotong, Yunnan 27[degrees]12' 103[degrees]25' HZ Heze, Shandong 35[degrees]8' 115[degrees]15' LC Liaocheng, Shandong 36[degrees]15' 115[degrees]34' TA Taian, Shandong 36[degrees]11' 117[degrees]07' WF Weifang, Shandong 36[degrees]25' 119[degrees]3' YT Yantai, Shandong 37[degrees]19' 121[degrees]14' QD Qingdao, Shandong 36[degrees]19' 120[degrees]23' Code Location Number of Sampling dates individuals used DL Dalian, Liaoning 107 Sep 2007 HLD Huludao, Liaoning 51 Sep 2007 CZ Changzhi, Shanxi 228 Jul 2008 JZ Jinzhong, Shanxi 10 Jul 2008 YC Yuncheng, Shanxi 33 Jul 2008 QHD Qinhuangdao, Hebei 12 Oct 2007 SJZ Shijiazhuang, Hebei 14 Nov 2007 BD Baoding, Hebei 10 Nov 2007 YL Yili, Xinjiang 8 Aug 2008 ZT Zhaotong, Yunnan 74 Sep 2012 HZ Heze, Shandong 22 Aug 2012 LC Liaocheng, Shandong 38 Oct 2012 TA Taian, Shandong 121 Aug 2012 WF Weifang, Shandong 90 Sep 2012 YT Yantai, Shandong 100 Dec 2012 QD Qingdao, Shandong 30 Jul 2012
Please note: Some tables or figures were omitted from this article.
|Printer friendly Cite/link Email Feedback|
|Author:||Zhang, Rui-Ming; Zhou, Hong-Xu; Guo, Dong; Tao, Yun-Li; Wan, Fang-Hao; Wu, Qiang; Chu, Dong|
|Date:||Jun 1, 2014|
|Previous Article:||Taxonomy and geographical distribution of Macdunnoughia (Lepidoptera: Noctuidae: Plusiinae) of China.|
|Next Article:||New record of the genus Homolophus (Opiliones: Phalangiidae) in Turkey and SEM studies on its morphology.|