Wolbachia infection in Trissolcus species ( Hymenoptera : Scelionidae ) *

Wolbachia is a maternally transmitted intracellular symbiont which causes reproductive distortions in the arthropods it infects. In recent years there has been an increasing interest in using Wolbachia as a potential tool for biological control by genetic manipulation of insect pests. In the present paper we report Wolbachia infection in several Trissolcus wasps (Hymenoptera: Scelionidae) which are important egg parasitoids of the sunn pest, Eurygaster integriceps Puton (Heteroptera: Scutellaridae). We used DNA sequence data for a gene encoding a surface protein of Wolbachia (wsp) not only to confirm Wolbachia infection but also to discriminate Wolbachia strains. Phylogenetic analyses indicated that Wolbachia strains in Trissolcus species were closely related to one another and belonged to supergroup B. Determination of the infection status of various populations, the possible role of Wolbachia in causing the incompatibility and knowledge of the reproductive compatibility of Trissolcus populations is important for the success of parasitoids in sunn pest management. 169 * Preliminary data of this study was presented at the 7 Hymenoptera Congress, Hungary, 2010. study Wolbachia infection status of various Trissolcus species was empirically demonstrated. MATERIAL AND METHODS The egg batches containing parasitoids of the sunn pest were kept at 25 ± 1°C with 65 ± 5% relative humidity. The winged adult parasitoids emerging from egg batches were divided into two groups, some of which were kept at –80°C for later molecular analysis whilst the rest were placed in tubes with 70% alcohol for taxonomic identification. The Wolbachia infection status of parasitoid species collected from various sites are presented in Table 1. The values (+ve and –ve) refer to the number of parasitoids sampled by PCR analysis and found positive and negative for the presence of Wolbachia, respectively. Percentage infected shows the Wolbachia infection observed in the total sampled population. Adult parasitoids were homogenized in lysis buffer (50 mM Tris, 50 mM EDTA, 0.4 M LiCl, 1% CTAB, 2% PVPP and 0.5% Tween 20) with a sterile pestle in order to isolate genomic DNA. The homogenate was incubated at 65°C for 15 min and extracted with chloroform-isoamyl alcohol (24 : 1) followed by isopropanol precipitation. The pellet was washed with 70% ethanol and resuspended in sterile water. Polymerase chain reactions (PCRs) were performed in a total volume of 50 μl using 1 μl of DNA template and GoTaq Flexi DNA polymerase (Promega, Madison, WI, USA) according to the manufacturer’s instructions: 5× buffer, 10 mM each dNTP, 10 mM each primer and 1.25 u/μl DNA polymerase. We used universal primers of wsp gene in PCR amplifications (Braig et al., 1998), wsp 81 F (5’TGG TCC AAT AAG TGA TGA AGA AAC 3’) and wsp 691 R (5’ AAA AAT TAA ACG CTA CTC CA 3’). The sequences obtained with these general primers allowed us to design species specific primers: Tris wsp F (5’ TGG TCC AAT AAG TGA TGA AGA AAC T 3’) and Tris wsp R (5’ CAC CAT AAG AGC CGA AAT AAC G 3’). A third primer set (12 SAI: 5’ CTA GGA TTA GAT ACC CTA TT 3’and 12 SBI: 5’ AAG AGC GAC GGG CGA TG 3’) was used to assess the quality of the template DNA. 12S primers are considered universal for insect mtDNA amplifying an approximately 400 bp DNA fragment (Simon et al., 1991). PCR products were visualized by gel electrophoresis on 1.5% agarose gels stained with ethidium bromide. In the case of negative amplification, PCRs were repeated with higher DNA template concentrations to ensure that the dilutions used were not below the sensitivity of the PCR technique. Data were excluded in cases in which the 12S control primers were unable to amplify a product from a given DNA extraction. Wolbachia specific PCR products were purified with Wizard SV Gel and PCR Clean up System (Promega). The purified PCR fragments were cloned into PGEM T vector systems (Promega). Sequencing reactions were performed with DTCS Quick Start Kit (Beckman Coulter, Brea, CA, USA), cleaned with Agencourt CleanSeq Kit (Agencourt Bioscience, Brea, CA, USA) and analyzed with CEQ 8800 Genetic Analysis System (Beckman Coulter). Sequence analysis was performed using blast at NCBI (www.ncbi.nih.gov/BLAST) and DNA sequences were aligned with ClustalW. The amplified fragments were confirmed to encode the WSP protein through conceptual translational of their DNA sequences and comparison of the translation to closely related WSP protein sequences. The obtained sequences were used to generate phylogenetic tree of Wolbachia harbored within Trissolcus species. Phylogenetic analyses were conducted in MEGA4 (Tamura et al., 2007). Pairwise distances were calculated using the Maximum Composite Likelihood method in MEGA4 (Tamura et al., 2004, 2007). The number of base substitutions per site between sequences was calculated. All results were based on the pairwise analysis of five sequences. The evolutionary history was inferred using the NeighborJoining method (Saitou & Nei, 1987). A bootstrap consensus tree inferred from 1000 replicates is taken to represent the evolutionary history of the taxa analyzed (Felsenstein, 1985). Branches corresponding to partitions reproduced in less than 170 92 3 35 Carpocoris pudicus Poda 39°33 ́38 ̋N, 33°15 ́14 ̋E 39°41 ́12 ̋N, 32°54 ́56 ̋E Ankara, Turkey T. flavipes Thompson 100 0 48 Aelia rostrata Boheman A. rostrata Boheman 39°26 ́23 ̋N, 32°31 ́09 ̋E 39°30 ́05 ̋N, 32°26 ́31 ̋E 38°15 ́33 ̋N, 32°25 ́08 ̋E 38°10 ́34 ̋N, 32°24 ́46 ̋E Ankara, Turkey Konya, Turkey T. rufiventris Mayr 0 12 0 E. maura (Linnaeus, 1758) E. maura (Linnaeus, 1758) 37°35 ́00 ̋N, 33°06 ́01 ̋E 39°29 ́11 ̋N, 35°24 ́25 ̋E Konya, Turkey Yozgat, Turkey T. vassilievi Mayr 0 70 0 E. integriceps Puton E. maura (Linnaeus, 1758) E. maura (Linnaeus, 1758) 37°05 ́29 ̋N, 35°37 ́33 ̋E 37°13 ́20 ̋N, 35°05 ́26 ̋E 37°31 ́17 ̋N, 33°17 ́24 ̋E 39°33 ́38 ̋N, 33°06 ́36 ̋E 39°47 ́06 ̋N, 32°44 ́04 ̋E Adana, Turkey


INTRODUCTION
The sunn pest, Eurygaster integriceps Puton (Hemiptera: Scutelleridae) is a major pest of wheat and barley in wide areas of the Near and Middle Eastern, Western and Central Asia, Northern Africa, and Eastern and Southern Europe (Brown, 1962;Critchley, 1998;Parker et al., 2002).The pest feeds on both the vegetative stages of the plant as well as maturing grain.Sunn pest infestations in some areas can lead to 100% crop loss in the absence of control measures.The current management strategy for this pest mainly relies on chemical control.In addition to the high cost, insecticides pollute the environment, as well as killing non-target insects, whilst resistance has developed to various types of insecticides in this species (Critchley, 1998;Bandani et al., 2005;Sukhoruchenko & Dolzhenko, 2008).Thus new control methods are needed to diminish reliance on insecticides for control of this serious pest.Among natural control agents, the most promising of them are hymenopterans egg parasitoids.Of these, the most important belong to the genus Trissolcus Ashmead and Telenomus Haliday (Hymenoptera: Scelionidae) (Brown, 1962;Kozlov & Kononova, 1983;Orr, 1988;Kocak & Kilincer, 2003;Bouhssini et al., 2004).Common species play a potential role in suppressing sunn pest populations below the economic threshold for control (Barbulescu, 1971;Critchley, 1998).However, the success of the parasitoid in terms of percentage parasitism of the host varies among regions as well as from year to year.Several studies have been conducted in order to improve the efficiency of the parasitoid (Kivan & Kilic, 2002;Trissi et al., 2005;Iranipour et al., 2010Iranipour et al., , 2011;;Fathi et al., 2011).Since the success of a bio-logical control is dependent on many factors, often including mass rearing of the parasitoid in the laboratory, whilst its release can sometimes be fraught with difficulties, including genetic compatibility between parasitoid and host (Hopper et al., 1993).
In this study we detected Wolbachia as an endosymbiont of Trissolcus species, for the first time.We scanned Trissolcus species including T. festivae Viktorov, T. grandis Thompson, T. simoni Mayr, T. semistriatus Nees, T. vassillievi Mayr, T. rufiventris Mayr and T. flavipes Thompson in terms of Wolbachia infection.Wolbachia surface protein gene (wsp) sequences of different parasitoids have been previously reported.As a result of this study Wolbachia infection status of various Trissolcus species was empirically demonstrated.

MATERIAL AND METHODS
The egg batches containing parasitoids of the sunn pest were kept at 25 ± 1°C with 65 ± 5% relative humidity.The winged adult parasitoids emerging from egg batches were divided into two groups, some of which were kept at -80°C for later molecular analysis whilst the rest were placed in tubes with 70% alcohol for taxonomic identification.The Wolbachia infection status of parasitoid species collected from various sites are presented in Table 1.The values (+ve and -ve) refer to the number of parasitoids sampled by PCR analysis and found positive and negative for the presence of Wolbachia, respectively.Percentage infected shows the Wolbachia infection observed in the total sampled population.
Adult parasitoids were homogenized in lysis buffer (50 mM Tris, 50 mM EDTA, 0.4 M LiCl, 1% CTAB, 2% PVPP and 0.5% Tween 20) with a sterile pestle in order to isolate genomic DNA.The homogenate was incubated at 65°C for 15 min and extracted with chloroform-isoamyl alcohol (24 : 1) followed by isopropanol precipitation.The pellet was washed with 70% ethanol and resuspended in sterile water.
Polymerase chain reactions (PCRs) were performed in a total volume of 50 µl using 1 µl of DNA template and GoTaq Flexi DNA polymerase (Promega, Madison, WI, USA) according to the manufacturer's instructions: 5× buffer, 10 mM each dNTP, 10 mM each primer and 1.25 u/µl DNA polymerase.We used universal primers of wsp gene in PCR amplifications (Braig et al., 1998), wsp 81 F (5'TGG TCC AAT AAG TGA TGA AGA AAC 3') and wsp 691 R (5' AAA AAT TAA ACG CTA CTC CA 3').The sequences obtained with these general primers allowed us to design species specific primers: Tris wsp F (5' TGG TCC AAT AAG TGA TGA AGA AAC T 3') and Tris wsp R (5' CAC CAT AAG AGC CGA AAT AAC G 3').A third primer set (12 SAI: 5' CTA GGA TTA GAT ACC CTA TT 3'and 12 SBI: 5' AAG AGC GAC GGG CGA TG 3') was used to assess the quality of the template DNA.12S primers are considered universal for insect mtDNA amplifying an approximately 400 bp DNA fragment (Simon et al., 1991).PCR products were visualized by gel electrophoresis on 1.5% agarose gels stained with ethidium bromide.In the case of negative amplification, PCRs were repeated with higher DNA template concentrations to ensure that the dilutions used were not below the sensitivity of the PCR technique.Data were excluded in cases in which the 12S control primers were unable to amplify a product from a given DNA extraction.Wolbachia specific PCR products were purified with Wizard SV Gel and PCR Clean up System (Promega).The purified PCR fragments were cloned into PGEM T vector systems (Promega).Sequencing reactions were performed with DTCS Quick Start Kit (Beckman Coulter, Brea, CA, USA), cleaned with Agencourt CleanSeq Kit (Agencourt Bioscience, Brea, CA, USA) and analyzed with CEQ 8800 Genetic Analysis System (Beckman Coulter).
Sequence analysis was performed using blast at NCBI (www.ncbi.nih.gov/BLAST) and DNA sequences were aligned with ClustalW.The amplified fragments were confirmed to encode the WSP protein through conceptual translational of their DNA sequences and comparison of the translation to closely related WSP protein sequences.The obtained sequences were used to generate phylogenetic tree of Wolbachia harbored within Trissolcus species.Phylogenetic analyses were conducted in MEGA4 (Tamura et al., 2007).Pairwise distances were calculated using the Maximum Composite Likelihood method in MEGA4 (Tamura et al., 2004(Tamura et al., , 2007)).The number of base substitutions per site between sequences was calculated.All results were based on the pairwise analysis of five sequences.The evolutionary history was inferred using the Neighbor-Joining method (Saitou & Nei, 1987).A bootstrap consensus tree inferred from 1000 replicates is taken to represent the evolutionary history of the taxa analyzed (Felsenstein, 1985).Branches corresponding to partitions reproduced in less than  1.The Wolbachia infection status of parasitoid species collected from various sites.The values (+ve and -ve) refer to the number of parasitoids sampled by PCR analysis and found positive and negative for the presence of Wolbachia, respectively.Percentage (%) infected shows the Wolbachia infection observed in the total sampled population.50% bootstrap replicates are collapsed.The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches (Felsenstein, 1985).The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree.The evolutionary distances were computed using the Maximum Composite Likelihood method (Tamura et al., 2004) and are in units of the number of base substitutions per site.All positions containing gaps and missing data were eliminated from the dataset.
Alignments were checked in terms of intragenic recombination using the software package (Martin et al., 2005).Programs used in the RDP2 software package were RDP, GeneConv, Bootscan, MaxChi, Chimaera, and Sister Scanning.Analyses were run with default settings except for window and step sizes.

RESULTS AND DISCUSSION
Seven Trissolcus species including T. festivae Viktorov, T. grandis Thompson, T. simoni Mayr, T. semistriatus Nees, T. vassillievi Mayr, T. rufiventris Mayr, and T. flavipes Thompson were screened for Wolbachia infection by PCR analysis.A 500 bp fragment of the wsp gene was amplified and sequenced from the endosymbionts of five Trissolcus species with species specific primers.Results showed that infections could be detected in T. festivae, T. grandis, T. simoni, T. rufiventris, and T. flavipes, which are the most common species attacking eggs of Eurygaster integriceps (Kocak & Kilincer, 2002;Trissi et al., 2004).This finding represents the first record of Wolbachia infection in Trissolcus species.
Cloning and sequencing of PCR bands revealed a gene encoding WSP protein isolated from diverse arthropods according to BLAST analysis (www.ncbi.nih.gov/BLAST).The sequences have been submitted to Gen-Bank with the accession numbers for wsp sequences as follows: HQ447075 for T. grandis, HQ447076 for T. simoni, HQ447077 for T. festivae, HQ447078 for T. flavipes, and HQ447079 for T. rufiventris.According to pairwise analysis, all Trissolcus wsp sequences belong to the same bacterial strain.GenBank homology searches of Trissolcus wsp sequences indicated that the symbionts of all Trissolcus species were most closely related to the Wolbachia symbiont of other parasitic wasps.A phylogenetic tree of all the Wolbachia strains found in infected Trissolcus species was constructed.All procedures used for phylogenetic reconstruction (maximum parsimony, maximum likelihood, and neighbor-joining methods) place all Trissolcus Wolbachia strains in a monophyletic group with those of B group Wolbachia with bootstrap values of 100 (maximum likelihood and neighbor-joining analysis).Fig. 1 shows the phylogenetic tree obtained by neighbor-joining method with 1000 bootstrap replicates.The trees based on maximum likelihood and parsimony analyses are not shown, but do not significantly deviate from these results.Furthermore, intragenic recombination within the wsp gene was analyzed using RDP, GeneConv, Bootscan, MaxChi, Chimaera, and Siscan which are implemented in the RDP2 program.According to these analyses, four recombination events were detected involving three species; T. rufiventris, T. grandis, and T. flavipes (Table 2).The major and minor parents were identified as Acromyrmex insinuator (Schultz, Bekkevold & Boomsma 1998) (Hymenoptera: Formicidae) and Glossina austeni Newstead (Diptera: Glossinidae), respectively.
Previous studies have reported that prokaryotic endosymbionts other than Wolbachia can cause reproductive alterations in insects (Lawson et al., 2001;Zchori-Fein et al., 2001;Hunter et al., 2003;Hagimori et al., 2006;Perlman et al., 2006).In the present work, we have reported only Wolbachia infection in Trissolcus species, since other potential secondary endosymbionts of Trissolcus species remain as yet unknown.It will be interesting to know how these multiple symbionts interact with their hosts and each other.
It has been suggested that mixing Wolbachia infected and uninfected populations may cause reproductive failure in biocontrol programs of parasitoids (Mochiah et al., 2002;Perlman et al., 2006).If parasitoid strains with Wolbachia infection are released in an area where a Wolbachia free population occurs, sterile mating will diminish the reproductive potential of the introduced parasitoid.Mating between males from populations that harbor Wolbachia and females free from Wolbachia infection could theoretically contribute to failure in biological control programmes to eradicate pest insects.Differences in distribution and Wolbachia infection types in parasitoids may give clues which enable parasitoid strains to be used more effectively.It is particularly important to determine the infection status of different populations when they are mixed either in the laboratory for mass production or when they are released in the field where a population is already established (Mochiah et al., 2002).On the other hand, Wolbachia has also been shown to induce parthenogenesis in parasitic Hymenoptera which can be considered as an advantage in biological control (Stouthamer et al., 1990;Zchori-Fein et al., 1992;Stouthamer, 1997).
Biological control programmers should be aware of Wolbachia infections and their effects on parasitoid populations which are important for the establishment of laboratory cultures.This is because mixed populations of differing Wolbachia infectivity could result in a severe reduction of population growth in the laboratory.Further studies need to be done to determine the distribution and prevalence of Wolbachia infection in Trissolcus species.In addition, further crossing experiments should be performed between the same strains or between an infected female and an uninfected male or a treated male in order to determine the influence of Wolbachia infection on Trissolcus reproduction.

Fig. 1 .
Fig. 1.Phylogenetic analysis of Wolbachia infections in Trissolcus species in relation to other insect species.Phylogenetic tree was constructed with MEGA 4 Software (Tamura et al., 2007) using neighbor-joining method.

TABLE 2 .
Summary of recombination events identified by the Recombination Detection Program (RDP2).