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Dataset of the paper entitled methods for high-throughput screening of novel agents against the maize pest, Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae)
<p>Title: Methods for high-throughput screening of novel agents against the maize pest, Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae) </p> <p>Authors: Sri Ita Tarigan, Gyorgy Turoczi, Jozsef Kiss, Stefan Toepfer</p> <p>Abstract: <br>The western corn rootworm, <em>Diabrotica virgifera virgifera</em> (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 <sub>50</sub>, ED <sub>80</sub> 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.</p> <p>Data:</p> <p>The data file is related to the screening of commercial insecticides against eggs, first instar larvae (L1) and adults of the maize pest, <em>Diabrotica virgifera virgifera</em> 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.</p> <p>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 23-25<sup>0</sup>C 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.</p> <p>To assess the effect and dose-responses of commonly used insecticides on neonates of <em>D. v. virgifera</em>, 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 2/3<sup>rd </sup>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 cm<sup>2</sup> 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. </p> <p>To access the effect and dose-responses of common insecticides on <em>D.v.virgifera</em> 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 cm<sup>3</sup>). 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 cm<sup>3</sup>). 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 23-25<sup>0</sup>C, 50–90% r.h, L: D 12:12. Adult mortality were recorded on days 1,3, 5, 7 of experiment. </p> <p>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 <sub>50,80</sub>) were calculated. </p> <p>The raw data as well as the standardised data are available as a csv file on zenodo. </p> <p> </p> <p> </p>
Figure 1 Predation success ofG. aculeifer, S in Predation capacity of soil-dwelling predatory mites on two major maize pests
Figure 1 Predation success ofG. aculeifer, S. scimitus andM. robustulus on WCR and WW first instar larvae during the 10-minutes predation assays. n=20. NS = no significant difference among predator species (p-value> 0.05). The error bars represent the 95% confidence interval for the predation success.
Data from:Climate-specific dynamics of fall armyworm on maize: Implications for pest monitoring and management
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Data from: ‘Becoming a species by becoming a pest’ or how two maize pests of the genus Ostrinia possibly evolved through parallel ecological speciation events
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Data from: Species composition and community structure of small pest rodents (Muridae) in cultivated and fallow fields in maize growing areas in Mayuge district, Eastern Uganda
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Figure 6 from: Li Q, Hu H, Triapitsyn S, Yi L, Lu J (2018) Anagrus dmitrievi sp. n. (Hymenoptera, Mymaridae), an egg parasitoid of Zyginidia eremita (Hemiptera, Cicadellidae), a pest of maize in Xinjiang, China. ZooKeys 736: 43-57. https://doi.org/10.3897/zookeys.736.20883
Figure 6 Records of Zyginidia eremita in Xinjiang. The large star denotes the type locality of Anagrus dmitrievi.
Figure 2 from: Li Q, Hu H, Triapitsyn S, Yi L, Lu J (2018) Anagrus dmitrievi sp. n. (Hymenoptera, Mymaridae), an egg parasitoid of Zyginidia eremita (Hemiptera, Cicadellidae), a pest of maize in Xinjiang, China. ZooKeys 736: 43-57. https://doi.org/10.3897/zookeys.736.20883
Figure 2 Anagrus dmitrievi (female, holotype): A ovipositor (arrows pointing to distal setae on one of its external plates, or second valvifers) B fore and hind wings.
Figure 1 from: Li Q, Hu H, Triapitsyn S, Yi L, Lu J (2018) Anagrus dmitrievi sp. n. (Hymenoptera, Mymaridae), an egg parasitoid of Zyginidia eremita (Hemiptera, Cicadellidae), a pest of maize in Xinjiang, China. ZooKeys 736: 43-57. https://doi.org/10.3897/zookeys.736.20883
Figure 1 Anagrus dmitrievi (female, holotype): A slide B antennae (complete antenna without mps on F4; F4 of the incomplete antenna with 1 mps) C body D mesoscutum (arrows pointing to adnotaular setae on its midlobe).
Figure 4 from: Li Q, Hu H, Triapitsyn S, Yi L, Lu J (2018) Anagrus dmitrievi sp. n. (Hymenoptera, Mymaridae), an egg parasitoid of Zyginidia eremita (Hemiptera, Cicadellidae), a pest of maize in Xinjiang, China. ZooKeys 736: 43-57. https://doi.org/10.3897/zookeys.736.20883
Figure 4 A parasitized eggs of Zyginidia eremita by Anagrus dmitrievi in a maize leaf B an adult female of A. dmitrievi right after emergence C an adult male of A. dmitrievi.
Figure 1 from: Li Q, Hu H, Triapitsyn S, Yi L, Lu J (2018) Anagrus dmitrievi sp. n. (Hymenoptera, Mymaridae), an egg parasitoid of Zyginidia eremita (Hemiptera, Cicadellidae), a pest of maize in Xinjiang, China. ZooKeys 736: 43-57. https://doi.org/10.3897/zookeys.736.20883
Figure 1 - Anagrus dmitrievi (female, holotype): A slide B antennae (complete antenna without mps on F4; F4 of the incomplete antenna with 1 mps) C body D mesoscutum (arrows pointing to adnotaular setae on its midlobe).
Figure 6 from: Li Q, Hu H, Triapitsyn S, Yi L, Lu J (2018) Anagrus dmitrievi sp. n. (Hymenoptera, Mymaridae), an egg parasitoid of Zyginidia eremita (Hemiptera, Cicadellidae), a pest of maize in Xinjiang, China. ZooKeys 736: 43-57. https://doi.org/10.3897/zookeys.736.20883
Figure 6 - Records of Zyginidia eremita in Xinjiang. The large star denotes the type locality of Anagrus dmitrievi .
Figure 4 from: Li Q, Hu H, Triapitsyn S, Yi L, Lu J (2018) Anagrus dmitrievi sp. n. (Hymenoptera, Mymaridae), an egg parasitoid of Zyginidia eremita (Hemiptera, Cicadellidae), a pest of maize in Xinjiang, China. ZooKeys 736: 43-57. https://doi.org/10.3897/zookeys.736.20883
Figure 4 - A parasitized eggs of Zyginidia eremita by Anagrus dmitrievi in a maize leaf B an adult female of A. dmitrievi right after emergence C an adult male of A. dmitrievi .
Figure 2 from: Li Q, Hu H, Triapitsyn S, Yi L, Lu J (2018) Anagrus dmitrievi sp. n. (Hymenoptera, Mymaridae), an egg parasitoid of Zyginidia eremita (Hemiptera, Cicadellidae), a pest of maize in Xinjiang, China. ZooKeys 736: 43-57. https://doi.org/10.3897/zookeys.736.20883
Figure 2 - Anagrus dmitrievi (female, holotype): A ovipositor (arrows pointing to distal setae on one of its external plates, or second valvifers) B fore and hind wings.
Predation test results and dynamics between three species of soil-dwelling predatory mites and early stages of maize pest.
<p>PREDATORY MITES</p> <p> The three species used in this experiment were stored in climatic chambers at 25°C +/- 0,5°C and 70% +/- 10 RH% with constant obscurity. A mix of <em>Aleuroglyphus ovatus</em> stages was used as food and extra water was provided three times a week in a 100 mm x 94 mm bugdorm-5002 with 30µm nylon screen port sold by Bugdorm©.</p> <p><em>Macrocheles robustulus</em></p> <p> Koppert Biological systems provided <em>Macrocheles robustulus</em>. Their product is called Macro-mite©. We maintained them on vermiculite for 2 months with a mix of <em>A. ovatus</em> stages.</p> <p><em>Gaeolaelaps aculeifer</em></p> <p><em> </em><em>Gaeolaelaps aculeifer</em> is produced by EWH Bioproduction, Denmark. The population was maintained during 8 months on a substrate made of 1/3 third blond sphagnum peat and 2/3 of fine vermiculite and fed with a mix of <em>A. ovatus</em> stages.</p> <p><em>Stratiolaelaps scimitus</em></p> <p> <em>Stratiolaelaps scimitus</em> individuals used in this experiment are produced by Bioline AgroSciences. The product is called Hypoline©. This population has been maintained on blond sphagnum peat and fed with a mix of <em>A. ovatus</em> stages for 2 years.</p> <p>PREYS</p> <p> We experimented eggs and first instar larvae for both <em>Diabrotica virgifera virgifera</em> and <em>Agriotes sordidus </em>as potential prey. We also added <em>Aleuroglyphus ovatus</em> eggs as a positive control of predation activity since astigmatid mites are known to be a suitable food source for those species (Rueda-Ramirez et al. 2018).</p> <p><em>Diabrotica virgifera virgifera</em> eggs</p> <p> WCR diapausing eggs were provided by the Centre of Agriculture and Bioscience International (CABI), Hungary. They were stored at 7°C +/- 0,5°C below their temperature of development (Meinke et al. 2009). We sieved the eggs from their substrate and selected only turgescent eggs to offer them to the predatory mites.</p> <p><em>Diabrotica virgifera virgifera</em> first instar larvae</p> <p> We placed WCR eggs on the plaster of Paris in a climatic chamber at 25°C +/- 0,5°C and 70% +/- 10 RH%. We added water twice a week to keep the plaster of Paris moist. We checked daily if eggs hatched and introduced the first instar larvae in the predation device.</p> <p><em>Agriotes sordidus</em> eggs</p> <p> Arvalis provided <em>Agriotes sordidus</em> eggs and first instar larvae by sending us a couple of adults ready to lay eggs in Petri dishes filled with a sample of soil where they have been collected. Both eggs and first instar larvae have been extracted from this dirt.</p> <p><em>Aleuroglyphus ovatus</em> eggs</p> <p><em> </em><em>A. ovatus</em> eggs are produced by Bioline AgroSciences. Eggs were sterilized before presentation to the predatory mites.</p> <p><em>Ephestia kuehniella </em>eggs<br> </p> <p><em> E. kuehniella </em>eggs are produced by Bioline Agrosciences. Eggs were sterilized before presentation to the predatory mites.</p> <p> PREDATION DEVICE</p> <p> Predation tests have been inspired by El Adouzi, Bonato, et Roy 2017; Lovis et al. 2011 and Nordenfors et Hoglund 2000 protocols by isolating each mite individually. However, we chose to carry out the predation tests in 2 mL Eppendorf tubes containing each 1 mL of dried plaster of Paris to maintain a high percentage of humidity necessary to soil-dwelling predatory mites survival (El Adouzi, Bonato, et Roy 2017). Adult mites of both sexes were individually isolated and starved for 7 days in the tubes before the predation tests. In total, 240 predatory mites have been isolated with 1/3 of each species to present them to 4 different types of prey. Twenty predation tests were made by prey/predator couple. <br> During the 7-days period of starvation, we added 100µL of water every 3 days to maintain a suitable relative humidity necessary for soil-dwelling predatory mites survival. We also drilled the top of the tube and covered it with a 106 µm mesh width nylon tissue. This size of mesh allowed for water and gas exchange while preventing mites from leaving the tube. These tubes were stored in a climatic chamber at 25°C +/- 0,5°C with 70% +/- 10% RH. All three species were active after this period of storage and starvation.</p> <p> We introduced 20 times one prey in a tube containing a predatory mite and observed predation activity during a maximum of 10 minutes or less if predation happens before that timing. We observed each mite feeding or non-feeding activity through the tube with a binocular. We used an indirect source of light, controlled at 100 lux (measured with the Digital Illuminance meter TES 1335), to minimize natural behavior disruption of these lucifugous species. During each assay, the timing and number of contacts between the predator and the prey before predation were noted. We considered predation activity when mites impaled the prey with their chelicerae. We chose to observe predation on a short duration because some of the prey could be impacted by plaster of Paris abrasive texture if it dries up.</p>
Data from: Estimation of the dispersal of a major pest of maize by cline analysis of a temporary contact zone between two invasive outbreaks
Dispersal is a key factor in invasion, and in the persistence and evolution of species. Despite the importance of estimates of dispersal distance, dispersal measurement remains a real methodological challenge. In this study, we characterized dispersal by exploiting a specific case of biological invasion, in which multiple introductions in disconnected areas lead to secondary contact between two differentiated expanding outbreaks. By applying cline theory to this ecological setting, we estimated σ, the standard deviation of the parent-offspring distance distribution, of the western corn rootworm, Diabrotica virgifera virgifera, one of the most destructive pests of maize. This species is currently invading Europe, and the two largest invasive outbreaks, in Northern Italy and Central Europe, have recently formed a secondary contact zone in Northern Italy. We identified vanishing clines at 12 microsatellite loci throughout the contact zone. By analyzing both the rate of change of cline slope and the spatial variation of linkage disequilibrium at these markers, we obtained two σ estimates of about 20 km.generation−1/2. Simulations indicated that these estimates were robust to changes in dispersal kernels and differences in population density between the two outbreaks, despite a systematic weak bias. These estimates are consistent with the results of direct methods for measuring dispersal applied to the same species. We conclude that secondary contact resulting from multiple introductions is very useful for the inference of dispersal parameters and should be more widely used in other species.
Figure 3 from: Li Q, Hu H, Triapitsyn S, Yi L, Lu J (2018) Anagrus dmitrievi sp. n. (Hymenoptera, Mymaridae), an egg parasitoid of Zyginidia eremita (Hemiptera, Cicadellidae), a pest of maize in Xinjiang, China. ZooKeys 736: 43-57. https://doi.org/10.3897/zookeys.736.20883
Figure 3 Anagrus dmitrievi (male, paratypes): A antenna B fore wing C genitalia.
Figure 5 from: Li Q, Hu H, Triapitsyn S, Yi L, Lu J (2018) Anagrus dmitrievi sp. n. (Hymenoptera, Mymaridae), an egg parasitoid of Zyginidia eremita (Hemiptera, Cicadellidae), a pest of maize in Xinjiang, China. ZooKeys 736: 43-57. https://doi.org/10.3897/zookeys.736.20883
Figure 5 Life cycle of Zyginidia eremita: A, B, egg C–G nymphs H–J adults.
Figure 5 from: Li Q, Hu H, Triapitsyn S, Yi L, Lu J (2018) Anagrus dmitrievi sp. n. (Hymenoptera, Mymaridae), an egg parasitoid of Zyginidia eremita (Hemiptera, Cicadellidae), a pest of maize in Xinjiang, China. ZooKeys 736: 43-57. https://doi.org/10.3897/zookeys.736.20883
Figure 5 - Life cycle of Zyginidia eremita : A, B, egg C–G nymphs H–J adults.
Figure 3 from: Li Q, Hu H, Triapitsyn S, Yi L, Lu J (2018) Anagrus dmitrievi sp. n. (Hymenoptera, Mymaridae), an egg parasitoid of Zyginidia eremita (Hemiptera, Cicadellidae), a pest of maize in Xinjiang, China. ZooKeys 736: 43-57. https://doi.org/10.3897/zookeys.736.20883
Figure 3 - Anagrus dmitrievi (male, paratypes): A antenna B fore wing C genitalia.
Data from: Estimation of the dispersal of a major pest of maize by cline analysis of a temporary contact zone between two invasive outbreaks
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