**1. Introduction**

Maize (*Zea mays* L.) is one of the most important crops worldwide. About 200 million hectares is planted, with an average yield of 22 tons/hectare, resulting in 1150 million tons of maize harvested worldwide [1]. The western corn rootworm (WCR) *Diabrotica virgifera virgifera* is the worst pest in the United States and a major alien invasive pest in Europe [2,3]. The main damage caused by WCR to maize plants is by its larval stage that feeds on corn roots, which affects important physiological processes of the plant. The resulting damage leadstostalklodgingandyieldlosses,whichinturnleadstoeconomicdamagetocrops[4].

Suppression with chemical insecticides is an important managemen<sup>t</sup> tool for this pest [5], but WCR has rapidly developed resistance to the insecticides used for control [6]. The first noted case of resistance to insecticides was to cyclodiene insecticides (aldrin and heptachlor) in 1959 in Nebraska [7,8]. So far, WCR has evolved resistance to organophosphates (methyl parathion), carbamates (carbaryl) [6,9], and pyrethroids (bifenthrin and

**Citation:** Kadoi´c Balaško, M.; Mikac, K.M.; Benítez, H.A.; Bažok, R.; Lemic, D. Genetic and Morphological Approach for Western Corn Rootworm Resistance Management. *Agriculture* **2021**, *11*, 585. https://doi.org/10.3390/ agriculture11070585

Academic Editor: Stanislav Trdan

Received: 13 May 2021 Accepted: 22 June 2021 Published: 24 June 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

tefluthrin) [10,11]. In addition to insecticides, WCR has developed resistance to crop rotation [12–14] and to the *Bt* toxin in genetically modified maize [15]. Crop rotation remains the most effective control tactic against WCR. However, resistance to crop rotation has been documented in Illinois and other neighboring states [12]. Spencer et al. [16] observed that some of the WCR populations in northern Indiana and east central Illinois feed on soya bean foliage and flowers, as well as lay eggs in soya bean fields. This behavioral change in the WCR populations in the eastern Corn Belt has eliminated the effectiveness of crop rotation as a rootworm managemen<sup>t</sup> option. As a consequence, the use of soil and foliar insecticides for WCR has increased to protect corn following soya bean. It was estimated that each year WCR costs US farmers at least USD 1 billion through yield losses and treatment costs [17], but after adaptation to crop rotation, these losses are estimated to be higher [18]. Transgenic maize expressing *Bacillus thuringiensis* (*Bt*) was introduced in 2003 in the United States [15]. However, resistance to maize expressing Cry3Bb1 was reported in Iowa in 2009 [19]. Afterwards, resistance to Cry3Bb1 was detected in fields throughout Iowa [20,21] but also in WCR populations found in Illinois, Nebraska, and Minnesota [22–24]. Selected rootworm populations developed resistance to the toxins Cry34/Cry35Ab1, Cry3Bb1, and mCry3A under laboratory and greenhouse conditions [25–28]. Cross-resistance was found in WCR field populations between the Cry3Bb1, mCry3A, and eCry3.1Ab toxins [21–23,29]. WCR populations evolved resistance to all four currently available Bt toxins (Cry3Bb1, mCry3A, eCry3.1Ab, and Cry34/35Ab1) [19,23,29–31], and consequently, the challenge of managing has become more difficult.

Resistance is a dynamic phenomenon, meaning that mechanisms already known can change over time. Ongoing monitoring is essential to determine whether managemen<sup>t</sup> recommendations remain valid or need to be revised in light of changing circumstances or newly acquired knowledge [32]. WCR resistance to insecticides and managemen<sup>t</sup> strategies is a serious and growing problem in maize production, and before it becomes an even more widespread and major problem, there is a need to explore and implement novel methods (such as single nucleotide polymorphisms and geometric morphometrics) for the early detection of resistance or adaptation that causes WCR resistance.

Population genetic markers can be used to provide genetic data for WCR that is useful when investigating changes in genetic structure and differentiation [3,33,34]. Different types of molecular markers (allozymes, mtDNA sequencing, AFLPs, microsatellites, and SNPs) have already been used in North American WCR populations. The result showed high genetic diversity and a general lack of population structure across the US Corn Belt [35–37].

Several studies on WCR resistance mechanisms have been performed [38–40]. Coates et al. [41] attempted the use of SNPs as population genetic markers in WCR in the US and showed that both markers (microsatellites and SNPs) gave similar results. This does not sugges<sup>t</sup> that SNPs are less effective at separating genetic variation in the species, but it is likely a result of low numbers of SNPs and low genome coverage because the authors used 12 biallelic loci among 190 individuals. Wang et al. [40] found that cylcodiene resistance is correlated with SNPs in the gamma-aminobutyric acid (GABA) receptor. Flagel et al. [42] used SNPs to identify candidate gene families for insecticide resistance and to understand how population processes have shaped variation in WCR populations. Their WCR transcriptome assembly included several gene families that have been implicated in insecticide resistance in other species and that have provided a foundation for future research. Flagel et al. [43] discovered and validated genetic markers in WCR associated with resistance to the *Bt* toxin Cry3Bb1. They found that the inheritance of Cry3Bb1 resistance is associated with a single autosomal linkage group and is almost completely recessive. Niu et al. [44] found that SNP markers identified in a single autosomal linkage group (LG8, 115–135 cm) were correlated with resistance to Cry3Bb1 in field populations of WCR. Although the linkage of these genes to Cry3Bb1 resistance was strong, the causal gene for Cry3Bb1 resistance was not confirmed and remains to be reported.

Geometric morphometrics (GM) (i.e., phenotype size and shape analysis) is a technique that can be used to show hindwing shape and size differences among rootworm populations [45]. By analyzing wing size and shape, it is possible to reveal the invasive adaptation of the adults' traits to different environmental influences. Numerous studies have been performed on the WCR hindwings using geometric morphometry [46–49]. Mikac et al. [46] provided preliminary evidence of wing shape and size differences in WCR from rotated versus continuous maize. Most recently, Mikac et al. [45] determined morphological differences in wing shape in populations adapted to crop rotation and *Bt* maize compared with a non-resistant WCR population. This study showed evidence of differential wing shape in relation to resistance development and highlights the importance of wing size and shape as a reliable, inexpensive, ye<sup>t</sup> effective biomarker for resistance detection in corn rootworm. The research of Mikac et al. [45] looked at the *Bt*-resistant individuals as a whole, so it is necessary to extend their research to each *Bt* toxin separately. A deeper understanding of maize rootworm wing shape and flight morphology, wing geometry, aspect ratio, and flight efficiencies will help identify which resistant phenotypes are most likely to invade geographic areas where they are not ye<sup>t</sup> present.

According to Bouyer et al. [50], changes in an organism's genotype takes much longer to manifest than in its phenotype, thus making geometric morphometrics a much more useful tool than genetics for detecting changes in populations in the short term. That suggests morphology can retain useful information on genetic structure and has the benefit over molecular methods of being inexpensive, easy to use, and able to yield a lot of information quickly. However, resistance cannot be fully understood without genetic data. Genetic studies are an important tool for developing improved methods for detecting resistance, for studying resistance mechanisms, and for choosing approaches to resistance managemen<sup>t</sup> [51]. Several studies sugges<sup>t</sup> that results are more accurate when both methods are combined. Morphological traits can provide additional information about underlying population genetics, and morphology can retain useful information about genetic structure [52–56].

This is the first study that combines both genetic and geometric morphometric techniques on the same WCR populations and same individuals. The aim of this study was to define genetic variables between known phenotypes and to explore phenotypic markers related to changes in the genome. We hypothesized that by combining genetic and morphological markers, it would be possible to determine and predict resistance to *Bt* toxins and crop rotation in the field.

#### **2. Materials and Methods**
