Experimental and Applied Acarology

, Volume 57, Issue 1, pp 37–51 | Cite as

Isolation and characterization of polymorphic microsatellite markers in Tetranychus urticae and cross amplification in other Tetranychidae and Phytoseiidae species of economic importance

  • B. Sabater-MuñozEmail author
  • S. Pascual-Ruiz
  • M. A. Gómez-Martínez
  • J. A. Jacas
  • M. A. Hurtado


Tetranychus urticae Koch is a cosmopolitan phytophagous mite considered as the most polyphagous species among spider mites. Population genetic studies using molecular markers such as microsatellites have proven to be extremely informative to address questions about population structure, phylogeography and host preferences. The aim of this study was to increase the available molecular tools to gain insight into the genetic structure of T. urticae populations of citrus orchards, which might help in their management. Five microsatellite DNA libraries were developed using probes with the motifs CT, CTT, GT and CAC following the FIASCO protocol. Positive clones, those that included the insert with the microsatellite, were detected using the PIMA-PCR technique. Combinations of primers were designed on 22 out of 32 new microsatellites loci and their polymorphism was tested in four populations sampled along the eastern coast of Spain. Eleven successful amplifications were obtained. Cross amplification was tested in the tetranychids Aphlonobia histricina, Eutetranychus banksi, E. orientalis, Oligonychus perseae, Panonychus citri, Tetranychus evansi, T. okinawanus and T. turkestani, and the phytoseiids Amblyseius swirskii, A. cucumeris, A. andersoni, Euseius stipulatus, Neoseiulus barkeri, N. californicus, Phytoseiulus persimilis and Typhlodromus phialatus. Eight successful cross amplifications were obtained.


Citrus clementina Microsatellites SSRs IPM 



This work was partially funded by Ministerio de Ciencia e Innovación (MICINN) projects AGL2005-07155-C03/AGR and AGL2008-05287-C04/AGR and by the Fundació BANCAIXA—Universitat Jaume I projects P1-1A2005-03 and P11B2008-02.


  1. Abad-Moyano R (2009) Biological control bases of Tetranychus urticae Koch in clementine orchards. PhD Thesis, Departamento de Ecosistemas Agroforestales, ETSIA, Universidad Politécnica de Valencia. p 141Google Scholar
  2. Abercrombie LG et al (2009) Permanent genetic resources added to mol ecol resour database 1 January 2009–30. Mol Ecol Resour 9:1375–1379CrossRefGoogle Scholar
  3. Agrawal AA (2000) Host-range evolution: adaptation and trade-offs in fitness of mites on alternative hosts. Ecology 81:500–508CrossRefGoogle Scholar
  4. Aguilar-Fenollosa E, Ibáñez-Gual MV, Pascual-Ruiz S, Hurtado M, Jacas JA (2011a) Effect of ground-cover management on spider mites and their phytoseiid natural enemies in clementine mandarin orchards (II): bottom-up regulation mechanisms. Biol Control 59:158–170CrossRefGoogle Scholar
  5. Aguilar-Fenollosa E, Ibáñez-Gual MV, Pascual-Ruiz S, Hurtado M, Jacas JA (2011b) Effect of ground-cover management on spider mites and their phytoseiid natural enemies in clementine mandarin orchards (ii): top-down regulation mechanisms. Biol Control 59:171–179CrossRefGoogle Scholar
  6. Ansaloni T, Aucejo S, Jacas JA (2007) Estimating the intrinsic rate of increase of Tetranychus urticae: which is the minimum number of immature individuals to consider? Exp Appl Acarol 41:55–59PubMedCrossRefGoogle Scholar
  7. Aucejo S, Foó M, Gimeno E, Gómez-Cadenas A, Monfort R, Obiol F, Prados E, Ramis M, Ripollés JL, Tirado V, Zaragozà L, Jacas J, Martínez-Ferrer MT (2003) Management of Tetranychus urticae in citrus in Spain: acarofauna associated to weeds. IOBC WPRS Bull 26:213–220Google Scholar
  8. Aucejo-Romero S, Gómez-Cadenas A, Jacas JA (2004) Effects of NaCl-stressed citrus plants on life history of Tetranychus urticae (Acari: Tetranychidae). Exp Appl Acarol 33:55–67PubMedCrossRefGoogle Scholar
  9. Augustinos AA, Stratikopoulos EE, Drosopoulou E, Kakani EG, Mavragani-Tsipidou P, Zacharopoulou A, Mathiopoulos KD (2008) Isolation and characterization of microsatellite markers from the olive fly, Bactrocera oleae, and their cross-species amplification in the Tephritidae family. BMC Genomics 9:618. doi: 10.1186/1471-2164-9-618 PubMedCrossRefGoogle Scholar
  10. Bailly X, Migeon A, Navajas M (2004) Analysis of microsatellite variation in the spider mite Tetranychus turkestani (Acari: Tetranychidae) reveals population genetic structure and raises questions about related ecological factors. Biol J Linn Soc 82:69–78CrossRefGoogle Scholar
  11. Bech N, Novoa C, Allienne JF, Boissier J (2010) Transferability of microsatellite markers among economically and ecologically important galliform birds. Gen Mol Res 9(2):1121–1129CrossRefGoogle Scholar
  12. Belkhir K, Borsa P, Chikhi L, Raufaste N, Bonhomme F (2012) 1996–2004 GENETIX 4.05, logiciel sous windows TM pour la génétique des populations. Laboratoire Génome, populations, interactions, CNRS UMR 5171, Université de Montpellier II, Montpellier (France). Available at
  13. Ben-David T, Melamed S, Gerson U, Morin S (2007) ITS2 sequences as barcodes for identifying and analyzing spider mites (Acari: Tetranychidae). Exp Appl Acarol 41:169–181PubMedCrossRefGoogle Scholar
  14. Bodenheimer FS (1951) Citrus entomology Dr. W Junk PublishersGoogle Scholar
  15. Brookfield JFY (1996) A simple new method for estimating null allele frequency from heterozygote deficiency. Mol Ecol 5:453–455PubMedGoogle Scholar
  16. Canales-Aguirre CB, Ferrada S, Hernández CE, Galleguillos R (2010) Usefulness of heterologous microsatellites obtained from Genypterus blacodes (Schneider 1801) in species Genypterus off the Southeast Pacific. Gayana 74(1):74–77Google Scholar
  17. Carbonnelle S, Hance T, Migeon A, Baret P, Cros-Arteil S, Navajas M (2007) Microsatellite markers reveal spatial genetic structure of Tetranychus urticae (Acari : Tetranychidae) populations along a latitudinal gradient in Europe. Exp Appl Acarol 41:225–241PubMedCrossRefGoogle Scholar
  18. Fry JD (1989) Evolutionary adaptation to host plants in a laboratory population of the phytophagous mite Tetranychus urticae Koch. Oecologia 81:559–565CrossRefGoogle Scholar
  19. Fry JD (1992) On the maintenance of genetic variation by disruptive selection among hosts in a phytophagous mite. Evolution 46:279–283CrossRefGoogle Scholar
  20. Gould F (1979) Rapid host range evolution in a population of the phytophagous mite Tetranychus urticae Koch. Evolution 33:791–802CrossRefGoogle Scholar
  21. Grbić M et al (2011) The genome of Tetranychus urticae reveals herbivorous pest adaptations. Nature 479:487–492PubMedCrossRefGoogle Scholar
  22. Hinomoto N, Osakabe M, Gotoh T, Takafuji A (2001) Phylogenetic analysis of green and red forms of the two-spotted spider mite, Tetranychus urticae Koch (Acari: Tetranychidae), in Japan, based on mitochondrial cytochrome oxidase subunit I sequences. Appl Entomol Zool 36:459–464CrossRefGoogle Scholar
  23. Hinomoto N, Todokoro Y, Higaki T (2011) Population structure of the predatory mite Neoseiulus womersleyi in a tea field based on an analysis of microsatellite DNA markers. Exp Appl Acarol 53:1–15PubMedCrossRefGoogle Scholar
  24. Hmimina M, Allam L, Ougass Y, Marmouche A (1995) Circonstances des pullulations de Tetranychus urticae Koch (Tetranychidae: Acarina) en verger d’agrumes. IOBC Bull 18:28–35Google Scholar
  25. Hurtado MA, Ansaloni T, Jacas JA, Navajas M (2008a) Structure of Tetranychus urticae (Acari: Prostigmata) populations occurring in Spanish orchards (Citrus reticulata Blanco) and its relevance for pest management. IOBC WPRS Bull 38:243Google Scholar
  26. Hurtado MA, Ansaloni T, Cros-Arteil S, Jacas JA, Navajas M (2008b) Sequence analysis of the ribosomal internal transcribed spacers region in spider mites (Prostigmata: Tetranychidae) occurring in citrus orchards in Eastern Spain: use for species discrimination. Ann Appl Biol 153:167–174Google Scholar
  27. Li T, Chen X-L, Hong XY (2009) Population genetic structure of Tetranychus urticae and its sibling Species Tetranychus cinnabaribus (Acari: Tetranychidae) in China as inferred from microsatellite data. Ann Entomol Soc Am 102(4):674–683CrossRefGoogle Scholar
  28. Lunt DH, Hutchinson WF, Carvalho GR (1999) An efficient method for PCR-based isolation of microsatellite arrays (PIMA). Mol Ecol 8:891–893CrossRefGoogle Scholar
  29. McMurtry JA (1985) Citrus. Spider Mites. In: Helle W, Sabelis MW (eds) Their Biology, Natural Enemies and Control, Vol IB. Elsevier, Amsterdam, pp 339–347Google Scholar
  30. Navajas M (1998) Host plant associations in the spider mite Tetranychus urticae (Acari: Tetranychidae): insights from molecular phylogeography. Exp Appl Acarol 22:201–214CrossRefGoogle Scholar
  31. Navajas M, Fenton B (2000) The application of molecular markers in the study of diversity in acarology: a review. Exp Appl Acarol 24:751–774PubMedCrossRefGoogle Scholar
  32. Navajas MJ, Thistlewood HMA, Lagnel J, Hughes C (1998a) Microsatellite sequences are under-represented in two mite genomes. Insect Mol Biol 7:249–256PubMedCrossRefGoogle Scholar
  33. Navajas M, Lagnel J, Gutierrez J, Boursot P (1998b) Species-wide homogeneity of nuclear ribosomal ITS2 sequences in the spider mite Tetranychus urticae contrasts with extensive mitochondrial COI polymorphism. Heredity 80:742–752PubMedCrossRefGoogle Scholar
  34. Navajas M, Lagnel J, Fauvel G, De Moraes G (1999) Sequence variation of ribosomal internal transcribed spacers (ITS) in commercially important phytoseiidae mites. Exp Appl Acarol 23:851–859PubMedCrossRefGoogle Scholar
  35. Navajas M, Perrot-Minnot MJ, Lagnel J, Migeon A, Bourse T, Cornuet JM (2002) Genetic structure of a greenhouse population of the spider mite Tetranychus urticae: spatio-temporal analysis with microsatellite markers. Insect Mol Biol 11:157–165PubMedCrossRefGoogle Scholar
  36. Nishimura S, Hinomoto N, Takafuji A (2003) Isolation, characterization, inheritance and linkage of microsatellite markers in Tetranychus kanzawai (Acari: Tetranychidae). Exp Appl Acarol 31:93–103PubMedCrossRefGoogle Scholar
  37. Olivatti AM, Boni TA, Silva-Júnior NJ, Resende LV, Gouveia FO, Telles MPC (2011) Heterologous amplification and characterization of microsatellite markers in the Neotropical fish Leporinus friderici. Genet Mol Res 10(3):1403–1408PubMedCrossRefGoogle Scholar
  38. Pérez-Sayas C, Pina T, Gómez-Martínez MA, Jacas JA, Hurtado MA (in preparation) molecular detection of tetranychidae citrus pests on phytoseiidae predators by multiplex PCR (I): detection periodGoogle Scholar
  39. Rousset F (2008) GENEPOP ‘007: a complete re-implementation of the GENEPOP software for windows and linux. Mol Ecol Res 8(1):103–106CrossRefGoogle Scholar
  40. Rychlik W (1992) OLIGO 4.06, primer analysis software. National Biosciences Inc., PlymouthGoogle Scholar
  41. Sabater-Muñoz B et al (2006) Large-scale gene discovery in the pea aphid Acyrthosiphon pisum (Hemiptera). Genome Biol 7(3):R21PubMedCrossRefGoogle Scholar
  42. Sambrook J, Fritsch EF, Maniatis T (eds) (1989) Molecular cloning a laboratory manual. Cold Spring Harbor Press, Cold Spring Harbor, NYGoogle Scholar
  43. Souliotis P, Tsagkarakou A, Nomikou M (1997) Field observations and eco-ethological aspects of Phytoseiid mites in greek citrus groves. Acarologia XXXVIII: 29–37Google Scholar
  44. Staden R, Judge DP, Bonfield JK (2003) Analysing sequences using the staden package and EMBOSS. In: Krawetz SA, Womble DD (eds) Introduction to bioinformatics. A theoretical and practical Approach, Human Press Inc., Totawa, NJ 07512Google Scholar
  45. Sunnucks P, Hales DF (1996) Numerous transposed sequences of mitochondrial cytochrome oxidase I-II in aphids of the genus Sitobion (Hemiptera: Aphididae). Mol Biol and Evol 13:510–524Google Scholar
  46. Swirski E (1977) Integrated control of mites in Israel, In: Carpens ‘O (ed) I Congreso Mundial de Citricultura, CEBAS Murcia-Valencia 1973′ 2: 477–480Google Scholar
  47. Talhouk AS (1975) Citrus pests throughout the world. Technical Monograph No. 4. Ciba-Geigy Agrochemicals, BaselGoogle Scholar
  48. Telles MPC, Peixoto FP, Lima JS, Resende LV, Vianello RP, Walter MEMT, Collevatti RG (2011) Development of microsatellite markers for the endagered Neotropical tree species Tibouchina papyrus (Melastomataceae). Genet Mol Res 10(1):321–325PubMedCrossRefGoogle Scholar
  49. Tixier M-S, Kreiter S, Barbar Z, Ragusa S, Cheval B (2006a) The status of two cryptic species: Typhlodromus exhilaratus Ragusa and Typhlodromus phialatus Athias-Henriot (Acari: Phytoseiidae): consequences for taxonomy. Zool Scr 35:115–122CrossRefGoogle Scholar
  50. Tixier M-S, Kreiter S, Ferragut F, Cheval B (2006b) Morphological and molecular evidences for the synonymy of Kampimodromus hmiminai McMurtry and Bounfour and K. adrianae Ferragut and Pena-Estevez (Acari: Phytoseiidae). Can J Zool 84(8):1216–1222CrossRefGoogle Scholar
  51. Uesugi R, Osakabe M (2007) Isolation and characterization of microsatellite loci in the two-spotted spider mite, Tetranychus urticae (Acari : Tetranychidae). Mol Ecol Resour 7:290–292Google Scholar
  52. Uesugi R, Kunimoto Y, Osakabe M (2009a) The fine-scale genetic structure of the two-spotted spider mite in a commercial greenhouse. Exp Appl Acarol 47:99–109PubMedCrossRefGoogle Scholar
  53. Uesugi R, Sasawaki T, Osakabe M (2009b) Evidence of a high level of gene flow among apple trees in Tetranychus urticae. Exp Appl Acarol 49:281–290PubMedCrossRefGoogle Scholar
  54. Vacante V (1986) Influence of white mineral oil treatments on eastern sicily. CEC Experts Meeting. Acireale 1985. In ‘Integrated Pest Control in Citrus Groves’. Balkema, Rotterdam, pp 423–431Google Scholar
  55. Xie L, Hong XY, Xue XF (2006) Population genetic structure of the two-spotted spider mite (Acari: Tetranychidae) from China. Ann Entomol Soc Am 99:959–965CrossRefGoogle Scholar
  56. Zane L, Bargelloni L, Patarnello T (2002) Strategies for microsatellite isolation; a review. Mol Ecol 11:1–16PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2012

Authors and Affiliations

  • B. Sabater-Muñoz
    • 1
    Email author
  • S. Pascual-Ruiz
    • 2
  • M. A. Gómez-Martínez
    • 2
  • J. A. Jacas
    • 2
  • M. A. Hurtado
    • 2
  1. 1.Unidad Asociada Entomología UJI-IVIA, Centro de Protección Vegetal y BiotecnologíaInstituto Valenciano de Investigaciones Agrarias (IVIA)Moncada, ValenciaSpain
  2. 2.Unitat Associada d’Entomologia UJI-IVIA, Departament de Ciències Agràries i del Medi NaturalUniversitat Jaume I (UJI)Castelló de la PlanaSpain

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