*2.6.2 Meteorus*

In 1990, in Tamaulipas and Nuevo Leon, Mexico, it was reported t*o Meteorus* prob. *laphygmae*, *M.* prob. *versicolor,* and four more species of *Meteorus*, unknown up to that time [48]. Other species of *Meteorus* have been reported over time, but there are few studies regarding their genomic sequences.

In Durango, Mexico, the genus *Meteorus* is mostly distributed in Santiago, Papasquiaro, and Durango, probably due to variations in climate and altitude (**Figure 1**). It belongs to the region of Las Quebradas and the other municipalities to the region of valleys and smooths.

From 2012 to date, studies have been carried out in various locations in municipalities located in the center and north of the state of Durango, which has allowed us to know the diversity of fall armyworm parasitoids. **Figure 1** shows the sampled municipalities.

**Figure 1.** *Distribution of the Braconidae subfamily in Durango, Mexico.*

#### *Genetic Delimitation of Fall Armyworm Parasitoids Isolated in Maize in Durango, Mexico DOI: http://dx.doi.org/10.5772/intechopen.112612*

*Ch. insularis* is the parasitoid that is mostly distributed in Durango and Mexico [54]; however, in the last two years, in Durango *Meteorus* sp., it is the parasitoid that presents greater capacities to be massively reproduced in the laboratory due to its development on an artificial diet (data not yet published).

#### **2.7 Morphological delimitation**

#### *2.7.1 Chelonus*

Specimens with morphological characters to belong to this genus were separated using the taxonomic keys of [50]. Species identification was carried out by PhD. Alejandro Gonzalez-Hernandez, through the comparison of the preserved material with reference specimens from the Collection of Entomophagous Beneficial Insects of the Facultad de Ciencias Biologicas de la Universidad Autonoma de Nuevo Leon, Mexico.

#### *2.7.2 Meteorus*

The obtained parasitoids were labeled and preserved in 70% alcohol. The Meteorinae (Euphorinae) material was studied at the Insect Museum (MI-FA) of the Universidad Autonoma de Tamaulipas, where it was mounted and labeled using the EntoPrint program with the respective collection data. For the determination of the subfamily and genus, we used the keys of [50] while for the determination of the species the keys of [55, 56]. For Durango, Dgo., Mexico, it has been reported to *Meteorus arizonensis* Muesebeck (Hymenoptera: Braconidae) y *Meteorus laphygmae* Viereck (Hymenoptera: Braconidae); however, their morphological characters do not coincide 100% with the taxonomic keys because they present color patterns in the mesosome that could indicate that they are other species. In this regard, [57] pointed out upon the unreliable color variability in identifying species. In fact, the color pattern is a variable that might be affected by environmental conditions [58].

The color patterns in *M. arizonensis* and *M. laphygmae* should not be considered as distinctive to identify a species; in *Meteorus,* there were nine morphotypes or different color patterns in the mesosome of the specimens of this species, even so, genetically they all belong to the same species; however, in this regard, [59] indicate that this property (melanism) increases the flight activity of wasps at low ambient temperatures of *M. pulchicornis* (Wesmael); they subjected this parasitoid (coconuts) to different temperatures (15, 20, 25 and 30°C); it was observed that at the lowest temperatures, the body of the parasitoid darkened more, which could indicate that the color change in some morphological characters of the parasitoids is due to the change in their body temperature and the environment in which they develop and not that they are different species. Similar situation with *Chelonus*, where coloration patterns in the metasome indicate that *Ch. insularis* and *Ch. sonorensis* belong to the same species [29, 32].

#### **2.8 Genetic delimitation**

#### *2.8.1 DNA extraction*

Twenty-seven individuals belonging to *Meteorus laphygmae* with five individuals, followed by *M. arizonensis* with four specimens, *Chelonus insularis* with 14 specimens, *Ch. sonorensis* with two specimens, and *Ch. cautus* with three specimens, separated according to their morphological characteristics, were used.

Total genomic DNA was isolated per individual using the Promega DNA extraction kit, following the manufacturer's instructions with some modifications. Briefly, the digestion time was modified taking a total of 16 h at 56°C in a dry bath with continuous shaking. The next step was cleaning the aqueous phase with salt precipitation of detergent, proteins, and lipids followed by an organic solvent cleanup (adding 350 μl of chloroform-isoamyl alcohol 24:1), mixed it by inversion for 20 s each sample and centrifuge it by five minutes to recover the aqueous phase in a new tube (1.5 ml).

The DNA precipitation was reached by adding 1.5 volumes of cool isopropanol, followed by storing samples at −20°C for 12–16 h. Samples were cleaned with cool ethanol 80% two times. After washing samples with ethanol, those were dried and hydrated with 60 μl of milli-Q water.

From the isolated DNA, a fragment of the mitochondrial cytochrome c oxidase I (COI) gene was amplified in the individuals of the species analyzed using oligonucleotides HCO-2198 (5′-TAA ACT TCA GGG TGA CCA AAA AAT CA-3′) (forward) and LCO-1490 (5′-GGT CAA CAA ATC ATA AAG ATA TTG G-3′) (reverse) reported by [60]. PCR conditions were 1 min 30 s at 94°C, denaturation 35 cycles at 94°C (1 min), alignment at 50°C (1 min), extension at 72°C for 1 min, and a final extension step at 72°C (15 min) in a thermal cycler (Model 9600, Labnet International, Edison, NJ) using 50–150 ng of DNA, 0.40 pmol of each oligonucleotides, 2.5 mM of MgCl 2, 0.2 mM of each dNTPs (Promega, Madison, WI), 1× of polymerase chain reaction buffer, and 1 unit of Taq polymerase (Promega) in a final volume of 50 μl [53, 61].

Amplified PCR products in 1% agarose gels stained with ethidium bromide were visualized by electrophoresis and observed at 430 nm in a UV transilluminator. The double-chain products were purified using a Wizard SV Gel and PCR Clean-up purification system. The amplified products were sequenced on a Genetic Analyzer Applied Biosystems 310 using the method of big dye terminator (Applied Biosystems Inc., Foster City, CA). The sequence files were edited and aligned using Chromas Pro ver. 2.1.10.1.

The sequences were translated into proteins to confirm the identity of the fragments [62]. Multiple alignments used Clustal X [63] with gap opening costs = 50, gap extension = 6.6, divergent delay of sequences = 30%, and DNA transition weight = 0.5 [64]. Genetic diversity in the species was measured as haplotype diversity (h), number of private haplotypes (P), and nucleotide diversity (p) analyzed using Arlequin version 3.5.1.21 and DnaSP version 5.1 [65]. Arlequin version 3.5.1.21 [66] was used for analysis of molecular variance (AMOVA) of population structure. Genetic differences between individuals were analyzed. Sum of the squares of deviation (SSD) and index of Harpending's-Raggedness [67] were calculated to evaluate the fit of the observed data using a model of sudden demographic expansion or a model of geographic range expansion. The mismatch distribution was compared with expected distributions by models of sudden population expansion [68] and spatial expansion [69, 70].

To reconstruct phylogenetic relationships, we used Bayesian inferences. We used *Campoletis sonorensis* Cameron, *C. flavicincta* (Ashmead), and *Homolobus truncator* Say as outgroups to polarize the characters within samples of *Meteorus* spp. and *Chelonus* spp.

Bayesian analysis used the GTR model with invariant rate heterogeneity. A posterior probability analysis [71] was performed using the program MrBayes version 3.0b4 [72]. Bayesian posterior probability calculations were implemented in a range of ten million generations, sampling every 1000 generations, and discarding the first

#### *Genetic Delimitation of Fall Armyworm Parasitoids Isolated in Maize in Durango, Mexico DOI: http://dx.doi.org/10.5772/intechopen.112612*

1000 trees sampled (as burn-in). Support for nodes was determined by posterior probabilities [72, 73]. For Bayesian analysis, three independent runs were conducted to get an impression of the robustness of the phylogenetic reconstruction.

The amplified fragments were between 695 and 710 bp, the sequences were editing and adjusting them to 650 bp. Regarding the AMOVA, the maximum distribution of variance was observed when two groups were formed: group 1: *Ch. insularis* + *Ch. sonorensis* and group 2: *Ch. cautus*; this indicates that the groups are different from each other (FSC = 0.02289, p = 0.011 at 95% confidence), there is no difference within them (FST = 0.97679, p = 0.43 at 95% confidence) (**Figures 2** and **3**), these were compared with group 3: *Ch. insularis* from different states of Mexico, the key assigned in the GeneBank® is indicated [53].

The unpaired distribution of DNA (Mismatch distribution) showed two peaks that reinforce the existence of two groups corresponding to group 1 and group 2, and each of them presented a sudden population increase.

#### *2.8.2 Phylogeny*

The median-joining networks of *Meteorus* and *Chelonus* haplotypes did not reveal divergent clusters of haplotypes by phenotype or color. Rather, the COI networks are star-shaped [74], whereas the network shows no structure, indicating that the phylogenetic information given by these sequences is adequate for phylogenetic inference. The trees generated by the Bayesian analyses are mostly unresolved within analyzed species, and the clusters that are formed may contain sequences from different localities and different morphospecies.

The phylogenetic affinities showed that the analyzed specimens of *Ch. insulares* and *Ch. sonorensis* are in a single group, where both are mixed (without forming different groups); in turn, this group is separated from another group of sequences belonging to *Ch. insularis* from other parts of Mexico. In the case of *Ch. cautus*, it is observed that the sequences obtained in this work tend to form a single group that is separated from

**Figure 3.** *Phylogenetic tree showing the groups of Chelonus sp. de Durango, compared to other species.*

*Chelonus insularis* + *Ch. sonorensis;* it is reinforced by genetic distances between the two morphospecies of *Chelonus* (G. D. = 0.005) that occur in Durango, Mexico suggests that there is a reproductive isolation among populations that occurs in central and southern parts of Mexico contrasted with species from Durango. In the case of *M. laphygmae* and *M. arizonensis,* the phylogenetic affinities are like that described earlier, it means that the two morphospecies of *Meteorus* from Durango form a single genetic group or *M. laphygmae* + *M. arizonensis* represents a single species with a genetic distance closed to zero (G. D. = 000001) and the genetic distances with other valid species of the same genus are up to 5%, as shown in **Table 2** (G. D. higher than 0.050).
