Print version ISSN 1519-566X
Neotrop. entomol. vol.38 no.2 Londrina Mar./Apr. 2009
Genetic similarity among Agistemus pallinii Matioli et al (Acari: Stigmaeidae) found in citrus orchards in Viçosa, Minas Gerais State, Brazil
Similaridade genética entre Agistemus pallinii Matioli et al (Acari: Stigmaeidae) encontrados em pomares de citros em Viçosa, MG
André L MatioliI; Angelo PalliniII; Mara G TavaresIII
IInstituto Biológico, Lab de Entomologia Econômica, Rodovia Heitor Penteado, km 3, C. postal 70, 13.001-970, Campinas, SP; firstname.lastname@example.org
IIDepto. de Biologia Animal; email@example.com
IIIDepto. de Biologia Geral; firstname.lastname@example.org. Univ. Federal de Viçosa - UFV, 36.570-000, Viçosa, MG
Stigmaeidae are very important predators of mite and insect pests on several crops in Brazil. It is considered the second most important family of predatory mites in citrus orchards in Brazil. However, their identification, especially that of the members of the genus Agistemus, is rather difficult based only on morphology. Hence, this study describes the use of random amplified polymorphic DNA (RAPD-PCR) markers to determine the genetic similarity of an Agistemus pallinii Matioli et al population found in 2004 in a citrus orchard in Viçosa, Minas Gerais State, Brazil, preying on Panonychus citri (McGregor). Amplifi cations were performed with 12 random primers (OPAA8, OPAA19, OPAB1, OPAB5, OPAB18, OPAC9, OPAC17, OPAC19, OPAD10, OPAE9, OPAE12 and OPAE17), which generated 119 bands, with 53.8% polymorphism. The coefficients of genetic similarity among the individuals ranged from 0.68 to 0.99, indicating a high genetic similarity among them. The 3D projection analysis clustered the majority of individuals confirming their high similarity. Though individuals of A. pallinii are minute (± 360 µm long), the PCR-RAPD technique can still be used for their identification, complementing morphological analyses or for comparison of populations collected in different geographic regions. This is the first molecular study carried out with stigmaeid mites.
Key words: Predatory mite, genetic variability, molecular marker
Stigmaeidae são importantes predadores de ácaros e insetos pragas em várias culturas no Brasil. Essa família é considerada a segunda mais importante de ácaros predadores em citros no Brasil. Entretanto, o status taxonômico desses ácaros, principalmente do gênero Agistemus, é difícil de ser analisado com base em caracteres morfológicos. Assim, este trabalho descreve o uso de marcadores polimórfi cos amplificados ao acaso (PCR-RAPD) para investigar a similaridade genética de uma população de Agistemus pallinii Matioli et al encontrada em 2004 em pomar de citros em Viçosa, MG, predando Panonychus citri (McGregor). As amplificações foram realizadas com 12 iniciadores randômicos (OPAA8, OPAA19, OPAB1, OPAB5, OPAB18, OPAC9, OPAC17, OPAC19, OPAD10, OPAE9, OPAE12 e OPAE17), os quais geraram 119 bandas, sendo 53,8% polimórficas. Os coeficientes de similaridade genética entre os indivíduos variaram de 0,68 a 0,99, indicando alta similaridade entre eles. A análise de projeção gráfica 3D mostrou que a maioria dos indivíduos analisados formou um único grupo, confirmando a alta similaridade genética entre os mesmos. Assim, mesmo considerando o pequeno tamanho de A. pallinii (± 360 µm), a técnica de PCR-RAPD pode ser utilizada para a sua identificação, em suporte às análises morfológicas ou em comparações de populações coletadas em diferentes regiões geográficas. O presente trabalho representa o primeiro estudo molecular realizado com ácaros da família Stigmaeidae.
Palavras-chave: Ácaro predador, variabilidade genética, marcador molecular
Stigmaeidae mites are important predators on several crops in Brazil, such as citrus, coffee, apple, rubber tree and guava, controlling phytophagous and also fungivorous mites (Matioli et al 1998, Ferla & Moraes 2003).
Identification of these mites is primarily based on morphologic and taxonomic characters and by crossbreeding species. However, these protocols are sometimes insufficient, especially for Agistemus, making necessary the use of other methods to identify them.
The application of molecular markers to studies on mites recently yielded new insights into their population structures and taxonomic relationships. Molecular markers, such as those generated by RAPD-PCR proved very useful in distinguishing species and in identifying genetic variations within and among populations (Perrot-Minnot & Navajas 1995, De Guzman et al 1997, Edwards et al 1997, Hance et al 1998, Yli-Mattila et al 2000). Edwards et al (1998), for example, used this method to compare three similar species of phytoseiid mites: Typhlodromalus limonicus Garman & McGregor, T. manihoti Moraes and T. tenuiscutus McMurtry & Moraes, concluding that this technique could be used to distinguish other cryptic species, in addition to interbreeding experiments.
Though the Stigmaeidae is considered the second most important predatory mite family in citrus orchards, this method has not yet been applied to their identifi cation or to the comparison of populations collected in different geographic regions. Therefore, in here we report the use of random amplified polymorphic DNA (PCR-RAPD) markers to investigate the genetic similarity of an Agistemus pallinii Matioli et al population found in citrus orchards in Viçosa, Minas Gerais State, Brazil, preying on Panonychus citri McGregor (Acari: Tetranychidae).
Material and Methods
Mites were sampled in a citrus orchard in Viçosa-MG, Brazil, in 2004, and reared under controlled conditions (25ºC, 12:12h L:D, and 75% RH). Brevipalpus phoenicis (Geijskes) and P. citri, supplemented with Typha sp. pollen, were offered as a rearing diet (Matioli et al 2007), using citrus leaves as a substrate.
DNA extraction of 33 A. pallinii females followed Groot et al (2005). Individual specimens were placed in a centrifuge tube (0.5 ml) kept in ice, and females were homogeneized in 3 µl of proteinase K (20 mg/ml). Subsequently, 50 µl of a 5% Chelex100® solution was added, samples were briefloy vortexed, incubated at 37ºC for 1h and quickly vortexed again after 0.5h. Subsequently, the samples were incubated at 96ºC for 8 min and briefloy agitated. The extracted DNA was used immediately or stored at -20ºC for two or three weeks for later use.
DNA amplification. The DNA of individual mites was amplified by PCR-RAPD as described by Williams et al (1990) with some modifications. Each PCR reaction was set up in a total volume of 25 µl with 1X reaction buffer, 0.1 mM dNTPs, 0.4 µM 10-base primer (Operon Technologies, Alameda, CA, USA), 0.5 U Taq polymerase (Phoneutria, Belo Horizonte, MG, Brazil) and 3 µl DNA template (unknown concentration), and runin a PTC-100 thermocycler (MJ Research) programmed at 92ºC for 30 s, 35ºC for 1 min, 3 min and 42 s. to increase to 72ºC; 72ºC for 2 min (45 cycles); 72ºC for 7 min (1x).
Amplification products were separated in 1.2% agarose gels containing 0.2 µg/ml of ethidium bromide in TBE buffer (Tris-borate 90 Mm, pH 8.0, EDTA 10mM). Bands were visualized under UV light and the images were analyzed using the AlphaDigidoc 1201 system (AlphaDigidocTM). A negative control without genomic DNA was used in each amplification to check for possible contaminants.
Initially, DNA of six specimens of A. pallinii was used in amplification tests to verify the amplification patterns of the RAPD primers. In these amplifications 56 primers were tested and 34 produced amplifications patterns that could be used in subsequent analyses. However, due to the small amounts of DNA available, the subsequent amplifications were carried out only with 12 primers: OPAA8, OPAA19, OPAB1, OPAB5, OPAB18, OPAC9, OPAC17, OPAC19, OPAD10, OPAE9, OPAE12 and OPAE17 (Table 1).
The amplicons produced were scored as present (1) or absent (0), and the constructed data matrix was used for the calculation of the Jaccard's similarity index (Sij) among the pairs of individuals. The genetic similarity (Sij) was then converted to genetic distances (Dij) using the arithmetic complement of similarity given by the expression: Dij = 1Sij. This matrix was submitted to a 3D graphic dispersion analysis. The projection efficiency of the distances in the plane was estimated according to the correlation between the original distances and those showed in the graph. All analyses were carried out using the Genes program (Cruz 2005).
Results and Discussion
The method used in the present work for the extraction of DNA of individual mites was appropriate for the RAPD-PCR amplifications, despite the small size of the mites and, consequently, the small amount of DNA. Our data corroborated that of Yli-Mattila et al (2000), showing the suitability of Chelex 100® in extracting DNA from Euseius finlandicus Oudmans (Acari: Phytoseiidae). The main limitation was the small number of possible amplifications that can be carried out with the same sample.
Amplifications carried out with the 12 selected primers produced 119 bands, with a range of 4 to 15 bands/primer (Table 1). The mean number of bands produced per primer (9.9) was close to that found for three species of Typhlodromalus (8.1) (Edwards et al 1997) and different strains of Tetranychus sp (8.7) (Hance et al 1998).
In general, the PCR-RAPD patterns generated were clear and similar in number among individuals, as the one obtained with the primer OPAE12 (Fig 1), allowing the efficient characterization of the population of A. pallinii analyzed in the present study. However, as the absence of one determined band in only one specimen was considered as a polymorphism, 53.8% of the bands generated (64 bands) were polymorphic in the analyzed population.
The RAPD technique had already been used for population analyses and identification of different mite species and/or strains. Hernandez et al (1998) obtained RAPD patterns capable of differentiating strains of Boophilus microplus (Canestrini) susceptible from resistant ones. Edwards et al (1998) examined variable RAPD markers in three Typhlodromalus species and found several bands that could be used to distinguish phytoseiid species, while Yli-Mattila et al (2000) tested 20 primers in order to determine specific RAPD products to distinguish between strains and mite species.
The mean coefficient of similarity obtained for paired comparisons among the 33 of A. pallinii was 0.85 (SD = 0.066), with values ranging from 0.68 to 0.99. The highest similarity (0.99) was observed between 8 and 27, while the lowest (0.68) similarity was between specimens 23 and each of 29 to 32.
This high similarity could be confirmed in the dispersion graphic analysis (Fig 2) where the distance between points is proportional to the dissimilarity between genotypes. Thus, the majority of the analyzed specimens grouped together confirming the high similarity between them, while the more dissimilar individuals (28, 29, 30, 31, 32, 33 and 23) were more distant graphically. The correlation value (81.5%) obtained from this analysis further showed a good adjustment between the genetic distances and the distances graphically represented.
Strong characters to distinguish among species of Agistemus are limited, most consisting of ratios, easily influenced by the mounting of the mites. Thus, the RAPD-PCR technique can be a useful tool for species identification. Another advantage is that the species identity can be determined quicker. However, this technique must not replace the taxonomy based on morphology. Hance et al (1998), for example, used this technique to compare strains of Tetranychus sp. to complement fecundity and biometry studies. Additionally, the loss of information due to the dominance of the technique can be minimized by increasing the number of loci analyzed (Miller 2000). Through cloning and sequencing of RAPD products it is also possible to develop more specific primers (SCARs). Amplification with these primers could result in a more precise identification of species as demonstrated with the identification of different strains of E.finlandicus (Yli-Mattila et al 2000).
This is the first molecular study carried out with predatory mites of the genus Agistemus (Stigmaeidae), whose species is difficult to determine based on morphologic characters only. The RAPD technique revealed to be a promising tool for the identification of species as well as in intra and inter specific diversity studies and serving as a basis for future studies related to other Agistemus species.
We thank Drs. Eddie Ueckermann (ARC-Plant Protection Research, Pretoria South Africa), Gilberto Jose de Moraes (Departamento de Entomologia, Fitopatologia e Zoologia, ESALQ/USP, Piracicaba, SP, Brazil), Mario Eidi Sato and Suzete A. L. Destéfano (Instituto Biológico, Campinas, SP, Brazil) for reviewing an early version of this manuscript. CNPq and UFV are thanked for financial and logistic support.
Cruz C D (2005) Programa Genes. Versão Windows: aplicativo computacional em genética e estatística. Viçosa, MG. [ Links ]
De Guzman L I, Rinderer T E, Stelzer J A (1997) DNA evidence of the origin of Varroa jacobsoni in the Americas. Biochem Genet 35: 327-335. [ Links ]
Edwards O W, Melo E L, Smith L, Hoy M A (1998) Discrimination of three Typhlodromalus species (Acari: Phtytoseiidae) using random amplified polymorphic DNA markers. Exp Appl Acarol 21: 101-109. [ Links ]
Ferla N J, Moraes G J (2003) Biologia de Agistemus flooridanus Gonzalez (Acari, Stigmaeidae). Rev Bras Zool 20: 261-264. [ Links ]
Groot TV M, Janssen A, Pallini A, Breeuwer J A J (2005) Adaptation in the asexual false spider mite Brevipalpus phoenicis: evidence for frozen niche variation. Exp Appl Acarol 36: 165-176. [ Links ]
Hance T, Neuberb P, Noel-Lastelle C (1998) The use of fecundity, lobe biometry and the RAPD-PCR technique in order to compare strains of Tetranychus sp. Exp Appl Acarol 22: 649-666. [ Links ]
Hernandez R, Chen A C, Davey R B, Ivie G W, Wagnenr G G, George J E (1998) Comparison of genomic DNA in various strains of Boophilus microplus (Acari: Ixodidae). J Med Entomol 35: 895-900. [ Links ]
Matioli A L, Leite G L D, Pallini Filho A, Picanço M (1998) Distribuição espacial e temporal e efeitos de diferentes tratos culturais em ácaros associados a laranja Pêra-Rio. Agro-Ciencia (Chile) 14: 395-405. [ Links ]
Matioli A L, Tavares M G, Pallini A (2007) Agistemus pallinii n.sp. (Acari: Stigmaeidae) from citrus orchards in Brazil. Int J Acarol 33: 245-251. [ Links ]
Miller L M (2000) Classifying genealogical origins in hybrid populations using dominant markers. J Hered 91: 46-49. [ Links ]
Perrot-Minnot M J, Navajas M (1995) Biparental inheritance of RAPD markers in males of the pseudo-arrhenotokous mite Typhlodromus pyri. Genome 38: 838-844. [ Links ]
Williams J G K, Kubelik A R, Livak K, Rafalski J A, Tingey S V (1990) DNA polymorphism amplified by arbitrary primers are useful as genetic markers. Nucl Acids Res 18: 6531-6535. [ Links ]
Yli-Mattila T, Paavanen-Huhtala S, Fenton B, Tuovinen T (2000) Species and strain identification of the predatory mite Euseius finlandicus by RAPD-PCR and ITS sequences. Exp Appl Acar 24: 863-880. [ Links ]
Received 19/IX/07. Accepted 11/III/09.
Edited by Denise Návia - EMBRAPA/CENARGEN