domain: sde sde.be sdv

text:
Aleksandar Ivezi
email: aleksandarivezic@yahoo.com

Paul Rugman-Jones

Thibaut Malausa

Nicolas Ris

Aleksandra Ignjatovi C- Cupina

Molecular identification of Trichogramma species parasitizing Ostrinia nubilalis in corn and pepper in south-east border of Europe

Keywords: Biological control, COI, PCR, Wolbachia

The current study extends the faunistic survey of Trichogramma species parasitizing ECB to all agricultural growing regions in Serbia. Specimens of Trichogramma were reared from parasitized egg masses of ECB collected from field-grown corn and pepper crops. The number of egg masses parasitized varied by location. Using sequences of mitochondrial cytochrome oxidase I (COI) gene, we examined intra and interspecific variation in Trichogramma parasitizing the ECB egg masses. Seventy specimens were successfully sequenced of which, 65 were identified as T. brassicae. The remaining five were identified as T. evanescens, and this second species was only collected from pepper.

Introduction

Corn (Zea mays L) is one of the most important grain crops in the world. In Serbia, it is grown on 1-1,2 million hectares. More than half of it (52%) is on the territory of the province of Vojvodina and the rest is in the areas below the rivers Sava and Danube. Although, the production of corn is affected by several harmful insects, the most important pest of corn in Serbia is the European corn borer (hereinafter: ECB) Ostrinia nubilalis (H€ ubner 1796) (Lepidoptera, Pyralidae) [START_REF] Camprag | Jugoslavija: Savez dru stava za za stitu bilja Jugoslavije[END_REF]. ECB is a highly polyphagous pest and can reproduce on many host plants. However, its preferred host is maize, and the large scale cultivation of maize has contributed to its rapid spread over large areas. In addition to the damage it causes in maize, ECB has been gaining increasing importance as a pest of pepper fruits in Serbia [START_REF] Sekuli C R, Kere Si T, Cubranovi C M, Radonji C K | European corn borer (Ostrinia nubilalis Hbn.) -a pest of pepper[END_REF].

Various control methods of ECB have been promoted worldwide, with the application of chemical insecticides being the most commonly employed tactic. However, because ECB larvae spend most of their life inside the plant, chemical control is not easy, and there has always been interest in alternative control methods [START_REF] Suverkropp | Temperature influences walking speed and walking activity of Trichogramma brassicae (Hym. Trichogrammatidae)[END_REF]. Due to the growing awareness of the impact of various chemical pesticides on the environment, biological control methods are becoming more favored and increasingly important. One such method is the augmentative release of commercially available species of the genus Trichogramma Westwood, 1833 (Hymentoptera: Trichogrammatidae).

Several species of Trichogramma have been identified as promising biological control agents of ECB [START_REF] Beglyarov | Seasonal colonization of entomophages in the USSR[END_REF][START_REF] Bigler | Mass production of Trichogramma maidis Pint. et Voeg. and its field application against Ostrinia nubilalis Hbn. in Switzerland[END_REF][START_REF] Van Schelt | Some aspects on the storage and application of Trichogramma maidis in corn[END_REF][START_REF] Hassan | The mass rearing and utilization of Trichogramma to control lepidopterous pests: achievements and outlook[END_REF]. Trichogramma are egg parasitoids that attack more than 200 lepidopteran host species, including the pest groups of borers, webworms, loopers, leafworms, fruitworms, cutworms, bollworms and armyworms [START_REF] Knutson | The Trichogramma Manual[END_REF]. As a result, they have been widely used in inundative and inoculative biological control programs in more than 30 countries, in agricultural crops (e.g. corn, cotton, sugarcane, fruit trees, vegetables) and natural forests [START_REF] Hase | Beitrage zur Lebensgeschichte der Schlupfwespe Trichogramma evanescens Westwood (in.Germany)[END_REF][START_REF] Sweetman | The principles of biological control[END_REF][START_REF] Stinner | Efficacy of inundative releases[END_REF][START_REF] King | Biological control of bollworm and tobacco budworm in cotton by augmentative releases of Trichogramma[END_REF][START_REF] Li | Worldwide use of Trichogramma for biological control on different crops: a survey[END_REF][START_REF] Smith | Biological control with Trichogramma: advances, successes, and potential of their use[END_REF][START_REF] Van Lenteren | Success in biological control of arthropods by augmentation of natural enemies[END_REF][START_REF] Van Lenteren | Environmental risk assessment of exotic natural enemies used in in-inundative biological control[END_REF]. Indeed, Trichogramma have been successfully used for inundative biological control of lepidopteran pests for more than 120 years [START_REF] Smith | Methods and timing of releases of Trichogramma to control lepidopterous pests[END_REF][START_REF] Van Lenteren | Success in biological control of arthropods by augmentation of natural enemies[END_REF]. Furthermore, it has been estimated that over 16 million ha globally receive parasitoid inundative releases [START_REF] Hassan | Selec ¸ão de esp ecies de Trichogramma para o uso em programas de controle biol ogico[END_REF][START_REF] Van Lenteren | Environmental risk assessment of exotic natural enemies used in in-inundative biological control[END_REF]. This history includes releasing Trichogramma wasps to control ECB populations. In Europe, Trichogramma spp. are released against ECB in French [START_REF] Meissle | Pests, pesticide use and alternative options in European maize production: current status and future prospects[END_REF] and German [START_REF] Hassan | Mass-production and utilization of Trichogramma: 2. Four years successful biological control of the European corn borer[END_REF] cornfields [START_REF] Hassan | Selec ¸ão de esp ecies de Trichogramma para o uso em programas de controle biol ogico[END_REF] and [START_REF] Van Lenteren | Environmental risk assessment of exotic natural enemies used in in-inundative biological control[END_REF] reported that over 16 million ha receive parasitoid inundative releases.

Trichogramma are common natural enemies in agricultural areas of the Mediterranean [START_REF] Pintureau | Syst ematique evolutive du genre Trichogramma Westwood (Hymenoptera: Trichogrammatidae) en Europe[END_REF][START_REF] Silva | Molecular differentiation of five Trichogramma species occurring in Portugal[END_REF][START_REF] Oztemiz | Trichogramma species egg parasitoids of Lepidoptera in the eastern Mediterranean region of Turkey[END_REF]) and several species have previously been identified as promising biological control agents of ECB, including T. maidis [START_REF] Bigler | Mass production of Trichogramma maidis Pint. et Voeg. and its field application against Ostrinia nubilalis Hbn. in Switzerland[END_REF][START_REF] Van Schelt | Some aspects on the storage and application of Trichogramma maidis in corn[END_REF], T. evanescens [START_REF] Hassan | The mass rearing and utilization of Trichogramma to control lepidopterous pests: achievements and outlook[END_REF] and T. brassicae, [START_REF] Burgio | Control of European corn borer in sweet corn by Trichogramma brassicae Bezd. (Hym., Trichogrammatidae)[END_REF]. Seven species of Trichogramma were previously recorded from the former Yugoslavia, T. evanescens [START_REF] Boucek | A faunistic review of the Yugoslavian Chalcidoidea (Parasitic Hymenoptera)[END_REF][START_REF] Injac | The role of natural enemies on reduction of the Mamestra brassicae L. population density in the region of Belgrade[END_REF], T. minutum [START_REF] Boucek | A faunistic review of the Yugoslavian Chalcidoidea (Parasitic Hymenoptera)[END_REF], T. embryophagum [START_REF] Voegel E | Preliminary experiments using Trichogramma sp. against the codling moth in apple orchards in the Parisian region[END_REF], T. pretiosum [START_REF] Danon | Biological control of the European corn borer, Ostrinia nubilalis (H€ ubner), by Trichogramma pretiosum Riley[END_REF], T. brassicae, T. cacoeciae and T. oleae [START_REF] Pintureau | Syst ematique evolutive du genre Trichogramma Westwood (Hymenoptera: Trichogrammatidae) en Europe[END_REF][START_REF] Pintureau | Les esp eces europ eennes de Trichogrammes[END_REF], but little is known about their distribution in current day Serbia. Resident populations have been recorded in Serbia [START_REF] Krnjaji | Dinamika populacija jajnih parazitoida kupusne sovice (Mamestra brassicae L.) sa osvrtom na mogu cnost kori s cenja Trichogramma evanescens Westwood u suzbijanju steto cina iz reda Lepidoptera[END_REF][START_REF] Tancik | Natural parasitism of the second generation European Corn Borer Eggs Ostrinia nubilalis (Hubner) (Lepidoptera, Pyralidae) by Trichogramma spp. in sweet corn fields in Vojvodina, Serbia[END_REF][START_REF] Ivezi C | Molecular identification of Trichogramma egg parasitoids of Ostrinia nubilalis in the north eastern Serbia[END_REF] but, identification to species level has only been done in isolated areas [START_REF] Krnjaji | Dinamika populacija jajnih parazitoida kupusne sovice (Mamestra brassicae L.) sa osvrtom na mogu cnost kori s cenja Trichogramma evanescens Westwood u suzbijanju steto cina iz reda Lepidoptera[END_REF][START_REF] Tancik | Natural parasitism of the second generation European Corn Borer Eggs Ostrinia nubilalis (Hubner) (Lepidoptera, Pyralidae) by Trichogramma spp. in sweet corn fields in Vojvodina, Serbia[END_REF][START_REF] Ivezi C | Molecular identification of Trichogramma egg parasitoids of Ostrinia nubilalis in the north eastern Serbia[END_REF]. Furthermore, little is known about intraspecific variation in these species [START_REF] Ivezi C | Molecular identification of Trichogramma egg parasitoids of Ostrinia nubilalis in the north eastern Serbia[END_REF]. In Trichogramma, traits such as fecundity, egg maturation rate and offspring sex-ratio, have been shown to be influenced by intraspecific genetic variation [START_REF] Wajnberg | Genetics of the behavioral ecology of egg parasitoids[END_REF][START_REF] Wajnberg | Intra-Population Genetic Variation in the Temporal Pattern of Egg Maturation in a Parasitoid Wasp[END_REF][START_REF] Coelho | Laboratory performance predicts the success of field releases in inbred lines of the egg parasitoid trichogramma pretiosum (hymenoptera: Trichogrammatidae)[END_REF]. All such traits could affect the performance of a particular strain (or genotype) as a biological control agent. Thus, before we can truly assess the potential for large scale use of these parasitoids in biological control in Serbia, it is essential to first identify and characterize the native populations of Trichogramma countrywide. It is expected that such autochthonous/ native species are likely to be the best adapted to environmental conditions in a specific ecosystem [START_REF] Whitman | Plant chemicals and the location of herbivorous arthropods by their natural enemies[END_REF].

The taxonomy and identification of Trichogramma has historically been problematic [START_REF] Stouthamer | Wolbachia pipientis: microbial manipulator of arthropod reproduction[END_REF]. Diagnostic morphological features are limited and those that are useful require specialized knowledge for interpretation (Nagaratti andNagaraja 1968, 1971;[START_REF] Pinto | Systematics of the Trichogrammatide with emphasis on Trichogramma[END_REF]. Morphological identification remains difficult mostly because of their minute size (<1 mm long) and the lack of reliable characters [START_REF] Stouthamer | Wolbachia pipientis: microbial manipulator of arthropod reproduction[END_REF]. Consequently, alternative identification methods such as the use of reproductive compatibility studies [START_REF] Pinto | Variation in reproductive compatibility in Trichogramma and its taxonomic significance (Hymenoptera: Trichogrammatidae)[END_REF] and the incorporation of molecular methods [START_REF] Stouthamer | Wolbachia pipientis: microbial manipulator of arthropod reproduction[END_REF]) have been developed. Sequences of the internal transcribed spacer 2 (ITS2) region of the nuclear ribosomal DNA are one accepted means of identifying Trichogramma species [START_REF] Stouthamer | Wolbachia pipientis: microbial manipulator of arthropod reproduction[END_REF]). In addition, sequences of the mitochondrial cytochrome oxidase subunit I (COI) are commonly used in 'DNA barcoding' studies to identify biological material to the species level [START_REF] Hebert | Biological identifications through DNA barcodes[END_REF]. Barcoding has been shown to be particularly useful for insects [START_REF] Zald Ivar-River On | DNA barcoding a highly diverse group of parasitoid wasps (Braconidae: Doryctinae) from a Mexican nature reserve[END_REF][START_REF] Malausa | DNA markers to disentangle complexes of cryptic taxa in mealybugs (Hemiptera: Pseudococcidae)[END_REF], including trichogrammatids [START_REF] Thiruvengadam | Differentiation of some indigenous and exotic trichogrammatids (Hymenoptera: Trichogrammatidae) from India based on Internal transcribed spacer-2 and cytochrome oxidase-I markers and their phylogenetic relationship[END_REF]. Currently, around 210 species of Trichogramma are formally recognized worldwide [START_REF] Pinto | A review of the New World genera of Trichogrammatidae (Hymenoptera)[END_REF], but this figure may change considerably with increasing use of molecular methods as tools for identification [START_REF] Pino | Trichogramma canariensis (Insecta: Hymenoptera: Trichogrammatidae) a parasitoid of eggs of the twinspot moth Chrysodeixis chalcites (Lepidoptera: Noctuidae) in the Canary Islands[END_REF]. Sequences of the COI gene also provide a very powerful tool with which to characterize within-species molecular diversity [START_REF] Mardulyn | Phylogenetic Signal in the COI, 16S, and 28S genes for inferring relationships among genera of Microgastrinae (Hymenoptera; Braconidae): evidence of a high diversification rate in this group of parasitoids[END_REF][START_REF] Correa | Molecular characterization of parasitoids from armored scales infesting citrus orchards in Corsica, France[END_REF].

A further factor that may influence the success of Trichogramma wasps in release programs is their mode of reproduction [START_REF] Stouthamer | Wolbachia pipientis: microbial manipulator of arthropod reproduction[END_REF]. The most common mode of reproduction in Trichogramma is arrhenotoky, in which females develop from fertilized eggs and males from unfertilized eggs [START_REF] Nazeri | Can Wolbachia infection improve qualitative characteristics of Trichogramma brassicae reared on cold stored eggs of the host?[END_REF]. However, thelytokous reproduction also occurs in some populations/species, in which unfertilized eggs also develop as females. In Trichogramma, thelytoky is typically induced by the endosymbiotic bacterium Wolbachia [START_REF] Louis | Research on the origin of unisexuality: thermotherapy cures both rickettsia and thelytokous parthenogenesis in a Trichogramma species (Hymenoptera: Trichogrammatidae)[END_REF][START_REF] Stouthamer | Molecular identification of microorganisms associated with parthenogenesis[END_REF]. All-female populations may be advantageous in the field of biological control since only females parasitize eggs, and, assuming similar levels of fecundity, all-female populations are expected to have higher growth rates, making them easier and cheaper to mass-rear [START_REF] Stouthamer | The use of sexual versus asexual wasps in biological control[END_REF]. Considering the influence that Wolbachia may have on host reproduction and fitness, the presence of this bacterium in Serbian populations of Trichogramma needs to be investigated to assure the success of potential biological control programs.

The aim of this study was to identify the resident Trichogramma species associated with ECB across all agricultural growing regions of Serbia. Trichogramma species were identified by sequencing the COI gene and those sequences were further used to investigate the genetic structure of populations collected in different parts of Serbia. Finally, we also tested these Serbian Trichogramma for the presence of Wolbachia.

Material and methods

Trichogramma populations were sampled at 21 different sites/localities in Serbia (Figure 1 and Table 1). These sites covered four natural geographic regions separated from each other by natural barriers and/or relatively large distances: (I) a cluster of sites in the Autonomous Province of Vojvodina, an area north of the River Danube and close to the borders with Croatia, Hungary and Romania; (II) a central region comprising three sites 50-100 km south of Belgrade; (III) an isolated eastern site close to the confluence of the borders with Romania and Bulgaria; and (IV) an isolated area in the south of the country, surrounded on three sides by mountains (Figure 1).

Sampling took place between late July and mid-September 2018; dates correspond with the activity of II and III generation of ECB [START_REF] Camprag | Jugoslavija: Savez dru stava za za stitu bilja Jugoslavije[END_REF]). Seventeen of the sites were sampled on a single occasion, but two samples were taken at both Vrbas (corn and pepper) and Nakovo (different dates), three samples were taken at Leskovac (different corn fields) and four samples were taken at Vr sac (two different pepper fields, on two different dates; Table 1). Thus, a total of 28 samples were taken. For each sample, 100 randomly selected plants of corn or pepper were inspected for the presence of ECB egg clusters. The total number of ECB egg clusters, and of parasitized egg clusters, was recorded. A single parasitized egg cluster from each sample was transported to the laboratory and maintained to enable the rearing of the parasitoids (Table 1). Following emergence, the wasps from each parasitized egg cluster were identified as male or female and preserved in labeled vials with 95% ethanol and stored at -20 C.

DNA extraction

Molecular studies were conducted at the National Institute of Agricultural Research (INRA), Sophia Antipolis, France. Genomic DNA was isolated from ethanol-preserved females using the QuickExtract DNA Extraction Solution 1.0 kit (LU-QE09050 LUCIGEN) following the manufacturer's protocol. Where possible, three female Trichogramma were randomly selected from those that emerged from each of the 28 laboratory-maintained ECB egg clusters and individually placed in 20 mL of extraction buffer and incubated for 15 min at 65 C and then for 2 min at 98 C. There were three exceptions: only two females emerged from samples IA013 and IA033; and, only one from IA023. In total, 80 individual females were extracted.

Molecular characterization of Trichogramma spp.

A fragment (about 700 bp) of the mitochondrial COI gene was amplified using the primers: LCO 1490 (5-GGTCAACAAATCATAAAGATATTGG-3) and HCO 2198 (5-TAAACTTCAGGGTGAC CAAAAAATCA-3) [START_REF] Folmer | DNA primers for amplification of mitochondrial cytochrome C oxidase subunit I from diverse metazoan invertebrates[END_REF]. PCR amplifications were carried out in a total volume of 25 mL with 1 mL of DNA. PCR was performed with the Multiplex PCR Master Mix (QIAGEN, # 206145) with a final concentration of 3 mM MgCl 2 and 0.125 lL of each of the two primer solutions (100 lM). The PCR conditions were as follows: 95 C for 15 min; 40 cycles at 94 C for 30 s, 50 C for 90 s, 72 C for 1 min; and a final elongation step at 72 C for 10 min. The presence and size of PCR products was confirmed using a QIAxcel DNA Fast Analysis Kit (QIAGEN S.A.S) on a QIAxcel Advanced System (QIAGEN S.A.S). Financial constraints allowed the PCR products to be Sangersequenced in only one direction (with primer HCO2198), by GENEWIZ (Essex, UK). The COI sequences obtained in the current study were aligned using MAFFT version 7.050 [START_REF] Katoh | MAFFT multiple sequence alignment software version 7: improvements in performance and usability[END_REF] and the Q-INS-i algorithm, employing default settings. The aligned COI sequences were trimmed to a uniform length (542 bp) and a haplotype network was constructed using the statistical parsimony method of [START_REF] Templeton | A cladistic analysis of phenotypic associations with haplotypes inferred from restriction endonuclease mapping and DNA sequence data III. Cladogram estimation[END_REF] in the software program TCS, version 1.21 [START_REF] Clement | TCS: a computer program to estimate gene genealogies[END_REF]. All haplotypes were translated and checked for stop codons and indels, to reduce the chances of including sequencing errors or NUMTs in subsequent analyses [START_REF] Song | Many species in one: DNA barcoding overestimates the number of species when nuclear mitochondrial pseudogenes are coamplified[END_REF], and subject to BLAST searches of the GenBank database (NCBI-National Center for Biotechnology Information) to identify matches (>99% similarity) with existing GenBank accessions. All sequences generated in this study were deposited in GenBank (accession nos. MT049959-MT050028).

Two species were identified in our samples, one common, one rare (see Results). Genetic variation was only examined in the common species. Trichogramma are not strong fliers and are thought to disperse on a local scale by hopping from plant to plant, walking, riding the wind and hitch-hiking on other insects (phoresy). Little is known about the dispersal abilities of natural Trichogramma populations but several studies have looked at dispersal of laboratory reared Trichogramma from a mass release site, and recorded localized spread of as much as 400 m, typically downwind (e.g. [START_REF] Bigler | Dispersal of Trichogramma evanescens Westw. and its impact on parasitism of eggs of Ostrinia nubilalis Hbn[END_REF][START_REF] Smith | Biological control with Trichogramma: advances, successes, and potential of their use[END_REF][START_REF] Kuske | Dispersal and persistence of mass released Trichogramma brassicae (Hymenoptera: Trichogrammatidae) in non-target habitats[END_REF][START_REF] Chapman | Dispersal of Trichogramma ostriniae (Hymenoptera: Trichogrammatidae) in Potato Fields[END_REF][START_REF] Fatouros | Phoresy in the field: natural occurrence of Trichogramma egg parasitoids on butterflies and moths[END_REF][START_REF] Sharma | Dispersal ability and parasitisation performance of Trichogramma spp. (Hymenoptera: Trichogrammatidae) in organic Basmati rice[END_REF]. This may facilitate passive dispersal of Trichogramma into neighboring crop fields and hence, localized genetic mixing across intensively farmed areas. In contrast, the ability of Trichogramma to actively migrate longer distances has not been rigorously investigated, but two separate studies that looked at dispersal of mass-released wasps into adjacent 'non-target' habitats both concluded that For each sample, 100 randomly selected plants of corn or pepper were inspected. Also see Figure 1 for geographic location of the sites.

any migration was limited to very short distances and likely dependent on the nature of that habitat, and the habitat preferences of the parasitoid [START_REF] Orr | Trichogramma nontarget impacts: a method for biological control risk assessment[END_REF][START_REF] Kuske | Dispersal and persistence of mass released Trichogramma brassicae (Hymenoptera: Trichogrammatidae) in non-target habitats[END_REF]. The food plant species of its hosts, may also directly or indirectly affect Trichogramma.

For example, simple differences in plant structural complexity may favor different parasitoid species or genotypes (reviewed by [START_REF] Romeis | Habitat and plant specificity of Trichogramma egg parasitoids -Underlying mechanisms and implications[END_REF]. In light of these factors, and since each of our 28 samples yielded a maximum of only three data points (DNA sequences), to facilitate a more meaningful analysis, the samples were divided into five population groups, based on geographic region (I-IV) and crop type (corn was sampled in all regions; pepper was only sampled in region I). This created five arbitrarily defined population groups, and genetic variation within these groups was examined in terms of haplotype number, haplotype diversity and nucleotide diversity (k), calculated using DnaSP Version 5.10.01 [START_REF] Librado | DnaSP v5: A Software for Comprehensive Analysis of DNA Polymorphism Data[END_REF]. Divergence between the population groups was estimated as the number of amino acid differences per site from averaging over all sequence pairs within and between groups (p-distance), calculated in MEGA version 6 [START_REF] Tamura | MEGA6: Molecular Evolutionary Genetics Analysis version 6.0[END_REF]). The geographic distribution of the mitochondrial haplotypes was also mapped using PhyloGeoViz Version 2.4.5 [START_REF] Tsai | PhyloGeoViz: a web-based program that visualizes genetic data on maps[END_REF] and visualized using GPS Visualizer (https://www. gpsvisualizer.com/map_input).

Wolbachia infection status

The DNA of each of the 80 extracted wasps was tested for the presence of Wolbachia using an established diagnostic marker [START_REF] Dedeine | Removing symbiotic Wolbachia bacteria specifically inhibits oogenesis in a parasitic wasp[END_REF]. We attempted to amplify a 400 bp fragment of the Wolbachia ftsZ gene using 1.2 lL of DNA in 15lL of final reaction volume, composed of 0.12 lL Taq (5 U/lL) (QIAGEN, # 201203), 1x PCR Buffer (QIAGEN), 1.5 mM MgCl 2 , 0.5 lM forward FtsZ-F (TTGCAGAGCTTG GACTTGAA) and reverse FtsZ-R (CATATCTCCGCC ACCAGTAA) primers [START_REF] Dedeine | Removing symbiotic Wolbachia bacteria specifically inhibits oogenesis in a parasitic wasp[END_REF], and 200 lM dNTP. The PCR conditions were as follows: 94 C for 5min, 35 cycles at 94 C for 30 s, 58 C for 1 min, 72 C for 1 min, with final elongation at 72 C for 10 min. The presence of amplicons was determined with the QIAxcel DNA Fast Analysis kit (QIAGEN SAS) on the QIAxcel analyzer. Wolbachia infected Psyttalia lounsburyi Silvestri, 1913 (Hymenoptera: Braconidae) and uninfected P. lounsburyi were used as positive and negative controls, respectively.

Results

Field parasitism rates

Monitoring of ECB in 2018, conducted on 21 different sites on the territory of Serbia, revealed 203 egg clusters of ECB. Of those 203 egg clusters, 75 were parasitized. On a regional scale, the highest percentage of parasitized ECB egg clusters was registered in region I, the Autonomous Province of Vojvodina, with parasitization rate of 41.7%. This was followed by region IV with parasitization of 20.6%, while the lowest levels of parasitization were registered in regions II and III, at 15.3% and 12.5%, respectively. Comparing individual sample sites, parasitization rates (the percentage of egg masses parasitized) ranged between 11.1% and 80.0%, depending on the locality, date of collection and examined crop. The highest percentages of parasitized egg clusters were registered in corn fields at sites in region I (Gakovo [80.0%], Banatska Topola [62.5%] and Nakovo [60.0%]). The lowest percentages of parasitized egg clusters were also registered in corn fields, but in regions II (Mladenovac [11.1%] and Gorovi c [14.3%]) and III (Negotin [12.5%]) (Table 1).

Monitoring of corn and pepper fields was performed during the period of oviposition of females of II and III generation of ECB. Our data showed significantly higher activity of Trichogramma wasps registered in the first half of September, corresponding with the activity of the III generation ECB. Monitoring of oviposition of II generation of ECB revealed 31.5% of parasitized egg clusters, while during the activity of III generation of ECB parasitization rate was 45.5%.

Genetic characterization of Trichogramma spp. associated with ECB

Twenty-eight parasitized egg clusters were sampled and individual females were used for molecular identification of the Trichogramma species present on the territory of Serbia in the south east border of Europe. A total of 80 emerged wasps were examined and presence of both male and female adults were registered in all sampled egg cluster. Seventy out of eighty tested samples yielded amplifiable DNA. Among those samples, a 542 bp fragment of the COI gene revealed the existence of 18 haplotypes, 15 of which (65 specimens) matched or clustered tightly with GenBank accessions (e.g. KC488658 and FM210198) identified as T. brassicae (Figure 2, Supporting Information Table S1). The remaining three haplotypes (5 specimens) were identified as T. evanescens, closely matching GenBank accessions MG932147 and MG932180 (Figure 2, Supporting Information Table S1). All five individuals identified as T. evanescens were sampled in pepper fields in Vojvodina (i.e. region I). Across both species, only two haplotypes, both T. brassicae, were identical (100% match) to those previously deposited in GenBank. The haplotype Tbr-H1 has previously been recorded in Iran (e.g. JX131627) and Tbr-H5 matched a commercial strain reared by Andermatt BioControl, Switzerland (FM210198) [START_REF] Pasquer | Microarraybased genetic identification of beneficial organisms as a new tool for quality control of laboratory cultures[END_REF].

Genetic variation was examined further only for the most common species, T. brassicae. Across the five arbitrary population groups, the 542 bp sequence of COI contained 46 polymorphic sites, which resulted in the 15 haplotypes. In terms of the actual number of haplotypes detected, corn fields in region I appeared to be the most polymorphic population group, with 9 haplotypes, followed by pepper fields in the same region, with 6. However, since this region was the most intensely sampled, this could be a reflection of sampling effort (Table 2). Despite the differences in sample sizes, haplotype diversity and nucleotide diversity were similar across the five population groups (Table 2). Only three haplotypes (Tbr-H1, Tbr-H2 and Tbr-H5) were numerically abundant, being present in 32, 9 and 7 individuals, respectively (Figure 2; Supporting Information Table S1). These haplotypes were also three of only four that were detected in more than a single population group, the fourth being Tbr-H6 (Figure 3). The remaining 11 haplotypes were restricted to a single population group, and indeed, 9 of those haplotypes were only detected once (i.e. in a single specimen). Perhaps the most surprising finding was that in the only region in which we collected from both corn and pepper (region I), among the 9 haplotypes detected in corn and 6 detected in pepper, only one of these (the numerically dominant Tbr-1) was detected in both crops (Supporting Information Table S1). Indeed there was very little overlap between the haplotypes detected in pepper in region I and those found in corn anywhere in Serbia. Despite this disparity, estimates of p-distance between all population groups, regardless of crop type, were similar (0.004-0.008; Table 3). Finally, none of the Trichogramma samples tested positive for Wolbachia.

Discussion

The use of Trichogramma wasps in biological control is common worldwide but its potential has not been explored for many pests and in many geographic Variation was estimated from a 542 bp sequences of COI and is expressed in terms of haplotype number (h), haplotype diversity (Hd) and nucleotide diversity (k), calculated using DnaSP Version 5.10.01. regions. Serbia is one of the countries at the south east border of Europe that does not currently use Trichogramma in its agricultural systems. However, monitoring of ECB on the territory of Serbia since 2010 has shown the presence of Trichogrammaparasitized egg clusters and significant rates of parasitism [START_REF] Krnjaji | Dinamika populacija jajnih parazitoida kupusne sovice (Mamestra brassicae L.) sa osvrtom na mogu cnost kori s cenja Trichogramma evanescens Westwood u suzbijanju steto cina iz reda Lepidoptera[END_REF][START_REF] Tancik | Natural parasitism of the second generation European Corn Borer Eggs Ostrinia nubilalis (Hubner) (Lepidoptera, Pyralidae) by Trichogramma spp. in sweet corn fields in Vojvodina, Serbia[END_REF][START_REF] Ivezi C | Molecular identification of Trichogramma egg parasitoids of Ostrinia nubilalis in the north eastern Serbia[END_REF]).

In the current study, we identified two different 2017). In the study of Tancik, the rate of egg parasitism varied from 9.3% to 73.6% during the period from 2004 until 2007 suggesting that, although, Trichogramma occurred constantly, their number greatly fluctuated from year to year. Clearly, Trichogramma have the potential to achieve high levels of parasitization in both corn and pepper in Serbia and their use as potential biological control agents warrants further investigation. A fundamental step in the development of any biological control program utilizing Trichogramma is the identification and choice of species and/or strains to use: not all Trichogramma species, populations or genotypes (strains) perform equally well, be it in terms of mass rearing or field dispersal and parasitism rates [START_REF] Van Driesche | Biological control[END_REF]. For example, key traits such as fecundity, egg maturation rate, and offspring sex-ratio, have been shown to be influenced by intraspecific genetic variation [START_REF] Wajnberg | Genetics of the behavioral ecology of egg parasitoids[END_REF][START_REF] Wajnberg | Intra-Population Genetic Variation in the Temporal Pattern of Egg Maturation in a Parasitoid Wasp[END_REF][START_REF] Coelho | Laboratory performance predicts the success of field releases in inbred lines of the egg parasitoid trichogramma pretiosum (hymenoptera: Trichogrammatidae)[END_REF]. Therefore, the first step for the implementation of these beneficial insects in the program of biological control of pests is the accurate identification and genetic characterization of those native species that might be considered for use as biological control agents. Across agricultural growing regions in Serbia, the current study found that T. brassicae was the predominant species parasitizing ECB eggs in both corn and pepper fields. A second species, T. evanescens, was also identified, but in much lower numbers, and only from pepper fields. This is very much in line with an earlier study that focused only on corn, and only on a single growing region, Kikinda [START_REF] Ivezi C | Molecular identification of Trichogramma egg parasitoids of Ostrinia nubilalis in the north eastern Serbia[END_REF]. That earlier study did recover a single specimen of T. evanescens from corn, but considered alongside the current study, it suggests that T. evanescens is naturally rare in corn. However, while T. brassicae was also the predominant species in pepper, T. evanescens was more likely to be encountered in this crop.

The findings of our study can be used to both justify and direct the rearing and augmentative (or inundative) release of Trichogramma in Serbia, perhaps most importantly in the regions with lower levels of natural parasitism. Outside of Serbia, in western regions of Europe, both T. brassicae and T. evanescens are used for biological control of ECB. Trichogramma brassicae is already commercially produced for biological control of ECB in maize in Switzerland, Germany and France [START_REF] Suverkropp | Temperature influences walking speed and walking activity of Trichogramma brassicae (Hym. Trichogrammatidae)[END_REF]). Indeed, one of the haplotypes identified in our study (Tbr-5) matches that of a strain that is already mass-reared for commercial sale and release in Europe [START_REF] Pasquer | Microarraybased genetic identification of beneficial organisms as a new tool for quality control of laboratory cultures[END_REF]. Similarly, T. evanescens has also been advocated as a biological control agent in many countries of Western Europe [START_REF] Hassan | Mass-production and utilization of Trichogramma: 2. Four years successful biological control of the European corn borer[END_REF][START_REF] Bigler | Mass production of Trichogramma maidis Pint. et Voeg. and its field application against Ostrinia nubilalis Hbn. in Switzerland[END_REF][START_REF] Hluch Y | The results of the testing a product based on Trichogramma sp. the European corn borer eggs Ostrinia nubilalis Hbn. and cotton bollworm Helicoverpa armigera Hbn. in maize[END_REF][START_REF] Tancik | Efficiency of Trichogramma evanescens Westwood in the control of European corn borer (Ostrinia nubilalis Hbn) on corn[END_REF]. The fact that these two species can be cheaply mass-produced on eggs of stored product pests like Sitotroga cerealella Olivier 1789 (Lepidoptera: Gelechiidea) or Ephestia kuehniella. Zeller 1879 (Lepidoptera: Pyralidae) strongly facilitate their commercial utilization in Serbia. If we must choose between these two species, then T. brassicae is probably the most parsimonious option, based simply on its natural abundance relative to T. evanescens in both corn and pepper. However, it would be interesting to continue the monitoring of both species in case that changes.

With 15 haplotypes, there was a high degree of genetic diversity in Serbian T. brassicae populations. Each of these haplotypes may be potentially considered for use in a biological control program. However, almost half of the T. brassicae specimens characterized were of just one haplotype, Tbr-H1. This haplotype occurred in four of the five population groups sampled, and in each of those, accounted for around 50% of the population. One interpretation of this finding is that this haplotype is particularly successful under natural (field) conditions, and may therefore be a good candidate for use in a biological control program. Future research should investigate the suitability of this haplotype for mass-rearing. This haplotype may be a particularly useful introduction in the east of the country (Region III) where it was not actually detected in our study. A mountain range separates the eastern region from the rest of the country, which may have acted as a natural barrier to the spread of this otherwise successful haplotype. That said, we recommend that sampling effort in the eastern (and central and southern) region should be increased to gain a more reliable picture of genetic diversity in those areas. Furthermore, a more rigorous approach would be to compare the suitability of several of the available haplotypes/strains [START_REF] Coelho | Laboratory performance predicts the success of field releases in inbred lines of the egg parasitoid trichogramma pretiosum (hymenoptera: Trichogrammatidae)[END_REF]).

An alternative approach to initiating a massrearing effort in Serbia could be to import T. brassicae from a mass-rearing facility outside of the country. It is interesting to note that one of the haplotypes detected in this study (Tbr-5) is shared with a strain of T. brassicae that is already in mass-rearing in Switzerland (or at least was a decade ago; [START_REF] Pasquer | Microarraybased genetic identification of beneficial organisms as a new tool for quality control of laboratory cultures[END_REF]). This similarity is currently based on just a relatively short fragment of one mitochondrial gene. Future work might seek to extend the genetic comparison of the two forms. With this in mind, it is also worth noting that 13 of the 15 Serbian T. brassicae haplotypes were new, in the sense that this study is the first time they have been deposited in GenBank. Any one of these haplotypes could potentially prove to be a better biological control agent than what is currently available, attracting foreign commercial interest.

One further factor that can influence the choice of Trichogramma strains is infection with the endosymbiotic bacterium Wolbachia [START_REF] Huigens | Natural interspecific and intraspecific horizontal transfer of parthenogenesis-inducing Wolbachia in Trichogramma wasps[END_REF]. Wolbachia can induce thelytokous parthenogenesis in Trichogramma creating heavily female-biased populations that can be beneficial to biocontrol (since only females parasitize eggs). For example, with no males produced, populations grow faster in mass-rearing, and since females are the ones that control the pest in the field, greater parasitism rates may be achieved [START_REF] Almeida | Trichogramma and its relationship with Wolbachia. Identification of Trichogramma species, phylogeny, transfer and costs of Wolbachia symbionts[END_REF]). As it happens, none of the Serbian wasps showed any presence of Wolbachia.

The results of this study are particularly interesting because they reveal new information about the biodiversity of Trichogramma spp. in an area without previous deliberate introduction of these insects. The obtained data will certainly benefit the potential commercial use of these insects and the implementation of conservation biocontrol strategies promoting the naturally high ECB parasitism rates achieved by resident Serbian Trichogramma populations. Molecular characterization of additional Trichogramma species from Serbia will help to clarify the Trichogramma fauna native to Serbia and it will help in a rational choice for testing and identifying the best species to be used for biological control.

Figure 1 .

 1 Figure 1. Locations in Serbia where corn and pepper crops were sampled in search of parasitized ECB egg masses. Sampling locations lay within one of four natural geographic regions: (I) an area north of the River Danube and close to the borders with Croatia, Hungary and Romania; (II) a central region 50-100 km south of Belgrade; (III) an isolated eastern site close to the confluence of the border with Romania and Bulgaria; and (IV) an isolated area in the south of the country, surrounded on three sides by mountains. Samples were collected in 2018. Map from Google Earth (earth.google.com/web/).

Figure 2 .

 2 Figure 2. Network of mitochondrial haplotypes present in Serbian populations of two Trichogramma species found parasitizing eggs of the European corn borer in corn and pepper fields; T. brassicae (white) and T. evanescens (grey). Haplotype size is proportional to the number of specimens sharing a haplotype. Small circles represent unobserved inferred haplotypes and lines connecting haplotypes represent a single nucleotide mutational change. (number alongside the line denotes the position of the mutation in the 542 bp fragment of COI).

Figure 3 .

 3 Figure 3. Geographic distribution of Trichogramma COI haplotypes parasitizing ECB egg masses in corn and pepper fields across Serbia. Sixty-five of 70 sequenced specimens were identified as T. brassicae, which in turn harbored 15 haplotypes.Only four haplotypes (singled out) were recorded in multiple regions and/or crops. A second species, T. evanescens, was found only in pepper fields in region I. Map was produced using PhyloGeoViz Version 2.4.5 and visualized using GPS Visualizer.

Table 1 .

 1 Details of twenty eight samples of Serbian corn or pepper crops taken in search of parasitized ECB egg masses.

			Sample			Total egg	Parasitized egg clusters
	Region	Site	number	Crop	Date of collection	clusters	No	%
	I	Despotovo	IA001	Corn	8 August 2018	5	1	20.0
	I	B a cka Topola	IA004	Corn	16 September 2018	12	5	41.7
	I	Njego sevo	IA008	Corn	4 August 2018	8	3	37.5
	I	Vrbas	IA010	Corn	27 August 2018	3	1	33.3
	I	"	IA011	Pepper	9 August 2018	4	2	50.0
	I	V r sac	IA015	Pepper	8 August 2018	13	6	46.1
	I	"	IA016	Pepper	8 August 2018	9	5	55.5
	I	"	IA017	Pepper	10 August 2018	3	1	33.3
	I	"	IA018	Pepper	10 August 2018	3	1	33.3
	I	Horgo s	IA023	Corn	15 August 2018	11	4	36.4
	I	Kanji za	IA024	Corn	15 August 2018	13	7	53.8
	I I	Srbobran Gospod -inci	IA027 IA028	Corn Pepper	22 August 2018 8 August 2018	10 5	3 1	30.0 20.0
	I	Bili c	IA031	Corn	13 September 2018	14	5	35.7
	I	Apatin	IA033	Corn	13 September 2018	6	3	50.0
	I	Gakovo	IA034	Corn	13 September 2018	5	4	80.0
	I	Zrenjanin	IA035	Corn	10 August 2018	14	6	42.8
	I	Kikinda	IA038	Corn	11 August 2018	5	1	20.0
	I	Nakovo	IA042	Corn	14 August 2018	3	1	33.3
	I	"	IA043	Corn	13 September 2018	5	3	60.0
	I	Banatska Topola	IA046	Corn	11 September 2018	8	5	62.5
	II	Kragujevac	IA048	Corn	13 September 2018	5	1	20.0
	II	Mladenovac	IA029	Corn	15 August 2018	9	1	11.1
	II	Gorovi c	IA053	Corn	16 September 2018	7	1	14.3
	III	Negotin	IA030	Corn	08 August 2018	8	1	12.5
	IV	Leskovac	IA012	Corn	15 August 2018	6	1	16.7
	IV	"	IA013	Corn	15 August 2018	4	1	25.0
	IV	"	IA014	Corn	15 August 2018	5	1	20.0

Table 2 .

 2 Genetic variation present in Trichogramma brassicae parasitizing ECB egg masses in different regions/crops in Serbia.

	Region/crop	N	h	Hd	k
	I/corn	32	9	0.7258	0.0040
	I/pepper	16	6	0.6167	0.0050
	II/corn	8	3	0.7500	0.0051
	III/corn	3	2	0.6667	0.0012
	IV/corn	6	3	0.7333	0.0052

Table 3 .

 3 Genetic divergence between the populations of Trichogramma brassicae parasitizing ECB egg masses in different regions/crops, in Serbia.

	Region/crop	I/corn	I/pepper	II/corn	III/corn	IV/corn
	I/corn	0.004
	I/pepper	0.005	0.005
	II/corn	0.005	0.006	0.005
	III/corn	0.008	0.008	0.006	0.001
	IV/corn	0.004	0.005	0.005	0.006	0.005
	Estimates of p-distance were calculated from a 542 bp sequence of the
	COI gene in MEGA version 6. Diagonal element represents within
	region/crop divergence.

Acknowledgement

The authors are grateful to MSc Branislav Trudi c, Melita Dejanovi c, Dragan Kerkez, Dragana Dragomirov, Mladen Djuran and Milena Mar ci c from the Forecasting and warning service in plant protection of Serbia for the help in the field work and Sylvie Warot from INRAE for the help in the laboratory work.

Funding

The study was supported by the Forecasting and warning service of plant protection in Serbia, by the grant 'BASF Srbija d.o.o.', by INRAE and Universit e Côte d'Azur (grant 'TRICHOBIO' and 'BOOST' from the UCA JEDI IDEX programme ANR-15-IDEX-0001) in France.

Disclosure statement

No potential conflict of interest was reported by the authors.