Development of Microsatellite Markers and Detection of Genetic Variation between Goniozus Wasp Populations Author(s): Sahand K. Khidr, Ian C.W. Hardy, Tania Zaviezo and Sean Mayes Source: Journal of Insect Science, 14(43):1-17. 2014. Published By: Entomological Society of America DOI: http://dx.doi.org/10.1673/031.014.43 URL: http://www.bioone.org/doi/full/10.1673/031.014.43 BioOne (www.bioone.org) is a nonprofit, online aggregation of core research in the biological, ecological, and environmental sciences. BioOne provides a sustainable online platform for over 170 journals and books published by nonprofit societies, associations, museums, institutions, and presses. Your use of this PDF, the BioOne Web site, and all posted and associated content indicates your acceptance of BioOne’s Terms of Use, available at www.bioone.org/page/terms_of_use. Usage of BioOne content is strictly limited to personal, educational, and non-commercial use. Commercial inquiries or rights and permissions requests should be directed to the individual publisher as copyright holder. BioOne sees sustainable scholarly publishing as an inherently collaborative enterprise connecting authors, nonprofit publishers, academic institutions, research libraries, and research funders in the common goal of maximizing access to critical research. Journal of Insect Science: Vol. 14 | Article 43 Khidr et al. Development of microsatellite markers and detection of genetic variation between Goniozus wasp populations Sahand K. Khidr1a, Ian C.W. Hardy1b*, Tania Zaviezo2c, and Sean Mayes1d 1School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough, LE12, 5RD, UK 2Departamento de Fruticultura y Enología, Facultad de Agronomía e Ingeniería Forestal, Pontificia Universidad Católica de Chile, Casilla 306 – 22, Santiago, Chile Abstract Molecular genetic markers reveal differences between genotypes according to the presence of alleles (the same or different) at target loci. Microsatellite markers are especially useful co- dominant markers that have been used in a wide range of studies to elucidate the population structure and dynamics of a range of organisms, including agriculturally beneficial insects such as parasitic wasps (parasitoids). In the present study, twelve primer pairs were designed for the south Asian , Goniozus nephantidis (Muesebeck) (Hymenoptera: Bethylidae), and 24 for its New World congener, Goniozus legneri Gordh, parasitoids of the larvae of the lepidopteran coconut pest Opisina arenosella Walker (Lepidoptera: Crytophasidae) and other lepidopteran pests, re- spectively, in order to investigate polymorphism within and between populations. The wasps fingerprinted were a total of 85 G. nephantidis and G. legneri, including individuals belonging to three putatively different strains of G. legneri. Annealing gradient tests (50-65°C) were conduct- ed to study the quality of the PCR amplification across an annealing temperature gradient using a mixed genotype DNA template from each species separately. Seven primer pairs, which ampli- fied clear products of approximately the expected size of G. nephantidis and 18 of G. legneri, were then selected for capillary analysis for fragment size determination on a Beckmann CEQ 8000. Neither G. nephantidis nor G. legneri were polymorphic within populations. However, there were six primer pairs that did show polymorphism between G. legneri populations that orig- inated from different geographical areas within South America (Uruguay and Chile). Furthermore, one primer pair revealed diversity between the two strains collected within Chile. One of the markers was subsequently used to provide unbiased assessment of primary sex ratio in G. legneri. Abbreviations: SSR, simple sequence repeat Correspondence: a [email protected], b [email protected], c [email protected], d [email protected], *Corresponding author Editor: Henry Hagedorn was editor of this paper. Received: 23 June 2012 Accepted: 14 March 2013 Published: 20 March 2014 Copyright: This is an open access paper. We use the Creative Commons Attribution 3.0 license that permits unrestricted use, pro- vided that the paper is properly attributed. ISSN: 1536-2442 | Vol. 14, Number 43 Cite this paper as: Khidr SK, Hardy ICW, Zaviezo T, Mayes S. 2014. Development of microsatellite markers and detection of genetic variation between Goniozus wasp populations. Journal of Insect Science 14:43. Available online: http://www.insectscience.org/14.43 Journal of Insect Science | http://www.insectscience.org 1 Journal of Insect Science: Vol. 14 | Article 43 Khidr et al. Introduction number of alleles per locus, which can identi- fy polymorphism, but they can detect high Among the natural enemies of agricultural levels of heterozygosity and have high muta- insect pests, hymenopteran parasitoids are one tion rates (Hancock 1999). As such, of the most important classes of biological microsatellite markers have been used to de- control agents and are also widely used in termine the genetic diversity and studies of evolutionary ecology and basic differentiation between populations through population biology (Godfray 1994; Jervis measuring the degree of heterozygosity in 2005; Hochberg and Ives 2000; Wajnberg et parasitoid species (e.g. 0.04–0.44 in Cotesia al. 2008; Hardy et al. 2013). The distributions melitaearum, 0.171–0.629 in Neotypus mela- and population structures of parasitoids are nocephalus, and 0.170–0.367 in Lysiphlebus influenced by a wide range of factors, such as hirticornis; Kankare et al. 2005; Anton et al. geological and geographical components, eco- 2007; Nyabuga et al. 2010) as well as the de- logical processes, and evolutionary and gree of gene flow and dispersal between genetic aspects (Zink 2002; Bond and Stock- populations (Avise 1994; McCoy et al. 2001; man 2008). Successful population genetic, Molbo et al. 2003; Kankare et al. 2005; Za- ecological, and evolutionary studies can be vodna et al. 2005; Drescher et al. 2010; achieved through the availability of suitable Nyabuga et al. 2010). molecular markers, which are important indi- cators of relationships between both In the present study, we designed a suite of individuals and populations (Carvalho 1998). microsatellites for screening two species of Such markers can reveal differences between bethylid wasps for genetic polymorphisms genotypes through the application of a range within and between populations. In principle of random markers not linked a priori to such markers could also prove useful for pest traits. control applications (Aebi et al. 2008; Ugelvig et al. 2008; Lozier et al. 2009; Zygouridis et Among the classes of widely used genetic al. 2009; Nicholls et al. 2010; Lavandero et al. markers are ‘microsatellite’ markers, also 2011), evaluating the effect of kinship on so- known as simple sequence repeats (SSRs). cial behaviors (Lizé et al. 2012), and for These consist of short, repeated units of measuring population parameters, such as lev- around two to six base pairs in length with an els of inbreeding, which have not been array that can be up to 200 bp long and are directly evaluated but are important in the un- found in both coding and non-coding regions derstanding of reproductive decisions (Hardy in all prokaryotic and eukaryotic genomes and Cook 1995; Hardy et al. 1998, 1999, (Tautz 1989; Arcot et al. 1995; Beukeboom 2000). The first direct application of these and Zwaan 2005). To date, they have been markers has been to provide assessment, as developed in a number of parasitoids, espe- described elsewhere (Khidr et al. 2013), of the cially braconids (Baker et al. 2003; Anton et sex of individual parasitoid eggs in order to al. 2006; Lozier et al. 2006). evaluate maternal sex allocation without the biasing influence of developmental mortality. Microsatellite markers, which are co- dominant (Loxdale and Lushai 1998), have The Bethylidae is a family of parasitoid hy- many advantages over other marker types be- menopteran wasps that has been thought to cause not only do they generally have a high comprise four extant subfamilies, Bethylinae, Journal of Insect Science | http://www.insectscience.org 2 Journal of Insect Science: Vol. 14 | Article 43 Khidr et al. Epyrinae, Pristocerinae, and Mestitiinae (Ev- capacity to inseminate their sisters (Hardy et ans 1964), with over 2000 described species al. 1999, 2000), and, as with many other (Gordh and Móczár 1990). Recently, the bethylids, the sex ratios of these species are higher level phylogeny of bethylids has been generally female biased with a low degree of estimated using molecular data from 33 spe- sex ratio variation between broods (sub- cies, resulting in a split of the sub-family binomial variance: Green et al. 1982; Hardy Mestitiinae into two separate sub-families, the and Mayhew 1998; Hardy et al. 1998; Khidr Mestitiinae and the Cephalonomiini (Carr et et al. 2013). al. 2010). Bethylid wasps attack almost exclu- sively the immature stages of coleopterans Materials and methods and lepidopterans, many of which are pests of important agricultural commodities such as Parasitoid origins and cultures coffee, coconut, sugarcane, apple, walnut, and The culture of G. nephantidis used in this almonds (Gordh 1982; Batchelor at al. 2005; study had been maintained in the laboratory Venkatesan et al. 2007; Zaviezo et al. 2007). for more than 20 years on the facultative host In this study, we have focused on Goniozus Corcyra cephalonica Stainton (Lepidoptera: nephantidis (Muesebeck) (Hymenoptera: Pyralidae), as