BioControl (2016) 61:639–647 DOI 10.1007/s10526-016-9752-1

Molecular characterization of parasitoids from armored scales infesting orchards in ,

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 (: ) (including A. melinus), four (including cryp- are important pests in citrus orchards worldwide. tic species) and one Ablerus (hyperparasitoid) species. Augmentative releases of 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 ( 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 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 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- (: 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 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 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, , San Nicolao, BCAs compared to parasitism induced by native or Valle-Di-Campoloro, Fraciccia, Borgo, San Giuliano, established parasitoid species remains poorly , 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 using DNA barcoding methods (see next Bayesian Information Criterion scores and Akaike paragraph). In parallel, a sample of 49 individuals Information Criterion, corrected value. The aligned from the AREFLEC rearing (institution in charge of dataset was then used to estimate maximum likelihood producing Aphytis melinus for biocontrol) were (ML) phylogenies under the selected DNA substitu- molecularly characterized in order to have a reference tion model using MEGA6. Bootstrap branch support sequence for the BCAs released by Corsican producers values were calculated with 500 ML replicates. COI in their fields. and 28S gene alignments for our sequence data where concatenated and analyzed using the same procedure DNA extractions and PCR amplification outlined above to estimate a consensus tree. The cytochrome oxidase subunit 1 (COI) is com- Genomic DNA was extracted with the prepGEM Insect monly used in DNA barcoding studies to identify DNA extraction kit (ZyGEM, Lane Hamilton, New biological material to the species level and it has been Zealand) without crushing the parasitoid body and shown to be particularly useful for (Zaldı´var- following manufacturer recommendations. Polymerase Rivero´n et al. 2010; Malausa et al. 2011). Moreover, Chain Reactions (PCR) were performed in a volume of the use of genetic markers such as the cytochrome 25 ll per well containing: 10.3 ll of purified water, oxidase I (COI) will allow us to characterize the 12.5 ll of QIAGEN PCR Mastermix (Hilden, Ger- within-species molecular diversity. The COI-gene many), 0.1 ll of forward primer and 0.1 ll of reverse divergence among parasitoids was estimated here primer. The primers used for this study were 50-AGAG through Maximum Composite Likelihood (Tamura AGAGTTCAAGAGTACGTG-30 and 50-TTGGTCCG et al. 2004). Estimates of divergence and the corre- TGTTTCAAGACGGG-30 for 28S-D2 (Malausa et al. sponding SE were obtained using the Maximum 2011) and 50-AYAATATAATRATTACWWTWCAT Composite Likelihood model in MEGA6 (Tamura GC-30 and 50-TTTWCCATTTAAWGTTA-TTATTC- et al. 2013). 30 for the COI region (Abd-Rabou et al. 2012). PCR conditions were as follows for both markers: an initial Parasitoid taxa delineation denaturation at 95 °C for 15 min, followed by 35 cycles of denaturation at 94 °C for 30 s, annealing for 90 s at We have used an integrative approach (including 58 °C, elongation at 72 °C for 60 s, and a final morphology, ecology and genetics) to delineate the 123 642 M. C. G. Correa et al.

Fig. 1 Maximum Likelihood trees for COI and 28S. Bootstrap per unit length. Accession numbers are indicated for sequences support values ([70) are shown on the corresponding nodes. previously available in GenBank and names in bold correspond Scales refer to genetic distances as percentage of substitutions to the sequences obtained in this study parasitoid taxa observed. Taxa delineation was based Aphytis (A. chrysomphali Mercet, 1912, A. hispanicus on: (i) similarities of COI and 28S sequences with (Mercet, 1912), A. lepidosaphes Compere, 1955, reliable GenBank accessions; (ii) association between A. melinus DeBach, 1959, A. yanonensis DeBach & parasitoids and their known host species (Rosen and Rosen, 1982), two Encarsia (E. inquirenda Silvestri, DeBach 1979; Rosen 1994), in particular for special- 1930 and E. perniciosi Tower, 1913) and one Ablerus ized ones (A. chrysomphali, A. lepidosaphes, A. meli- species. Some Genbank sequences were found to be nus, A. yanonensis); (iii) previous morphological mislabeled, such as JQ268916 which is labelled as identifications carried out by specialists in 2005 for A. melinus but must be in fact A. lepidosaphes the two species that were maintained in laboratory for (Fig. 1). mass-rearing (A. melinus and A. yanonensis) and The other two clades correspond to unknown those present in the field (A. chrysomphali and Encarsia strains, one of them being genetically close E. perniciosi) and (iv) the history of biological control to E. perniciosi (hereafter called Encarsia cf. perni- using Encarsia and Aphytis in Corsica (Tison et al. ciosi). Although most species analyzed presented a 2007; Malausa et al. 2008; Etienne and Jaloux 2014). single haplotype per marker, Aphytis melinus, A. his- panicus and A. chrysomphali presented two haplo- types each for the COI gene region. This Results mitochondrial polymorphism did not seem to be related with the number of individuals analyzed, since Molecular characterization the three species presented different sample sizes (134, 53 and 26 respectively—see Fig. 2). The AREFLEC The new sequences obtained have been deposited in sample for A. melinus presented both haplotypes (data GenBank with accession numbers KX065192– not shown). KX065214. A total of 870 individual parasitoids were Species delineation seemed to be unambiguous sequenced for the COI and/or 28S gene regions, with within this group, because genetic distances between sequences from both markers producing similar tree haplotypes of the same species were always below topologies (Fig. 1). Taken as a whole, ten highly 0.3 % (even when using GenBank data) and far lower supported clades were observed (with bootstrap sup- than the inter-specific genetic distances (Fig. 3). port being higher than 98 %). BLAST comparisons Indeed, divergences among different species within evidence high similarity for eight of these clusters with Aphytis ranged between 6.0 % (A. lepidosaphes and sequences from the GenBank database, including five A. hispanicus H2) and 9.5 % (A. melinus H2 and

123 Molecular characterization of parasitoids from armored scales infesting citrus orchards… 643

Fig. 2 Relative abundance of each parasitoid species on the different armored scales species sampled

A. chrysomphali H2) and between 4.6 % (between Aleria and Antisanti were composed of a single E. perniciosi and E. cf. perniciosi) to 11.7 % (be- species, Encarsia sp. in Aleria and A. hispanicus in tween E. inquirenda and E. sp) within Encarsia Antisanti. (Table 1; Fig. 3). Host specificity Species abundance and geographic distribution As shown in Fig. 4, both the genera Aphytis and Molecular identification of parasitoids emerging from Encarsia were able to parasitize members of the two field samples (i.e., excluding the AREFLEC rearing) Diaspididae subfamilies (Aspidiotinae and Diaspidi- revealed that the genera Aphytis and Encarsia were nae). Nevertheless, each parasitoid species was found present in 49.6 and 49.2 % of the samples respec- in association with one subfamily only, with the tively, with Ablerus representing only 1.2 % (ten exception of Encarsia inquirenda and Encarsia sp. individuals). At the species level, the most abundant which could emerge from both subfamilies. Interest- parasitoid found in Corsican populations was E. per- ingly, even if E. perniciosi and E. cf. perniciosi niciosi (n = 244), followed by A. melinus (n = 134), presented low genetic divergence levels (4.6 %), they A. lepidosaphes (n = 128) and E. cf. perniciosi were found to be preferentially associated with (n = 124) (Fig. 2). With regard to the geographic different host subfamilies (Fig. 3). These different distribution of parasitoids, the majority of the samples patterns reinforce the idea that these two lineages presented several species, with E. perniciosi being constitute different species. With regard to the main dominant in Borgo, Linguizzetta, Fraciccia and agronomic scale pests (i.e., A. aurantii and Ghisonaccia. The species E. cf. perniciosi was found U. yanonensis), three Aphelinidae species were found in most localities (excluding Borgo, Antisanti and to be mainly associated with A. aurantii (by decreas- Aleria), and represents 33 % of all parasitoids found in ing order of abundance E. perniciosi, A. melinus and Valle-Di-Campoloro. The reared BCA A. melinus was A. chrysomphali) while A. yanonensis was the main only present in Ghisonaccia and San Giuliano, where it parasitoid associated with U. yanonensis. was the most abundant species. A. lepidosaphes was found in almost every site, but was dominant only in Mignataja (the southernmost locality). In both Velone Discussion and San Nicolao, composed of organic crops, the dominant parasitoid was A. yanonensis, which agrees The main armored scale species affecting citrus with the fact that U. yanonensis was the most abun- orchards in Corsica were surveyed for parasitoid dant scale there. Finally, the smallest samples from emergence in order to identify natural enemies.

123 644 M. C. G. Correa et al.

Fig. 3 Boxplot showing the distribution of genetic distance Each box ends at the quartiles Q1 and Q3 and includes the values observed within species (intra-species), between species statistical median as a horizontal line inside the box. The within genera (inter-species) and among genera (inter-genera). ‘‘whiskers’’ extend to the farthest points that are not outliers Genetic distance is estimated as percentage of substitutions (i.e., that are within 1.5 times the inter-quartile range of Q1 and using the Maximum Composite Likelihood model in MEGA6. Q3)

Table 1 Genetic divergence observed among taxa Encarsia Aphytis Einq Ecfpe Eper Esp Achr H1 Achr H2 Ahis H1 Ahis H2 Amel H1 Amel H2 Alep Ayan

Encarsia Einq 0.012 0.016 0.017 0.025 0.025 0.024 0.023 0.026 0.026 0.028 0.026 Ecfpe 0.073 0.009 0.014 0.021 0.021 0.017 0.016 0.018 0.018 0.019 0.020 Eper 0.100 0.046 0.015 0.024 0.024 0.019 0.019 0.024 0.025 0.023 0.022 Esp 0.117 0.092 0.108 0.022 0.023 0.018 0.018 0.022 0.022 0.021 0.021 Aphytis Achr H1 0.173 0.140 0.159 0.147 0.002 0.013 0.013 0.015 0.015 0.015 0.015 Achr H2 0.176 0.143 0.161 0.149 0.002 0.013 0.014 0.016 0.016 0.015 0.015 Ahis H1 0.159 0.117 0.139 0.130 0.070 0.073 0.002 0.012 0.012 0.011 0.012 Ahis H2 0.157 0.115 0.137 0.128 0.073 0.075 0.002 0.011 0.012 0.011 0.012 Amel H1 0.175 0.124 0.159 0.160 0.090 0.093 0.067 0.065 0.002 0.013 0.012 Amel H2 0.178 0.126 0.161 0.158 0.093 0.095 0.069 0.067 0.002 0.013 0.012 Alep 0.176 0.122 0.155 0.146 0.085 0.087 0.062 0.060 0.076 0.078 0.014 Ayan 0.169 0.133 0.144 0.144 0.085 0.088 0.068 0.066 0.066 0.068 0.084 Divergence values within genera are highlighted in bold. SE estimate(s) are shown above the diagonal. Abbreviations for species are as follows (alphabetical order): Aphytis chrysomphali (Achr), Aphytis hispanicus (Ahis), Aphytis lepidosaphes (Alep), Aphytis melinus (Amel), Aphytis yanonensis (Ayan), Encarsia inquirenda (Einq), Encarsia cf. perniciosi (Ecfpe), (Eper), Encarsia sp. (Esp)

Molecular analyses of individual wasps revealed the and U. yanonensis). Although both genera (Aphytis presence of two main parasitoid genera (Aphytis and and Encarsia) include species commonly used as Encarsia) associated to four armored scale species BCAs against armored scales (Rosen and DeBach (A. aurantii, Parlatoria pergandei, Lepidosaphes spp. 1979; Rosen 1994; Heraty et al. 2007), their

123 Molecular characterization of parasitoids from armored scales infesting citrus orchards… 645

Fig. 4 Consensus tree obtained using both COI and 28S gene are shown on the corresponding nodes. Scales refer to genetic markers and showing the evolutionary distribution of parasitism distances as percentage of substitutions per unit length on armored scale subfamilies. Bootstrap support values ([70) systematics and morphology-based species identifica- pointed out by the genetic data. The only species tion remains extremely difficult. The lack of molecular showing a generalist behavior were E. inquirenda and information in public international databases is noto- Encarsia sp. Even if only 12 individuals were rious, and most species of aphelinid wasps are not obtained, Encarsia sp. specimens were recovered represented in GenBank. Our results show that the from Lepidosaphes spp., U. yanonensis and Parlato- joint analysis of 28S and COI gene markers efficiently ria pergandei scales. One species belonging to the separates different parasitoid taxa and allows for the genus Ablerus was also found to be associated to identification of new cryptic species within the different scale species in Corsica. Ablerus species are Encarsia clade (Encarsia cf. perniciosi and Encarsia known to hyperparasitize parasitoid wasps (Kattari sp). The molecular divergence values found within the et al. 1999; Ebrahimi 2014) and could hinder the genera Aphytis and Encarsia (7.6–8.9 %) are similar efficiency of parasitoid introductions. Therefore, their to those observed in previous studies using the COI presence in citrus orchards should be taken into gene (Hebert et al. 2003), with 9.4 % molecular account when evaluating biological control programs divergence being found for Aphytis species (Pina et al. against diaspidid scales. 2012) and 9.7 % for Encarsia (De Leo´n et al. 2010). Aonidiella aurantii, the California Red Scale, is the At the genus level, host ranges for Aphytis and most damaging armored scale on citrus worldwide Encarsia were significantly wide, and both genera (Grafton-Cardwell et al. 2008; Tena and Garcia-Marı´ appear to be able to parasitize the two armored scale 2011), so evaluating BCAs efficiency is of utmost subfamilies (Aspidiotinae and Diaspidinae). Never- importance. In a previous study by Tison et al. (2007), theless, most of the individual taxa (seven out of nine the two most common species found to parasitize parasitoid clades) showed a strong host-specificity and A. aurantii in Corsica were A. chrysomphali and attacked mainly a single armored scale species. While E. perniciosi. The endoparasitoid E. perniciosi has most E. cf. perniciosi samples (89 % of the individ- apparently adapted to parasitize A. aurantii only uals) were found parasitizing Lepidosaphes spp., the recently, probably after being introduced to control majority of E. perniciosi individuals emerged from the (Quadraspidiotus perniciosus)at A. aurantii scales, reinforcing the species distinction San Giuliano (Tison et al. 2007). Similarly, the

123 646 M. C. G. Correa et al. ectoparasitoid A. chrysomphali seems to have adapted Boyero JR, Vela JM, Wong E, Garcia-Ripoll C, Verdu MJ, to A. aurantii as a secondary host after being intro- Urbaneja A, Vanaclocha P (2014) Displacement of Aphytis chrysomphali by Aphytis melinus, parasitoids of the Cali- duced to control Chrysomphalus dictyospermi (Mor- fornia Red Scale, in the Iberian Peninsula. Span J Agric Res gan, 1889) (Tison et al. 2007). Even though 12:244–251 E. perniciosi remains the main species parasitizing De Leo´n JH, Neumann G, Follett P, Hollingsworth RG (2010) A. aurantii ([60 % of the parasitized individuals), our Molecular markers discriminate closely related species Encarsia diaspidicola and Encarsia berlesei (Hy- results differ from those of Tison et al. (2007) by the menoptera: Aphelinidae): biocontrol candidate agents for presence of A. melinus in a significant proportion of white peach scale in Hawaii. J Econ Entomol 103:908–916 the samples (23 %) and an increase on the incidence of Ebrahimi E (2014) Parasitoid and hyperparasitoid wasps of A. chrysomphali (from 3 to 10 %). This modification scale insects in Hayk Mirzayans Insect Museum, Iran. J Entomol Soc Iran 34:73–83 in the parasitoid composition most likely results from Etienne L, Jaloux B (2014) Incidence de laˆchers inondatifs du the recurrent releases of A. melinus realized by parasitoı¨de Aphytis melinus sur la biodiversite´ en vergers AREFLEC since 2009. It should be pointed out that d’agrumes en Corse. Universite´ d’Angers, France both A. melinus and A. chrysomphali were found to be Flint M, Kobbe B, Clark J, Dreistadt S, Pehrson J, Flaherty D, O’Connell NV, Phillips PA, Morse J (1991) Integrated pest highly specific, with almost 100 % of the sampled management for citrus, 2nd edn. University of California, individuals emerging from A. aurantii. Our results Oakland highlight the importance of molecular approaches to Gariepy TD, Haye T, Zhang J (2014) A molecular diagnostic identify effective and new BCA candidates to better tool for the preliminary assessment of host-parasitoid associations in biological control programmes for a new control armored scales. invasive pest. Mol Ecol 23:3912–3924 Go´mez-Marco F, Urbaneja A, Jaques JA, Rugman-Jones PF, Acknowledgments Thanks are due to the AREFLEC and Stouthamer R, Tena A (2015) Untangling the aphid-para- INRA colleagues that helped with sampling, and to the sitoid food web in citrus: can hyperparasitoids disrupt producers and technical advisors in Plant Protection that gave biological control? Biol Control 81:111–121 access to their orchards. Special thanks are due to the INRA-BPI Grafton-Cardwell EE, Ouyang Y, Striggow R, Vehrs S (2004) team for their patience and support. This work was funded by the Role of esterase enzymes in monitoring for resistance of ONEMA through the ECOPHYTO program of the Cors’Aphy California Red Scale, Aonidiella aurantii (Homoptera: Project. MC and FP received financial support from the Marie Diaspididae), to organophosphate and carbamate insecti- Curie FP7 IAPP Project ‘‘Colbics’’ #324475. FP also cides. J Econ Entomol 97:606–613 acknowledges support by the project CHALLENGEN Grafton-Cardwell EE, Lee JE, Stewart JR, Olsen KD (2006) (CTM2013-48163) of the Spanish Government. Role of two insect growth regulators in integrated pest management of citrus scales. J Econ Entomol 99:733–744 Grafton-Cardwell E, Ouyang Y, Striggow R, Vehrs S (2008) Armored scale insecticide resistance challenges San Joa- References quin Valley citrus growers. Calif Agric 55:20–25 Hardy NB (2008) Systematic studies of scale insects (Hemi- Abd-Rabou S, Shalaby H, Germain J, Ris N, Kreiter P, Malausa ptera: Coccoidea). University of California, Davis, USA T (2012) Identification of mealybug pest species (Hemi- Hebert PDN, Cywinska A, Ball SL, DeWaard JR (2003) Bio- ptera: Pseudococcidae) in Egypt and France, using a DNA logical identifications through DNA barcodes. Proc Biol barcoding approach. Bull Entomol Res 102:1–9 Sci 270:313–321 Agreste (2015) Agreste, statistique agricole annuelle. Retrieved Heraty J, Woolley J, Polaszek A (2007) Catalog of the Encarsia from http://www.agreste.agriculture.gouv.fr/enquetes/statis of the World (2007). http://cache.ucr.edu/*heraty/ tique-agricole-annuelle-saa/ Encarsia.cat.pdf Babcock CS, Heraty JM (2000) Molecular markers distin- Kattari D, Heimpel GE, Ode P, Rosenheim J (1999) Hyper- guishing Encarsia formosa and Encarsia luteola (Hy- parasitism by Ablerus clisiocampae Ashmead (Hy- menoptera: Aphelinidae). Ann Entomol Soc Am menoptera: Aphelinidae). Proc Entomol Soc Wash 93:738–744 101:640–644 Beardsley JW, Gonzalez RH (1975) The biology and ecology of Liang W, Meats A, Beattie GAC, Spooner-Hart R, Jiang L armored scales. Annu Rev Entomol 20:47–73 (2010) Conservation of natural enemy fauna in citrus Beltra` A, Addison P, A´ valos JA, Crochard D, Garcia-Marı´ F, canopies by horticultural mineral oil: comparison with Guerrieri E, Giliomee JH, Malausa T, Navarro-Campos C, effects of carbaryl and methidathion treatments for control Palero F, Soto A (2015) Guiding classical biological con- of armored scales. Insect Sci 17:414–426 trol of an invasive mealybug using integrative taxonomy. Malausa JC, Rabasse JM, Kreiter P (2008) Les insectes ento- PLoS ONE 10(6):e0128685 mophages d’interet agricole acclimates en France Ben-Dov Y, Miller DR, Gibson GAP (2010) Scale Net. metropolitaine depuis le debut du 20e`me siecle. Bull Retrieved from http://scalenet.info/ OEPP/EPPO 38:136–146

123 Molecular characterization of parasitoids from armored scales infesting citrus orchards… 647

Malausa T, Fenis A, Warot S, Germain JF, Ris N, Prado E, Tison G, Kreiter P, Giuge L, Thaon M, Jeanne Y, Lemay V, Botton M, Vanlerberghe-Masutti F, Sforza R, Cruaud C, Daoux F, Be´naouf G, Balajas J, Duval X, Borelli JG (2007) Couloux A, Kreiter P (2011) DNA markers to disentangle Pou rouge de Californie et agrumiculture corse. Phytoma- complexes of cryptic taxa in mealybugs (Hemiptera: La De´fense des ve´ge´taux 606:18–21 Pseudococcidae). J Appl Entomol 135:142–155 Zaldı´var-Rivero´n A, Martı´nez JJ, Ceccarelli FS, De Jesu´s- Monti MM, Nappo AG, Giorgini M (2005) Molecular charac- Bonilla VS, Rodrı´guez-Pe´rez AC, Rese´ndiz-Flores A, terization of closely related species in the parasitic genus Smith MA (2010) DNA barcoding a highly diverse group Encarsia (Hymenoptera: Aphelinidae) based on the mito- of parasitoid wasps (Braconidae: Doryctinae) from a chondrial cytochrome oxidase subunit I gene. Bull Ento- Mexican nature reserve. Mitochondrial DNA 21(Suppl mol Res 95:401–408 1):18–23 Munro JB, Heraty JM, Burks R, Hawks D, Mottern J, Cruaud A, Rasplus JY, Jansta P (2011) A molecular phylogeny of the Chalcidoidea (Hymenoptera). PLoS ONE 6(11):e27023 Margarita Correa applies molecular ecology for biological Murdoch WW, Swarbrick SL, Briggs CJ (2006) Biological control. control: lessons from a study of California Red Scale. Popul Ecol 48:297–305 Ferran Palero has specialized in DNA analyses and phylo- Pina T, Verdu´ MJ, Urbaneja A, Sabater-Mun˜oz B (2012) The genetic methods use of integrative taxonomy in determining species limits in the convergent coloration pattern of Aphytis spe- Noe´mie Dubreuil is specialized in biological control and cies. Biol Control 61:64–70 methods for rearing natural enemies. Rosen D (1994) Advances in the study of Aphytis (Hy- menoptera: Aphelinidae). Intercept Limited, Andover Laure Etienne is specialized in biological control and methods Rosen D, DeBach P (1979) Species of Aphytis of the world. for rearing natural enemies. Springer, Netherlands Rugman-Jones PF, Forster LD, Guerrieri E, Luck RF, Morse JG, Mathieu Hulak is specialized in biological control and Monti MM, Stouthamer R (2011) Taxon-specific multi- methods for rearing natural enemies. plex-PCR for quick, easy, and accurate identification of encyrtid and aphelinid parasitoid species attacking soft scale insects in California citrus groves. BioControl Gilles Tison is interested in biology, ecology and spatio- 56:265–275 temporal population dynamics of native and non-native pests Schmidt S, Polaszek A (2007) Encarsia or Encarsiella?—re- and their natural enemies. defining generic limits based on morphological and molecular evidence (Hymenoptera, Aphelinidae). Syst Sylvie Warot is specialized in molecular techniques and DNA Entomol 32:81–94 barcoding methods for biological control. Sorribas J, Garcia-Marı´ F (2010) Comparative efficacy of dif- ferent combinations of natural enemies for the biological Didier Crochard is specialized in molecular techniques and control of California Red Scale in citrus groves. Biol DNA barcoding methods for biological control. Control 55:42–48 Tamura K, Nei M, Kumar S (2004) Prospects for inferring very Nicolas Ris is interested in biology, ecology and spatio- large phylogenies by using the neighbor-joining method. temporal population dynamics of native and non-native pests Proc Natl Acad Sci USA 101:11030–11035 and their natural enemies. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: molecular evolutionary genetics analysis version Philippe Kreiter is interested in biology, ecology and spatio- 6.0. Mol Biol Evol 30:2725–2729 temporal population dynamics of native and non-native pests Tena A, Garcia-Marı´ F (2011) Current situation of citrus pests and their natural enemies. and diseases in the Mediterranean basin. IOBC/WPRS Bull 62:365–378

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