Molecular characterization of Iranian Trichogrammatids (Hymenoptera: Trichogrammatidae) and their Wolbachia endosymbiont

Journal of Asia-Pacific Entomology 15 (2012) 73–77 Contents lists available at SciVerse ScienceDirect Journal of Asia-Pacific Entomology journal homepage: www.elsevier.com/locate/jape Molecular characterization of Iranian Trichogrammatids (Hymenoptera: Trichogrammatidae) and their Wolbachia endosymbiont Javad Karimi a,⁎, Reyhaneh Darsouei a, Mojtaba Hosseini a, Richard Stouthamer b a b Department of Plant Protection, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran Department of Entomology, University of California, Riverside, CA 92521, USA a r t i c l e i n f o Article history: Received 19 May 2011 Revised 12 August 2011 Accepted 16 August 2011 Keywords: Iran ITS2 Sequencing Trichogramma Wolbachia wsp a b s t r a c t During 2009–2010, a field survey of native Trichogramma species was carried out in six provinces of Iran, including Khorasan Razavi, Tehran, Mazandaran, Guilan, Golestan, and Qom. In this study, a molecular method for identifying Trichogramma and for determining the prevalence of Wolbachia in those species was used. Based on ITS2 (internal transcribed spacer 2) sequence, 14 populations were identified as the species T. embryophagum, T. evanescens, or T. brassicae. Wolbachia infection in these Trichogrammatids was detected using wsp gene sequencing. The highest infection rates in Trichogramma were found in Mazandaran and Golestan provinces. There was no evidence of infection in Trichogramma species in Guilan and Qom provinces. Of the three infected populations, two populations of T. evanescens were infected with only one Wolbachia strain from sib subgroup and one population was superinfected. Here, we report the first data on molecular characterization of Iranian Trichogrammatids and their Wolbachia-endosymbionts. © Korean Society of Applied Entomology, Taiwan Entomological Society and Malaysian Plant Protection Society, 2011. Published by Elsevier B.V. All rights reserved. Introduction The parasitoid wasp family Trichogrammatidae consists of approximately 80 genera and 620 species worldwide (Pinto and Stouthamer, 1994). In the past, identification of Trichogramma was based on morphological parameters or characters such as body color, chaetotaxy, and, more recently, male genitalia (Pinto et al., 1989; Pinto, 1999; Stouthamer et al., 1999b). Modern approaches to identification include molecular tools. Precise identification of Trichogramma species is an important step for the use of these parasitoids in biological control programs against several lepidopterous pests. In particular, the correct identification of thelytokous isolates of Trichogramma is critical in biocontrol programs which use these agents (Pinto et al., 1997). For morphological identification, males need to be reared but they are often produced only at exceedingly low frequencies. Incorrect identification may lead to release of unsuitable species, resulting in subsequent failure of biocontrol (Stouthamer et al., 2000). In some cases, the release of the wrong species in an area where another closely related species is present, can lead to a long time suppression of both native and introduced species in biological control programs (Stouthamer et al., 2000). Given the economic importance of Trichogramma species as biocontrol agents of pests, especially of Lepidoptera (Hassan, 1988), simple, quick and widely applicable identification methods need to be developed. Novel approaches that use the DNA sequence of the internal ⁎ Corresponding author. E-mail address: [email protected] (J. Karimi). transcribed spacer 2 (ITS2) have provided a tool (Stouthamer et al., 1999b) that successfully distinguished closely related Trichogramma species. DNA sequences of the ITS2 of the ribosomal cistron are very useful for taxonomy (Agudelo-Silva, 1993; Hoy, 1994; Orrego and Pinto et al., 1997). In recent years, several molecular keys have been developed using ITS2 sequences of different Trichogramma species. Kumar et al. (2009) produced a molecular key for Trichogramma species in India using ITS2 PCR followed by restriction digest of the PCR products. Similarly, molecular keys have been developed using ITS2 sequences of Trichogramma species in Brazil (Ciociola et al., 2001) and in the Mediterranean area (Sumer et al., 2009). Wolbachia is an intracellular bacteria found in many species of arthropods. It manipulates the reproduction of their hosts in several ways, including induction of complete parthenogenesis (thelytoky) in several parasitoid wasp species and nematodes. It is estimated that at least 20% of all insect species are infected with Wolbachia (Werren and Windsor, 2000). The manipulation of the host's reproduction may play a role in host speciation and has potential applications in biological pest control (Stouthamer et al., 1999a). In Trichogramma, parthenogenesisinducing Wolbachia occur in ca. 9% of species. This bacterium may have positive, negative, or neutral effects on the biological-specific traits of Trichogramma (Stouthamer and Luck, 1993; Stouthamer et al., 1994; Varve et al., 1999; Tagami et al., 2002). According to Stouthamer (1993), potential advantages for biological control of wasps infected with parthenogenesis-inducing Wolbachia are: (a) production of no males, which increases population growth rate of the natural enemy, (b) decrease in the cost of mass rearing because only females are 1226-8615/$ – see front matter © Korean Society of Applied Entomology, Taiwan Entomological Society and Malaysian Plant Protection Society, 2011. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.aspen.2011.08.004 74 J. Karimi et al. / Journal of Asia-Pacific Entomology 15 (2012) 73–77 produced, (c), colonization is faster because thelytokous females do not have to waste time in searching for mates at low wasp population densities, and (d) depression of host populations to low level. These advantages encouraged us to look for Wolbachia in Trichogramma populations. This information will be useful in biocontrol programs because selection of the best Trichogramma species/strain for inundative releases is a key first step in a biological control plan. There is little information about Wolbachia and its prevalence in parasitic wasps like Trichogramma in Iran. Recently, Farrokhi et al. (2010) reported on the performance of PI-Wolbachia infected and uninfected T. brassicae collected from Northern Iran. All other studies on Iranian Wolbachia are limited to medically important insects, such as Phlebotomus papatasi and Culex pipiens (Parvizi et al., 2010). The purposes of our study were: identification of Iranian Trichogramma species from different climate zones of Iran using ITS2 sequence and determination of Wolbachia prevalence and phylogenetic status associated with the collected Trichogramma species. Material and method Sample collection Trichogramma were collected from parasitized Lepidopteran eggs in Khorasan Razavi, Tehran, Mazandaran, Golestan, Guilan, and Qom provinces of Iran during 2009 and 2010 (Fig. 1). The samples were from different hosts. For each parasitized egg, a single (mated or virgin) female was allowed to establish an isofemale line. These isofemale lines were used in our study. Specimens from these isofemale lines were used for morphological identification and voucher specimens were placed in the insect collection of Ferdowsi University of Mashhad, Iran. Identification of Trichogramma using sequencing of ITS2 DNA was extracted using a Bioneer kit (Bioneer Co. Daejeon, Korea). For each isofemale line, a sample of ten wasps kept at −20 °C for at least 12 h, then crushed using a micropestle in 200 μl lysis buffer and 20 μl proteinase K. The homogenate was incubated at 60 °C for 4 h, followed by 10 min at 95 °C. The supernatant was stored at −20 °C until use. The PCR reaction was performed using an Eppendorf thermocycler in 50 μl reaction volumes containing 2 μl DNA template, 5 μl Taq assay buffer, 1 μl dNTP's (each in 10 mM concentration), 1 μl forward and reverse primers (10 picomoles/μl), and 0.25 μl Taq polymerase (1 U). The primers used to amplify the ITS-2 region were 5′-TGTGAACTG CAGGACACATG-3′ (forward) and 5′-GTCTTGCCTGCTCTGAG-3′ (reverse). The ITS2 spacer was then amplified using PCR primers and the conditions described in Stouthamer et al. (1999b). PCR product was electrophoresed on 0.8% agarose gels along with a size ladder. Gels were stained using ethidium bromide. Fig. 1. Geographical distribution of sampling locations in different provinces of Iran. 75 J. Karimi et al. / Journal of Asia-Pacific Entomology 15 (2012) 73–77 Wolbachia detection We screened for the presence and identity of Wolbachia in different Trichogramma populations using the wsp gene. In addition, each of the isofemale lines sequenced for ITS2 was also screened for Wolbachia. The PCR reaction was performed in 25 μl reaction volumes using an Eppendorf thermocycler. Each reaction mixture contained 1 μl DNA template, 2.5 μl (10×) buffer, 0.75 μl MgCl2, 0.5 μl dNTPs, 0.5 μl forward and reverse primer (10 picomoles/μl), and 0.3 μl Taq polymerase (5 U). We used the wsp-forward (5′-TGGTCCAATAAGTGATGAAGAAAC-3′) and wsp-reverse primer (5′-AAAAATTAAA CGCTACTCCA-3′), as previously reported (Braig et al., 1998). The temperature profile consisted of an initial denaturation step at 94 °C for 30 s followed by 36 cycles (denaturation at 94 °C for 30 s, annealing at 50 °C for 45 s, and extension at 72 °C for 60 s), with a final extension at 72 °C for 5 min. For each DNA extraction, three control extractions were performed using a Drosophila melanogaster Wolbachia-positive line, a Trichogramma Wolbachia-negative line, and a non-DNA sample. After amplification, PCR products were purified and sequenced directly using standard Fluorescent cycle-sequencing by MilleGen Co. (France). Chromatograms were checked visually and sequences were aligned manually using BioEdit software (Hall, 1999). The resulting sense and antisense sequences were edited and used in alignment. Representative sequences for all known Wolbachia groups were retrieved from GenBank and included in phylogeny reconstruction. Sequences were aligned using the default settings of Clustal W (Thompson et al., 1994). Unweighted parsimony analysis of the alignments was conducted with PAUP*4.0b2 (Swofford, 1999). Gaps were treated as missing characters for the analyses and the reliability of trees was tested with a bootstrap test (Felsenstein, 1985). Parsimony bootstrap analysis included 1000 resamplings using the Branch and Bound algorithm. The most appropriate model of sequence evolution was determined using Hierarchical Likelihood Ratio Tests (hLRTs) in the program jModelTest 0.1.1 (Posada, 2008). Number of trees held at each step during stepwise addition was 1 and branch-swapping algorithm was tree-bisectionreconnection (TBR). Analysis was implemented into PAUP* for a 1000 replicate random addition heuristic search. Result and discussion The complete ITS2 gene sequence with portions of the flanking 5.8S and 28S rDNA genes was successfully sequenced for 14 isolates. The boundaries of the ITS2 were determined using the conserved sequence of the flanking regions by comparison with the sequence of Trichogramma. Three species of Trichogramma were identified based on the ITS2 sequences. ITS2 gene sequences were deposited in GenBank (Table 1). T. brassicae was the dominant species in our collection and 9 isofemale lines were identified as this species. Four and two isofemale lines of Trichogramma were identified as T. brassicae from Tehran and Qom provinces, respectively. Moreover, T. brassicae was commonly found in the Northern part of Iran, South of the Caspian Sea (Table 1). This species is commonly used as biocontrol agent against some key insect pests, including the European corn borer, Ostrinia nubilalis, the carob moth, Ectomyelois ceratoniae, and the rice stem borer, Chilo suppressalis (Ebrahimi et al., 1998). Ebrahimi et al. (1998) reported that T. brassicae as the most widespread Trichogramma species in Iran. The Iran 13 sample was deposited with accession number HM063427. It is typical for the T. embryophagum that is found in Iran. T. embryophagum was the least common species found in our study. This species is reared in an insectary located in the agricultural research center of Khorasan Razavi province (Mashhad, Torogh) in Northeastern Iran. Its sequence is identical to the Genebank accession number EU547670. Here, this species was named T. embryophagum and not T. cacoeciae because males are present in the culture. T. cacoeciae consists of only females and males are extremely rare (Pinto, 1999), while Table 1 Trichogramma samples used in this study, their geographic origin, GenBank accession numbers for their ITS2 sequences, and presence of Wolbachia. Assign Source Province G3 G7 G11 M2 M3 T2 T3 T4 T5 T.e Accession no. Isolate Wolbachia Mazandaran Guilan Mazandaran Qom Qom Tehran Tehran Tehran Tehran Khorasan HQ343301 HQ332598 HQ335390 HQ143679 HQ143680 HQ143675 HQ143676 HQ143678 HQ143677 HM063427 Iran10 Iran15 Iran7 Iran25 Iran23 Iran2 Iran3 Iran4 Iran5 Iran13 G5 G8 G10 T. brassicae T. brassicae T. brassicae T. brassicae T. brassicae T. brassicae T. brassicae T. brassicae T. brassicae T. embryophagum Behshahr Mazandaran T. evanescens Gorgan Golestan T. evanescens Gorgan Golestan T. evanescens M1 Qom HQ162663 Amol Langerud Amol Qom Qom Tehran Tehran Tehran Tehran Mashhad Qom Species T. evanescens HQ335391 Iran8 HQ332599 Iran6 HM214958 Iran20 Iran1 – – + FUM5 – – – – + FUM2 – + FUM7 + FUM1 + FUM3 +FUM4 and +FUM6 – T. embryophagum may have males. The thelytokous status of a species carrying the Wolbachia symbiont can make identification impossible because males are lacking. In such cases, antibiotic and heating treatments can eliminate the symbiont which may result in the production of males. Based on cytogenetic mechanisms, thelytoky in Trichogramma cacoeciae is different from other species such as T. embryophagum (Stouthamer et al., 1990; Vavre et al., 2004). T. cacoeciae exhibits complete parthenogenesis and males are absent in natural populations. Pinto (1999) observed only five males out of approximately 15,000 individuals under laboratory conditions. Males cannot be induced by antibiotic or heat treatment, and neither Wolbachia nor other symbionts have ever been found (Vavre et al., 2004). The wsp sequences can be accessed on GenBank under the numbers shown in Table 1. Amplification of wsp gene showed that seven Trichogramma populations (two populations of T. brassicae, four populations of T. evanescens, and one population of T. embryophagum) were infected with Wolbachia. The Wolbachia were characterized and named FUM1 to FUM7. Four Wolbachia strains, FUM1, FUM3, FUM5, and FUM7, belonged to the supergroup B and subgroup Sib (van Meer et al., 1999). The other strains, FUM2, FUM 4 and FUM6, belonged to supergroup A. Supergroup A and supergroup B of Wolbachia were found in different isofemale lines of T. evanescens. In addition, supergroup A Wolbachia were found in two different isofemale lines of T. brassicae. Both single infection and superinfection existed within Trichogramma. The strong similarity between the three FUM strains and Wolbachia in other hosts suggests that these strains have been transmitted horizontally between hosts. We aligned our wsp sequences with 45 Wolbachia wsp sequences in the GenBank. Fig. 2 shows the tree after bootstrapping 1000 times. The molecular methods applied in this study give quick results of Trichogramma identifications and can be used to survey laboratory colonies for contamination. The phylogeny of Wolbachia has been studied using number of different genes, i.e. 16S, 23S, ftsZ, SR2, and the wsp gene (Braig et al., 1998). Phylogenetic studies have led to the subdivision of the Wolbachia clade into 11 supergroups (Ros et al., 2008). Wolbachia from Trichogramma that induce parthenogenesis belong to supergroup B. Thelytokous populations of Trichogramma resulting from Wolbachia infection have different life-history characters than uninfected conspecifics. Consequently, finding Wolbachia infections and determining its effect on the host behavior could be useful. Characterization of the sib subgroup of Wolbachia was reported by van Meer et al. (1999) and Pintureau et al. (2002). It was later confirmed by de Almeida (2004). Supergroup B Wolbachia infection is the cause of thelytoky in at least 17 out of 190 described species of Trichogramma (de Almeida, 2004). 76 J. Karimi et al. / Journal of Asia-Pacific Entomology 15 (2012) 73–77 Fig. 2. Phylogenetic tree of wsp sequences of the Wolbachia strains from Trichogramma and closely related Wolbachia strains from other insects. Bootstrap values are given as percentage 1000 replicates. In this work, we attempted to detect Wolbachia strains in native Trichogramma populations in Iran. The phylogenetic relationship among all published strains of Wolbachia related to Trichogramma and some other insect groups throughout the world is analyzed based on wsp gene sequence. This data about Wolbachia and the ITS2-based identification of related Trichogramma are new for Iran. More analysis is necessary due to concerns about relationships of Wolbachia strains based on wsp gene. The wsp sequence is a common marker for Wolbachia detection and provides many more informative characters with which to determine evolutionary relationships between strains (Braig et al., 1998; Zhou et al., 1998). Screening of Wolbachia using the wsp gene is a preliminary approach. However, single-locus phylogeny of Wolbachia may be J. Karimi et al. / Journal of Asia-Pacific Entomology 15 (2012) 73–77 questionable because strains with similar wsp sequences often have different allelic profiles (Baldo et al., 2006). The wsp gene is divided into four hypervariable regions (HVRs) (Werren et al., 2008). They have high polymorphism (Baldo et al., 2006) and high levels of recombination have been observed in WSP and throughout Wolbachia genomes (Werren and Bartos, 2001; Baldo et al., 2006). These characters may not correctly show the true evolutionary and demographic histories of Wolbachia strains (Baldo et al., 2006). A multilocus sequence typing (MLST) is a new method which had recently been proposed for Wolbachia characterization. This approach may overcome the recombination concern and offer more data for comprehensive analyses (Baldo et al., 2006). Characterization of five housekeeping genes, gatB, coxA, hcpA, ftsZ and fbpA, is the principle data for MLST system. Therefore, the next step toward understanding the phylogeny of Wolbachia strains should be the analysis across a broader range of hosts, using sensitive methods for strain typing such as MLST, to determine precise relationships and endosymbionts. Understanding Wolbachia biodiversity could be of major importance in improving biological control using Trichogramma. For example, T. brassicae, which was released against the European corn borer, O. nubilalis, normally has a sexual mode of reproduction. A Wolbachia transfer inducing thelytokous reproduction in T. brassicae could increase the performance of parasitoids by producing only females (Pintureau et al., 2002). Low prevalence of Wolbachia may have been missed given the number of samples analyzed. Therefore, the probability of finding Wolbachia infection depends on the number of samples analyzed. Generally, information about Wolbachia infection rate may be questionable because: (1) not all populations of a species may be infected, and (2) infected and uninfected individuals usually coexist in a single population (Cook and Butcher, 1999). Data analyzed here are restricted to one part of the country. By increasing the number of samples when looking for Wolbachia, we can get a much better understanding of infection frequency in different populations. Acknowledgments We would like to express thanks to the Research Council of Ferdowsi University of Mashhad for the financial support of this study (grant 302 no. 14906). We thank everyone who helped us in this project. We especially thank those that helped us obtain samples: Sh. Farrokhi, M. R. Rezapanah, and J. Shirazi from Iranian Research Institute of Plant Protection and M. Tabari from the Iranian Rice Research Institute. The first author thanks R P de Almeida for his help and K. Minaei and M. Naderpour for their comments about the manuscript. References Baldo, L., Dunning Hotopp, J.C., Jolley, K.A., Bordenstein, S.R., Biber, S.A., Choudhury, R.R., Hayashi, C., Maiden, M.C.J., Tettelin, H., Werren, J.J., 2006. Multilocus sequence typing system for the endosymbiont Wolbachia pipientis. Appl. Environ. Microbiol. 72, 7098–7110. Braig, H.R., Zhou, W., Dobson, S.L., O' Neill, S.L., 1998. Cloning and characterization of a gene encoding the major surface protein of the bacterial endosymbiont Wolbachia pipientis. J. Bacteriol. 180, 2373–2378. Ciociola, J.R.A.I., Zucchi, R.A., Stouthamer, R., 2001. Molecular key to seven Brazilian species of Trichogramma (Hymenoptera: Trichogrammatidae) using sequences of the ITS2 region and restriction analysis. Neotrop. Entomol. 30, 259–262. Cook, J.M., Butcher, R.D.J., 1999. The transmission and effects of Wolbachia bacteria in parasitoids. Res. Popul. Ecol. 41, 15–28. de Almeida, R. P., 2004. Trichogramma and its relationship with Wolbachia: identification of Trichogramma species, phylogeny, transfer and costs of Wolbachia endosymbionts. PhD dissertation, Wageningen university, The Netherlands. Ebrahimi, E., Pintureau, B., Shojai, M., 1998. Morphological and enzymatic study of the genus Trichogramma in Iran. Appl. Entomol. Phytopathol. 66, 39–43. 77 Farrokhi, S., Ashouri, A., Shirazi, J., Allahyari, H., Huigens, M.E., 2010. A comparative study on the functional response of Wolbachia-infected and uninfected forms of the parasitoid wasp Trichogramma brassicae. J. Insect Sci. 10, 1–11. Felsenstein, J., 1985. Confidence intervals on phylogenies: an approach using the bootstrap. Evolution 39, 783–791. Hall, T.A., 1999. Bioedit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acid S. J. 41, 95–98. Hassan, S.A., 1988. Choice of the suitable Trichogramma species to control the European corn borer Ostrinia nubilalis Hbn. and the cotton bollworm Heliothis armigera Hbn: Colloques de l'INRA, 43, pp. 197–198. Hoy, M.A., 1994. Insect Molecular Genetics: An Introduction to Principles and Applications. Acad. Inc., San Diego, California. 546 pp. Kumar, G.A., Jalali, S.K., Venkatesan, T., Stouthamer, R., Niranjana, P., Lalitha, Y., 2009. Internal transcribed spacer-2 restriction fragment length polymorphism (ITS2-RFLP) tool to differentiate some exotic and indigenous trichogrammatid egg parasitoids in India. Biocontrol 49, 207–213. Orrego, C., Agudelo-Silva, F., 1993. Genetic variation in the parasitoid wasp Trichogramma (Hymenoptera: Trichogrammatidae) revealed by DNA amplification of a section of the nuclear ribosomal repeat. Fla. Entomol. 76, 519–524. Parvizi, P., Fardid, F., Amirkhani, A., 2010. Isolation process of two genes of wsp and 16S rRNA the intercellular bacteria, Wolbachia pipientis in Phelobotomus papatasi sandflea vector of zoonotic cutaneous leishmaniasis in Iran. Iran J. Med. Microbiol. 3, 53–60. Pinto, J.D., 1999. The systematics of the North American species of Trichogramma. Mem. Entomol. Soc. Wash. DC. 22, 287pp. Pinto, J.D., Stouthamer, R., 1994. Systematics of the Trichogrammatidae with emphasis on Trichogrammamm. In: Wajnberg, E., Hassan, S.A. (Eds.), Biocontrol with Egg Parasitoids. CAB International, pp. 1–36. Pinto, J.D., Velten, R.K., Platner, G.R., Oatman, E.R., 1989. Phenotypic plasticity and taxonomic characters in Trichogramma. Ann. Entomol. Soc. Am. 85, 413–422. Pinto, J.D., Stouthamer, R., Platner, G.R., 1997. A new cryptic species of Trichogramma (Hymenoptera: Trichogrammatidae) from the Mojave desert of California as determined by morphological, reproductive and molecular data. Proc. Entomol. Soc. Wash. 99, 238–247. Pintureau, B., Grenier, S., Heddi, A., Charles, H., 2002. Biodiversity of Wolbachia and of their effects in Trichogramma (Hymenoptera: Trichogrammatidae). Ann. Soc. Entomol. Fr. 38, 333–338. Posada, D., 2008. jModelTest: phylogenetic model averaging. Mol. Biol. Evol. 25, 1253–1256. Ros, V.D., Fleming, V.M., Feil, E.J., Breeuwer, J.A.J., 2008. How diverse is the genus Wolbachia? Multiple-gene sequencing reveals a putatively new Wolbachia supergroup recovered from spider mites (Acari: Tetranychidae). Appl. Environ. Microbiol. 54, 1036–1043. Stouthamer, R., 1993. The use of sexual versus asexual wasps in biological control. Entomophaga 38, 3–6. Stouthamer, R., Luck, R.F., 1993. Influence of microbe-associated parthenogenesis on the fecundity of Trichogramma deion and T. pretiosum. Entomol. Exp. Appl. 67, 183–192. Stouthamer, R., Luck, R.F., Hamilton, W.D., 1990. Antibiotics cause parthenogenetic Trichog ramma (Hymenoptera: Trichogrammatidae) to revert to sex. Proc. Natl. Acad. Sci. U. S. A. 87, 2424–2427. Stouthamer, R., Luko, S., Mak, F., 1994. Influence of parthenogenesis Wolbachia on host fitness. Norweg. J. Agric. Sci. Suppl. 16, 117–122. Stouthamer, R., Breeuwer, J.A.J., Hurst, G.D.D., 1999a. Wolbachia pipientis: microbial manipulator of arthropod reproduction. Ann. Rev. Microbiol. 53, 71–102. Stouthamer, R., Hu, J., Van Kan, F., Planter, G.R., Pinto, J.D., 1999b. The utility of internally transcribed spacer 2 DNA sequence of the nuclear ribosomal gene for distinguishing sibling species of Trichogramma. Biocontrol 43, 421–440. Stouthamer, R., Jochemsen, P., Platner, G.R., Pinto, J.D., 2000. Crossing incompatibility between Trichogramma minutum and T. platneri and its implications for their application in biological control. Environ. Entomol. 29, 827–837. Sumer, F., Tuncbilek, A., Oztemiz, S., Pintureau, B., Rugman-Jones, P., Stouthamer, R., 2009. A molecular key to the common species of Trichogramma of the Mediterranean region. Biocontrol 54, 617–624. Swofford, D., 1999. Paup 4.0b2a. Computer program distributed by the Sinauer Associates, Inc. Publisher, Sunderland, Massachusetts. Tagami, Y., Miura, K., Stouthamer, R., 2002. How does infection with parthenogenesis inducing Wolbachia reduce the fitness of Trichogramma. J. Invertebr. Pathol. 76, 267–271. Thompson, J.D., Higgins, D.G., Gibson, T.J., 1994. Clustal w improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673–4680. van Meer, M.M., Witteveldt, J., Stouthamer, R., 1999. Phylogeny of the arthropod endosymbiont Wolbachia based on the wsp gene. Insect Mol. Biol. 8, 399–408. Varve, F., Girin, C., Bouletreau, M., 1999. Phylogenetic status of a fecundity enhancing Wolbachia that does not induce thelytoky in Trichogramma. Insect Mol. Biol. 8, 67–72. Vavre, F., de Jong, J.H., Stouthamer, R., 2004. Cytogenetic mechanism and genetic consequences of thelytoky in the wasp Trichogramma cacoeciae. Heredity 93, 592–596. Werren, J.H., Baldo, L., Clark, M.E., 2008. Wolbachia: master manipulators of invertebrate biology. Nature Rev. 6, 741–751. Werren, J.H., Bartos, J.D., 2001. Recombination in Wolbachia. Curr. Biol. 11, 431–435. Werren, J.H., Windsor, D.M., 2000. Wolbachia infection frequencies in insects: evidence of a global equilibrium? Proc. R. Soc. Lond. B Biol. Sci. 267, 1277–1286. Zhou, W., Rousset, F., O'Neill, S., 1998. Phylogeny and PCR-based classification of Wolbachia strain using WSP gene sequences. Proc. R. Soc. Lond. B Biol.Sci. 265, 509–515.