Molecular Characterization of Parasitoids from Armored Scales Infesting Citrus Orchards in Corsica, France
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BioControl (2016) 61:639–647 DOI 10.1007/s10526-016-9752-1 Molecular characterization of parasitoids from armored scales infesting citrus orchards in Corsica, France Margarita C. G. Correa . Ferran Palero . Noe´mie Dubreuil . Laure Etienne . Mathieu Hulak . Gilles Tison . Sylvie Warot . Didier Crochard . Nicolas Ris . Philippe Kreiter Received: 23 January 2016 / Accepted: 6 July 2016 / Published online: 14 July 2016 Ó International Organization for Biological Control (IOBC) 2016 Abstract Armored scales (Hemiptera: Diaspididae) (including A. melinus), four Encarsia (including cryp- are important pests in citrus orchards worldwide. tic species) and one Ablerus (hyperparasitoid) species. Augmentative releases of Aphelinidae wasps (Hy- Host-specificity was found to be strong among menoptera) have been performed in Corsica, France to primary parasitoids, with Encarsia inquirenda Sil- control the California Red Scale (Aonidiella aurantii vestri, 1930 and an unidentified Encarsia being the (Maskell, 1879)) and the arrowhead scale (Unaspis sole taxa able to parasitize the two subfamilies yanonensis (Kuwana, 1923)), but biological control of (Aspidiotinae and Diaspidinae). armored scales requires the identification of their parasitoids to evaluate their potential as biological Keywords Diaspididae Á Armored scale Á control agents. In order to circumvent this issue, Parasitoid Á DNA barcoding Á Cryptic species parasitoids emerging from four armored scale species were characterized through DNA barcoding. All the parasitoids identified belong to the Aphelinidae (Hy- menoptera) and included a total of five Aphytis Introduction Handling Editor: Josep Anton Jaques Miret Armored scales (Hemiptera: Diaspididae) are arthro- pod pests found in fruit orchards worldwide, mostly Margarita C. G. Correa and Ferran Palero contributed equally affecting citrus crops including clementine, grape- to this work. fruits, lemons and oranges (Flint et al. 1991; Tena and Garcia-Marı´ 2011). Insecticides are commonly used Electronic supplementary material The online version of against diaspidid scales because infestations can cause this article (doi:10.1007/s10526-016-9752-1) contains supple- mentary material, which is available to authorized users. M. C. G. Correa Á F. Palero (&) Á S. Warot Á G. Tison D. Crochard Á N. Ris Á P. Kreiter INRA Centre de San Giuliano, Unite´ citrus, Poˆle INRA, Univ. Nice Sophia Antipolis, CNRS, UMR agronomique, 20230 San Giuliano, Corsica, France 1355-7254 Institut Sophia Agrobiotech, 06900 Sophia Antipolis, France F. Palero e-mail: [email protected] Centre d’Estudis Avanc¸ats de Blanes (CEAB-CSIC), Carrer d’Acce´s a la Cala Sant Francesc 14, 17300 Blanes, N. Dubreuil Á L. Etienne Á M. Hulak Spain AREFLEC, Corsic’Agropoˆle, route de Pianicce, Poˆle agronomique, 20230 San Giuliano, Corsica, France 123 640 M. C. G. Correa et al. fruit rejection (Beardsley and Gonzalez 1975; Boyero recent years as an important tool that facilitates an et al. 2014). Repeated use of synthetic organophos- accurate and fast identification of insect species phates has already led to resistance in Aonidiella (Gariepy et al. 2014). These molecular tools have aurantii (Maskell, 1879) (Hemiptera: Diaspididae) allowed scientists to perform phylogenetic studies (Grafton-Cardwell et al. 2004, 2006), concerns on (Schmidt and Polaszek 2007; Munro et al. 2011; Beltra` human health and significant fauna disruption (Liang et al. 2015) while also allowing for the identification of et al. 2010), so that alternative and efficient methods to hyperparasitoids and to study the competition between control armored scales are needed. Aphelinid wasps different and within the same trophic level (Rugman- (Hymenoptera: Aphelinidae) belonging to the Aphytis Jones et al. 2011;Go´mez-Marco et al. 2015). In and Encarsia genera are host-specific parasitoids particular, DNA barcoding methods have already been commonly used as biological control agents (BCAs) used to distinguish and clarify the status of some to control armored scales in citrus (Rosen and DeBach closely related species inside Aphytis and Encarsia 1979; Sorribas and Garcia-Marı´ 2010). (Monti et al. 2005; De Leo´n et al. 2010; Pina et al. In France, the island of Corsica represents the main 2012). Given the difficulties with morphology-based citrus growing region, comprising about 45 % of taxonomy in parasitoid wasps, and with more than the [4000 hectares planted in the country (Agreste 2200 scale species as potential hosts (Rosen and 2015). Insularity has kept citrus orchards free from DeBach 1979; Ben-Dov et al. 2010), hundreds of taxa pests in Corsica until recent years, but the intensifi- are likely to be still unknown (Hardy 2008). Therefore, cation of commercial exchanges among Mediter- the combined use of molecular methods and intensive ranean countries, together with human-related sampling should allow for the identification of new pressures (e.g., tourism), have caused the introduction BCA candidates from the field. and expansion of Diaspididae pests during the last With the aim of identifying and characterizing the decade. The main armored scale species present in the diversity of parasitoids attacking armored scales country include the California Red Scale A. aurantii, present in citrus orchards from Corsica, we have Lepidosaphes spp., Unaspis yanonensis (Kuwana, (i) collected field samples belonging to different 1923) and Parlatoria pergandei Comstock, 1881 armored scale species, (ii) isolated each armored scale (Ben-Dov et al. 2010). A. aurantii affects more than sample for parasitoid emergence, and (iii) identified one-third of the citrus orchards and has considerable the emerged parasitoids through morphology and economic impact in Corsica, so that several species of DNA barcoding methods. Furthermore, the combina- parasitoids have been released to control armored tion of host-specificity data and molecular phyloge- scales in the island (Tison et al. 2007). Following the netic analyses allowed us to spot the presence of example of successful releases of Aphytis melinus unidentified taxa that could be used in biological (DeBach, 1959) in California coastal valleys for control against armored scales. biological control of A. aurantii (Murdoch et al. 2006), augmentative releases of A. melinus have been performed in Corsica since 2009, with *170 hectares Materials and methods being treated in 2013. Releases of Aphytis yanonensis DeBach & Rosen 1982 have also been carried out to Sampling control U. yanonensis in the same area during 2010 and 2011 (Etienne and Jaloux 2014). Nevertheless, A total of 76 citrus orchards were sampled in 12 evaluation of parasitism induced by these introduced Corsican localities (Aleria, Ghisonaccia, San Nicolao, BCAs compared to parasitism induced by native or Valle-Di-Campoloro, Fraciccia, Borgo, San Giuliano, established parasitoid species remains poorly Linguizzetta, Mignataja, Antisanti, Folleli and documented. Velone-Orneto) during 2014 (Supplementary The small size (less than 2 mm in total length) and Table S1). Orchards sampled had different types of high diversity of aphelinid wasps thwart their identi- citrus species (Bitter orange, Citron, Clementine, fication and challenge their precise evaluation as Grapefruit, Kumquat, Lemon, Limes, Mandarin, BCAs (Babcock and Heraty 2000; Pina et al. 2012). Orange and Wekiwa Tangelo) and/or management DNA sequencing methods have become necessary in (conventional versus organic agriculture, in each case 123 Molecular characterization of parasitoids from armored scales infesting citrus orchards… 641 with or without releases of A. melinus). Diaspididae extension at 72 °C for 10 min. PCR products were samples were obtained by cutting off parts of the trees checked by electrophoresis through a QIAxcel infested with armored scales (fruits, leaves and/or Advanced System (QIAGEN, Hilden, Germany) and branches). Four species of armored scales were sent to Beckman Coulter Genomics (Takeley, United sampled in this study, two of them belonging to the Kingdom) for bidirectional sequencing. DNA Aspidiotinae subfamily (A. aurantii and Parlatoria sequences were corrected with Seqscape v3.0 (Applied pergandei) and the other two belonging to the Biosystems, Foster City, USA). Diaspidinae subfamily (Lepidosaphes spp. and U. yanonensis). Each sample was checked using a Phylogenetic relationships and genetic divergence magnifying glass and subdivided into sub-samples according to the number of pest species present. For DNA sequence alignments were conducted using the each sub-sample only one pest species was kept alive, program MUSCLE v3.6 (Edgar 2004) with default with individuals of other species being manually parameters and then checked by eye. BLAST searches destroyed. Plant material associated with each sub- (https://blast.ncbi.nlm.nih.gov/Blast.cgi) were carried sample was placed in emergence boxes and checked out to identify similarities between the sequences weekly for parasitoids emergence. After emergence, obtained in this work and those already available in parasitoids were introduced in Eppendorf tubes with GenBank. The MEGABLAST method was used absolute ethanol. The identification of parasitoids (recommended for highly similar sequences). Before followed a two-stage process: visual inspection to sort carrying out the likelihood-based analyses, model the individuals at the genus level (a posteriori, few selection of nucleotide substitution was performed mistakes were observed) and a molecular character- with MEGA6 (Tamura et al. 2013) according to ization