Susceptibility of Xylotrechus arvicola (Coleoptera: Cerambycidae) to Five Cry Toxins †

: The beetle Xylotrechus arvicola is a significant pest in vineyards ( Vitis vinifera ) in the wine-producing regions of the Iberian Peninsula. X. arvicola larvae bore the grapevine wood and make galleries, which cause structural damages to the plant and a decrease in the quality and quantity of its production. The susceptibility of X. arvicola larvae to five coleopteran toxic Cry proteins (Cry1B, Cry1I, Cry3A, Cry7A


Introduction
Xylotrechus arvicola Olivier (Coleoptera: Cerambycidae) is a significant grape pest (Vitis vinifera) in the Iberian wine-producing regions [1]. X. arvicola females lay the eggs on crevices or under the rhytidome of the vine [2]. The hatched larvae enter the stems producing galleries due to their feeding. In about 2 years, they pupate, resulting in the emergence of adults in about an additional month. The larvae cause structural damage to the stems, but also, the spread of fungi within the wood [3]. They can only be controlled with chemically synthesized systemic pesticides [4], which are legally complicated to use in vineyards.
The controlled evaluation of pesticides against this insect species is challenging, as the conditions for a laboratory rearing that fulfills the biological cycle has not yet been established. However, adult insects can be captured in the fields, and larvae maintained some time on a semisynthetic diet. This way, insecticides with different modes of action have been evaluated on both stages [4], but active substances with low environmental impact are still needed.
The most successful pesticide products in organic farming are based on Bacillus thuringiensis (Bt), a bacterium that produces pesticidal crystal proteins (Cry proteins). Each Cry protein has a narrow insect toxicity spectrum, and the most studied ones have been proteins with lepidopteran species as a target. However, several Cry proteins were reported toxic to a few coleopteran species or to have activity against both orders [5]. Still, most of the genera, within the Coleoptera order, with pest species have not yet been evaluated [6].
The aim of this research was to evaluate under laboratory conditions, and for the first time, the toxicological potential against a larval stage of the coleopteran cerambycid X. arvicola of different Cry proteins with known coleopteran activity

X. Arvicola Collection and Rearing
The protocol followed has been adapted from a previous study evaluating insecticide activity against X. arvicola adults: Insect adults were captured using the Crosstrap ® interception traps (Econex, Spain) [7] in vineyards located in Gordoncillo (León, Spain). Insects captured were paired and put into glass jars, letting them mate. The base was covered with filter paper. The insects had access to substrates for oviposition and bowls for drinking. Laid eggs were taken out and put into Petri dishes. X. arvicola neonate larvae used in the tests were obtained from these eggs. Adults and larvae, before and after the application of treatments, were kept in a chamber with controlled temperature (24 ± 1 °C), humidity (60 ± 5%), and photoperiod of 16:8 (light: darkness).

Cry Proteins
The Cry1Ba, Cry1Ia, Cr3Aa, Cry7Ab, and Cry23/37 proteins were prepared as reported by Rodríguez-González et al. [8]. Concisely, they were obtained from recombinant strains of B. thuringiensis and Escherichia coli that produced a single protein. The bacteria were washed and lysed in a Carbonate buffer. The quantity and quality of the Cry protein in preparations was evaluated by 12% SDS-PAGE analysis. Each solution was lyophilized to powder for storage.

Bioassays of Cry Proteins on Artificial Diet to X. Arvicola Larvae
Bioassays were carried out using newly X. arvicola neonate larvae (≤24 h) by the surface contamination method [9]. An artificial diet was used to fill 12-well bioassay trays (2 cm 2 /well) (Greiner CELLSTAR ® 12 well plates, Sigma-Aldrich Chemie GmbH, Steinheim, Germany). The diet was surface-sterilized for 10 min under UV light. Each well was inoculated with 100 μl of the protein solution obtained after suspending the lyophilized powder distilled water at a concentration of 100 μg/mL allowed to dry under a laminar flow hood. Once dried, each well contained approximately 1 μg/cm 2 of Cry protein, and one larva was transferred to each well and confined with a lid. Three replicates of 12 well plates (36 larvae in total) were used per treatment. Larval mortality was rated within 30 days. The larvae were considered dead if they did not react when prodded. As the control treatment, the diet inoculated with 100 μL of Na2CO3 50 mM, pH 10.5 buffer was used.

Statistical Analysis
Recorded mortalities were corrected with Abbott's formula [10] for each treatment. These data were used to calculate the means and standard error of the means (SEM) for the mortality values observed for each Bt Cry protein treatment.
Mortality rates from the five Cry treatments were put through one-way ANOVA. Differences among the treatments were examined by mean comparisons using the post-hoc Least Significant Difference (LSD) comparison test, considering statistically significant P value ≤ 0.05. Statistical analyses were performed using the SPSS version 26 software (IBM, 1968, NY, USA) (SPSS).

Results
A conventional bioassay was set up to evaluate X. arvicola larval susceptibility to Cry proteins. It provided positive results since treatment mortality were higher than the control (one-way ANOVA test, F = 2.097; df = 5, 66; p = 0.043) and provided small errors (Figure 1). All protein treatments showed statistically significant differences in mortality rates among them, except Cry1Ba and Cry7Ab treatments that showed similar mortality rates with the best larvicidal efficacy (killed 83% of treated larvae). Cry1Ia and Cry23/37 rendered intermediate larval mortality rates, with Cry1Ia showing higher rates. Cry3Aa (killed 50% of larvae) was the protein with the lowest mortality rate.

Figure 1.
Corrected mortality (% ± SE) of X. arvicola neonate larvae exposure to 1 μg/cm 2 of Cry proteins applied over artificial diet. The Abbott's formula was used for correction. Different letters on the bars indicate statistically significant differences (LDS post-test) among the mortality rates.

Discussion
The evaluation of insecticide active substances against coleopteran pests with a long and cryptic biological cycle is a challenge. The laboratory tests are the initial steps to find them, but it is arduous to set it up precisely. We have successfully applied the bioassay protocol used on X. arvicola larvae with other pesticides [11]., However, the accuracy of the results could be discussed, since some toxicological effects do not depend on the Cry proteins, which can degrade in a long treatment period. Nevertheless, we can assess the results since we assume the generally accepted fact that the main insect toxicity effect of Cry preparation relies on the Cry proteins in the sample, and, with the reported data, we have, at least, preliminary toxic information.
The cry proteins evaluated belong to very different classes and show high activity to X. arvicola larvae ranging from 50% to 83% of mortality after 30 days of evaluation. Chen et al. [12] reported similar results showing the toxic effect of the Bt strain Bt866 (with a cry3Aa gene) against two cerambycid species, Apriona germari and Anoplophora glabripennis.
Our current studies suggested that Cry proteins may minimize the damage caused by the X. arvicola larvae. It may be beneficial to develop these proteins as a bio-insecticide to apply them to vineyards during the emergence of the X. arvicola adults between June and July in the wine-producing regions with PDO [13].

Conclusions
The Cry proteins evaluated have demonstrated different toxicity activity against the insect pests assayed, with over 50% mortality rates in all cases. Cry1Ba and Cry7Ab showed the most aggressive responses. The larval stage tested is previous to drilling in the plant, which makes spray treatments feasible. The results can help in designing combinations of Cry proteins as biopesticides to apply them by the time these larvae hatch to increase vine wood protection.

Conflicts of Interest:
The authors declare no conflict of interest.