DOI: 10.5281/zenodo.11634320
title: Dataset of the paper entitled methods for high-throughput screening of novel agents against the maize pest, Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae)

description: Title: Methods for high-throughput screening of novel agents against the maize pest, Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae) 

Authors: Sri Ita Tarigan, Gyorgy Turoczi, Jozsef Kiss, Stefan Toepfer

Abstract: The western corn rootworm, Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae), poses a significant threat to maize crops in North America and Europe, necessitating development of novel, effective, and less disruptive crop protection agents. With recent bans on key insecticides and concerns about overuse of remaining options, there is an urgent need for accessible and comparable screening methods. We propose comparative high-throughput screening methods against the eggs, larvae and adults of this pest, emphasizing the importance of suitable positive controls tailored to the specific bioassay types. We evaluated seven common insecticides (imidacloprid, clothianidin, acetamiprid, novaluron, cypermethrin, chlorpyrifos-methyl, spinosad) against eggs, larvae, and adults as potential positive controls for each of the proposed assay methods. Dipping assays with ready-to-hatch eggs revealed several ingredients to cause mortality; but imidacloprid might be most suitable as a positive control due to its robust dose-response in reducing egg hatching and causing mortality of hatching neonates. Larval bioassays using artificial diet overlay assays revealed mortality caused by all insecticides, with imidacloprid and acetamiprid exhibiting best dose-mortality response curves as well as sublethal effects. Adult bioassays using artificial diet-core overlay assays revealed mortality caused by all insecticides, with cypermethrin or acetamiprid exhibiting best dose-mortality response curves. The provided ED 50, ED 80 values, and dose-response equations offer valuable insight for researchers in selecting appropriate positive controls for screening new crop protection agents or assessing resistance levels against different life stages of this pest.

Data:

The data file is related to the screening of commercial insecticides against eggs, first instar larvae (L1) and adults of the maize pest, Diabrotica virgifera virgifera using standard bioassays. We are proposing comparative high-throughput screening methods against the eggs, larvae and adults of this maize pest. This includes the crucial aspect of suitable positive controls tailored to the specific bioassay type. We evaluated seven common insecticides (imidacloprid, clothianidin, acetamiprid, novaluron, cypermethrin, chlorpyrifos-methyl, spinosad) against eggs, larvae, and adults as potential positive controls for each of the proposed assay method. To access effects and dose-responses of commonly used insecticides on eggs, we applied standard screening methods under controlled semi-sterile conditions.

For egg bioassays, eggs were transferred to the 200 ml of treatments in the eppendorf tubes and then soaked for 1 hour. Then 20µl with 10 to 20 eggs were pipetted onto a filter paper in a petri dish (150 mm×25 mm). Then 100 µl of sterilized tap water was added for moisture. The pipette tip was replaced between treatments. The eggs been transferred were counted per filter paper and dish (15± 8). The eggs were then incubated in the dishes at 23-250C for 7 days, when the experiment was terminated. Egg hatching, mortality of newly hatching larvae, and days until start of egg hatching were observed under stereo microscope and recorded.  Data were collected at 1,3, 5 and 7 days after treatments.

To assess the effect and dose-responses of commonly used insecticides on neonates of D. v. virgifera, we applied artificial diet-overlay bioassays under controlled semi-sterile conditions. Each insecticide was prepared in at least six concentrations. Each bioassay consisted of 3 to 6 polystyrene plates of 96 wells each (07-6096 of Biologix Ltd., USA, or Costar 3917 of Corning Inc., USA). Each well had a volume of 330 µl, with a diameter of 5 mm, a height of 10 mm, and a surface area of 0.34 cm². 190 µl of the diet were pipetted into each 330 µl well, filling each to approximately 2/3rd of its capacity. Plates containing the diet were left to dry in a laminar flow cabinet for 45 minutes and then stored overnight at temperatures ranging from 3 to 5°C. The following day, treatments were applied. This is, 17 µl of a treatment was applied to the 0.34 cm2 diet surface reaching good coverage and therefore forcing the after-placed larvae to feed through (10 to 100 µl pipette Biohit TM Proline). Each treatment was applied to 8 wells per plate. Following application, the plates were allowed to dry for a duration of 1 to 1.5 hours and were subsequently cooled for 1 hour in a refrigerator set at temperatures between 23 to 25°C. Each well received one neonate larva, carefully placed on the diet surface using a fine artist brush. A vigorous and visibly healthy larva was selected, lifted from the end of the abdomen with the brush, maneuvered towards a well surface, and allowed to crawl off the brush onto the diet. To avoid systematic errors, larvae were not arranged in treatment column order but rather in a rectangular pattern. After every 12 individual larvae, the brush was cleaned using 70% ethanol followed by sterile tap water. The filled plate was sealed with an optically clear adhesive qPCR seal sheet (#AB-1170, Termo Scientific, USA, or #BS3017000, Bioleader, USA), enabling data assessments without the need to open the plate. Four to five holes were carefully made with fine 00-insect pins into the seal per well to facilitate aeration. The plates, housing the larvae, were then incubated in a dark, ventilated incubator at a temperature of 23-25 °C and a relative humidity of 50 to 90% for a period of 5 days. We assessed mortality and stunting larvae within 3 and 5 days. 

To access the effect and dose-responses of common insecticides on D.v.virgifera adults, artificial diet-overlay bioassays with different doses were performed under controlled, semi-sterile conditions. Each insecticide was prepared in at least six concentrations. Active ingredients as specified on the product labels underwent serial dilutions using sterile tap water. Sterilized tap water was used as untreated control. In detail, each bioassay consisted of 6 polystyrene plates of 6 wells each (Eppendorf® 0030720016). Each treatment was applied to 3 wells of each plate per bioassay. The adult diet for a bioassay had been prepared 1-7days before treatment and adult infestation. The diet was prepared under semi-sterile conditions. The diet was poured out to 5-6 sterile 11 mm Petri dishes. The plates with diet were allowed to dry for up to 15 minutes under laminar flow cabinet then stored at 3 to 5°C overnight.The following day, a core of the diet was initially transferred to each well using flamed iron core-cutter (1 cm diameter) under a laminar flow. A core diet was placed each of the 6 wells of the plates. Approximately 40 µl of the treatments were then applied across the surface of diet core (0.34 cm3). The following day, a core of the diet was initially transferred to each well using flamed iron core-cutter (1 cm diameter) under a laminar flow. A core diet was placed each of the 6 wells of the plates. Approximately 40 µl of the treatments were then applied across the surface of diet core (0.34 cm3). Adult were subsequently transferred from the rearing cage into the wells of the 6-well plates containing the diet and treatments using a tube aspirator. For ease of transfer, the adults were cooled in a fridge for 4 to 7 minutes. Each well plate received 3 to 4 adults. Plates were sealed and incubated at 23-250C, 50–90% r.h, L: D 12:12. Adult mortality were recorded on days 1,3, 5, 7 of experiment. 

To allow comparisons between experiments, data were standardized to the data of the corresponding negative control, usually sterilized tap water, as follows: standardized data = 100 × (data in negative control - data in treatment)/maximum (data in control or in treatment). The distributions of the data were investigated using histograms and QQ normal and detrended normal probability. Skewness and kurtosis of residuals was also observed for normality of influences of treatments on eggs, neonates, or adults. Equality of variances was assessed using Levene’s test. Multiple comparisons were performed using the Tukey HSD post hoc test for data with equal variances and the Games-Howell post hoc test for data with unequal variances. For each tested insecticide, linear and logarithmic regression models were fit to the dose-response data. In case of significant linear or logartimic relathionships, doses leading to 50% or 80% of relative effects (ED 50,80) were calculated.  

The raw data as well as the standardised data are available as a csv file on zenodo.