Advanced Screening Methods for Assessing Motility and Hatching in Plant- Parasitic Nematodes

Advanced Screening Methods for Assessing Motility and Hatching in Plant- Parasitic Nematodes | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Method Article Advanced Screening Methods for Assessing Motility and Hatching in Plant- Parasitic Nematodes Alena Kadlecová, Romana Hendrychová, Tomáš Jirsa, Václav Čermák, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4235543/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 20 Jul, 2024 Read the published version in Plant Methods → Version 1 posted 10 You are reading this latest preprint version Abstract Background Plant-parasitic nematodes are economically important pests responsible for substantial losses in agriculture. Researchers focusing on plant-parasitic nematodes often need to assess basic parameters such as their motility, viability, and reproduction. Traditionally, these assays involve visually counting juveniles and eggs under a dissecting microscope, making this investigation time-consuming and laborious. Results In this study, we established a procedure to efficiently determine the motility of two plant-parasitic nematode species, Heterodera schachtii and Ditylenchus destructor , using the WMicrotracker ONE platform. Additionally, we demonstrated that hatching of the cyst nematode H. schachtii can be evaluated using both the WMicrotracker ONE and by assessing the enzymatic activity of chitinase produced during hatching. Conclusions We present easy and straightforward protocols for studying nematode motility and hatching that allow us to draw conclusions about viability and survival. Thus, these methods are useful tools for facilitating fast and efficient evaluation in various fields of research focused on plant-parasitic nematodes. The methods should also be compatible with other plant-parasitic nematode species. Plant-parasitic nematodes Heterodera schachtii cyst nematodes Ditylenchus destructor protocol methods screening motility viability hatching WMicrotracker ONE chitinase. Figures Figure 1 Figure 2 Figure 3 Figure 4 1. BACKGROUND Plant-parasitic nematodes (PPN) are significant pathogens affecting nearly all major agricultural crops ( 1 ). Given their economic importance, many nematologists have focused on monitoring PPN in the field or studying their biology and plant-parasite interactions to identify new avenues for plant breeders or potential molecular targets for nematicide development. Various substances or biological agents that can interfere with nematode survival or behaviour have also been studied ( 2 ). This research requires the performance of assays to evaluate basic characteristics such as survival, motility, and hatching of PPNs. Traditionally, these assays involve visually observing and counting nematodes and eggs under a dissecting microscope, making them time-consuming and laborious ( 3 ). Several high-throughput methods for performing similar assays have been optimized for the model nematode Caenorhabditis elegans ( 4 ) and, to some extent, for some species of mammalian parasitic nematodes ( 5 – 7 ). These methods are based on various principles, including automatic image acquisition and analysis ( 8 , 9 ), large object flow cytometry systems ( 10 ), and many others. While some of these methods require expensive machines, many can be performed using relatively affordable or common laboratory equipment. Surprisingly, attempts to adapt such methods for PPN appear to be relatively rare. In this work, we present a simple method for evaluating the motility of nematodes using the WMicrotracker ONE. This method provided reliable results for both motile infective juveniles (J2) of the sedentary cyst nematode Heterodera schachtii and the migratory endoparasitic nematode Ditylenchus destructor , suggesting that the platform is compatible with various PPN species. Furthermore, we describe two robust and easy methods for determining the hatching of H. schachtii – one utilizing the WMicrotracker ONE and the other based on measuring the activity of the enzyme chitinase. 2. METHODS 2.1. Nematode cultivation 2.1.1. Maintenance of Heterodera schachtii The stock culture of nematodes was maintained on mustard ( Sinapsis alba cv. Albatros) roots grown in vitro on modified Knop media according to previously published protocols ( 11 ). Mustard seeds were sterilized by successive treatment with 70% ethanol (1 min), 1.3% NaClO (5 min) and 96% ethanol (1 min) and washed 3 times with sterile double distilled water (ddH 2 O). Seeds were allowed to germinate on 0.8% H 2 O agar for 2 days in the dark at 25°C, and healthy seedlings (3 per plate) were then transferred onto 15 cm Petri dishes containing modified Knop medium supplemented with 3% sucrose ( 11 ). The plants were subsequently grown at 25°C under a 16-hour light regime. After 2–3 weeks, each plate was inoculated with approximately 300 H. schachtii J2. The infected plates were kept at 25°C in the dark. Mature cysts of the nematodes could be collected from the infected plates after approximately 2 months, when the worms had completed their life cycle, and females had transformed into mature cysts that were apparent on the roots. 2.1.2. Maintenance of Ditylenchus destructor The initial population of D. destructor was extracted from a hop plant (Central Bohemia/CZ) and maintained on carrot discs according to previously published protocols ( 12 ). Briefly, to prepare the discs, the carrots were surface-sterilized with 1% NaClO for 30 minutes, washed in sterile water in the hood, peeled and cut into ca. 1.5 cm pieces with sterilized equipment. The discs were placed on 10 cm Petri dishes and kept at 25°C in the dark until white callus specks were apparent (approximately 4 weeks). Plates that were not used immediately were stored in the refrigerator. The plates were inoculated with 50–60 nematodes per disc. In our experience, the best time to collect healthy populations for experiments is after 2–4 weeks at 25°C, when the discs start to develop brown colouration. 2.1.3. Collecting nematodes for experiments The motile H. schachtii J2 required for the tests were collected from funnels filled with 3 mM ZnCl 2 to increase the hatching rate of the nematodes ( 13 ). Approximately 300 cysts were placed in a sieve (60 µm mesh size) in the funnel so that it was approximately half covered with the liquid. The hatched J2 passed through the sieve and settled at the exit of the funnel, which led to a silicone tube closed with a clip. By opening the clip, the hatched J2 could be easily collected. The best time to collect a healthy population of J2 for the experiment was between 3 and 10 days after dissection of the cysts. Juveniles can be collected repeatedly from a funnel. For experiments with D. destructor , 5 ml of sterile ddH 2 O was added to plates containing infected carrot discs. Nematodes naturally migrate from the discs into the water. To increase the yield, the discs were partially submerged in water for approximately 30 min. The liquid containing nematodes was transferred from the plates to Eppendorf tubes. In case any debris from the carrot discs was collected, the nematodes were washed several times with water prior to the experiment (by allowing the nematodes to settle on the bottom of the tubes and exchanging the liquid). Populations containing a mixture of different developmental stages were used for the experiments. The concentration of nematodes was determined by counting the number of living nematodes in 3 10 µL drops. The suspension was further diluted with sterile ddH 2 O to achieve the desired final concentration. 2.2. Evaluation of nematode motility using WMicroTracker ONE The WMicrotracker ONE device (Phylumtech S.A.) emits an infrared beam that passes through the wells of a microtiter plate. Moving animals scatter light, and interference is subsequently detected. The instrument evaluates the activity in all wells continuously and then displays the number of these interferences (“activity counts”) per user-defined time interval (“bin”). All the data presented in this study show the number of activity counts detected in wells in 30-minute bins. The suspension was distributed into U-bottom 96-well plates (54 µL per well). The plates were kept in an incubator set to 20°C for 20–30 min prior to the measurement to allow the nematodes to settle on the bottom of the wells. Afterwards, the plates were placed into WMicrotracker ONE device, and the initial motility of the worms was recorded for 30 minutes. The device was operated according to the manufacturer’s instructions. Six microliters of the tested control chemicals (sodium hypochlorite and sodium azide at a concentration 10 times greater than the desired final concentration) or sterile ddH 2 O water was added to each well (at least 4 wells per condition), and the motility of the populations was remeasured using WMicrotracker ONE at different time points. Between the measurements, the experimental plates were sealed with parafilm or PCR seal, kept at 20°C, and gently shaken on an orbital shaker (150 rpm) to ensure aeration of the suspension. 2.3. Evaluation of Heterodera schachtii hatching 2.3.1. Measuring the movement of J2 emerging from cysts using WMicrotracker ONE The wells of a U-bottom 96-well plate were filled with 54 µL of sterile ddH 2 O or 3 mM ZnCl 2 . Three cysts were collected from the maintenance plate and placed into each well, while trying to ensure that cysts of similar size and colour were evenly distributed across the wells. After measuring the initial motility on WMicrotracker ONE (which should be close to 0 because no juveniles have yet emerged), 6 µL of the control chemical (ethanol at a concentration 10 times higher than the desired final concentration) or sterile ddH 2 O was added to each well. Due to the greater inherent variability of this assay, at least 8 wells per condition were used. The experimental plates were kept under the same conditions and remeasured at different time points, as described in section 2.2. 2.3.2. Measuring hatching using WMicrotracker ONE Approximately 300 cysts were collected from maintenance plates or retrieved from funnels previously used for other experiments and placed into a 100 ml glass bottle filled with 3–5 ml of sterile ddH 2 O or 3 mM ZnCl 2 . A medium-sized stirring bar was added to the bottle, and the cysts were crushed on a magnetic stirrer (1000 rpm, 5 min). The suspension was passed through a sieve (30 µm pore size) to remove smaller debris and some J2 that had already hatched inside of the cysts. The sieve was placed bottom up on a piece of mesh (116 µm pore size) and washed with 3–5 ml of ddH 2 O. The liquid passing through the mesh was collected. This step removes larger debris. The final suspension was enriched in eggs but was not completely clean, as some J2 and mid-sized debris were also collected. The concentration of eggs was determined by counting the number of intact eggs in three 10 µL drops under a microscope. Approximately 50 eggs per well were used. The experimental plates were prepared, stored, and measured as described in section 2.3.1. 2.3.3. Evaluation of hatching by the chitinase assay This assay is based on measuring the enzymatic activity of chitinase ( 14 ). The enzyme is produced by hatching juveniles to dissolve the chitin-containing eggshells. Its activity, which correlates to the number of viable juveniles, can be measured by the addition of a fluorogenic substrate. The egg suspension was prepared as described in section 2.3.2. A total of 400–800 eggs per well were used, and heat-killed eggs (55°C, 2 hours, these conditions were adapted from ( 26 )) served as a positive control. The experimental plates were stored as indicated in the previous sections for 7 days. Afterwards, 20 µM of the chitinase substrate (4-methylumbelliferyl β-D-N,N′,N″-triacetylchitotrioside, stock diluted in DMSO) was added, and the plates were incubated at 37°C for 1 hour. Alkaline buffer (1 M glycine, 1 N NaOH, pH = 10.6) was added at a 1:2 ratio (i.e., 30 µL to 60 µL of liquid in our case). The fluorescence was measured on a TECAN Infinite PRO plate reader (λex 365 nm, λem 460 nm, gain manually set to 65). 2.4. Data analysis The data were analysed in GraphPad Prism. All the data are displayed as the mean ± standard deviation (SD). The data were analysed by repeated measures two-way analysis of variance (RM ANOVA) followed by Dunnet’s post hoc test. To assess the quality of the assay, the Z factor ( 14 ) was calculated from the mean values and standard deviations using the following formula: $$Z=1-\frac{\left(3\times SD of sample\right)+\left(3\times SD of control\right)}{mean of sample-mean ofcontrol}$$ 3. RESULTS AND DISCUSSION Searching for new strategies for PPN control (i.e., screening of compounds, biological substances, etc.) is an important matter of research but is laborious and time-consuming. Here, we present several easy, robust, and straightforward assays with the potential to be developed into semi- or high-throughput screening tools. The assays described below are based on two different principles described in parts 2.2. and 2.3.3. 3.1. WMicrotracker ONE is a suitable tool for measuring the movement of plant-parasitic nematodes The WMicrotracker ONE was previously successfully used to analyse the locomotion and behaviour of not only the free-living model nematode C. elegans ( 15 ) but also multiple species of mammalian parasitic helminths ( 7 , 16 ), as well as insect and zebrafish larvae ( 17 ). Our experiments (Fig. 1 ) show that the platform can be used in a similar manner for PPN, including both migratory species and motile developmental stages of sedentary species, which are generally considered the most economically important ( 1 ). To validate the method, we exposed the experimental populations of D. destructor (mixed population) and H. schachtii (infective J2) to 2 toxic chemicals, sodium azide (10 mM) and sodium hypochlorite (1.4%). We chose to use these compounds as a positive control due to their reliable effect. During the selection process, we also tested the effect of a variety of commonly used anthelmintic drugs, such as levamisole, ivermectin and mebendazole, with unsatisfactory results (data not shown). These drugs are likely unable to efficiently penetrate the cuticle of nematodes ( 18 ). Better results would presumably be obtained if the nematodes were actively feeding and ingesting the substance during the exposure ( 19 ). It is important to consider this when, for example, testing new promising substances for possible nematicidal activity. We measured the motility of the treated and control nematodes for 2 hours immediately after the addition of the compounds and again 3 days after treatment (Fig. 1 ). As expected, both species showed a rapid decline in movement after exposure to both substances. After 30 minutes, the activity was reduced by 73.8% (from mean 258.9 activity counts to 67.7) in populations of D. destructor treated with NaN 3 , 84.9% (from mean 243.9 activity counts to 36.9) in D. destructor treated with NaClO, 98.9% (from mean 227.8 activity counts to only 2.4) in H. schachtii exposed to NaN 3 , and 79.7% (from mean 241.6 activity counts to 49) in H. schachtii in NaClO. Less than 1 mean activity count could be detected in populations of both nematode species treated with both substances after 3 days. At the same time, nematode motility in the controls remained consistent, with no decrease in activity counts detected in the short-term experiment and only a small decrease in motility (6–8% on average) after 3 days. According to our results, approximately 100–150 worms per well should be used for smaller and less active nematodes such as H. schachtii J2. For D. destructor and other more active PPN species, 30–50 nematodes per well are sufficient. Using plates with round bottoms allows nematodes to accumulate more closely and further stimulate each other’s movement by touch, resulting in higher detected activity counts than in plates with flat bottoms. This finding is in agreement with the information published on the website of Phylumtech S.A., the manufacturer of WMicrotracker ONE. The wells should contain approximately 40–100 µL of liquid in total so that the bottom of the plate is fully covered. Too large volumes of liquid in wells could hinder aeration of the suspension, especially during longer experiments. The assay is very efficient and easy to evaluate, especially in comparison to the visual counting of moving nematodes under a microscope. Nematodes cultivated in vitro can be harvested in relatively large quantities. This makes the assay suitable for high-throughput screening. To assess the quality of the assay, we calculated the Z factor ( 14 ). Values exceeding 0.5 (a threshold for the assay to be considered excellent) were achieved both overall and in all individual replicates, in longer experiments as well as in shorter assays, at 90 min and longer for H. schachtii and 120 min for D. destructor (supplementary table 1 ). This indicates that the assay is reliable and could presumably be used for screening larger libraries of compounds or other substances for nematicidal activity, similar to what was shown for nematodes parasitizing mammals ( 7 ). 3.2 Both the WMicrotracker ONE and the chitinase assay are suitable for evaluating the hatching of Heterodera schachtii The hatching rate of a PPN and the ability of a (putative) control agent to decrease it are important parameters. H. schachtii cysts can contain hundreds of eggs that remain vital in the soil for many years and are awaiting optimal conditions to hatch and infect a host plant ( 20 ). Thus, we established two streamlined protocols allowing us to reliably evaluate reproductive capacity of PPN. To obtain the best possible results, an important step in preparing these assays is acquiring a clean egg suspension. Unwanted debris can hinder the experiment in several ways. Poor visibility of the eggs in the suspension can make accurate determination of their concentration difficult. Larger pieces of debris tend to block the pipette, resulting in uneven distribution of the egg suspension. Reducing the amount of debris is especially crucial when using the WMicrotracker ONE device, where extra material present in wells could interfere with proper signal detection. The isolation of clean eggs from C. elegans can be performed simply by dissolving juveniles and adult nematodes in bleach ( 21 ). However, this process is not suitable for PPN, where eggs are usually extracted by passing the suspension through a series of sieves or using floating and centrifugation ( 3 , 22 ). In the case of H. schachtii , we mechanically crushed the cysts and then carefully passed the suspension through 2 sieves with different pore sizes (see section 2.3.2 for details). While the final suspension was not completely debris or juvenile free, it was sufficient to achieve reliable data of acceptable quality using both methods described. 3.2.1. The chitinase assay The chitinase assay was originally described for evaluating hatching in C. elegans ( 23 ), and a modified version of this assay was used to test the toxic effects of various compounds on C. elegans ( 24 , 25 ). As mentioned above, the principle of the assay involves the addition of the fluorogenic substrate 4-methylumbelliferyl β-D-N,N′,N″-triacetylchitotrioside. The reagent is cleaved by the nematode-produced enzyme chitinase, leading to the release of fluorescent 4-methylumbelliferone that can be detected. Our results show that the assay can also be used with PPN eggs (Figs. 2 , S1 and S2). As a positive control, we used heat-inactivated eggs We avoided using chemicals due to possible unwanted interactions. For instance, sodium azide, which was used as a control in previous assays, was reported to directly inhibit the activity of some chitinases ( 27 ). On the other hand, sodium hypochlorite, our other control substance, could negatively interact with ZnCl 2 , in which the eggs were incubated. Our rationale for using both water and ZnCl 2 was based on the reported ability of zinc salts to stimulate the hatching of several members of the genus Heterodera , including H. schachtii ( 13 , 28 ). Eggs of some species of PPN, including Heterodera spp., usually only start to hatch after they detect suitable environmental conditions and/or cues from nearby hosts. In our experiment, we observed a noticeable increase in the number of juveniles after 1 week of incubation of the eggs in both water and ZnCl 2 (Fig. S3), suggesting that hatching occurs even without additional stimuli. An increased number of juveniles correlated with increased detected activity of the enzyme chitinase. In the initial experiments (Fig. 2 A and S1), we determined that a concentration of 400 eggs per well was sufficient to achieve a reproducible, statistically significant difference between the positive and negative controls. The signal detected was approximately 1.7–2 times greater (increase of 71–125 RFU – relative fluorescence units) for the eggs incubated in both water and ZnCl 2 than for the heat-killed control. The difference was more obvious when more eggs were used, with 1600 eggs per well providing a signal more than 4 times greater than that of the positive control. Nevertheless, as the process of egg preparation is relatively laborious, using as few eggs as possible is desirable. To ensure that 400 eggs were indeed sufficient, we repeated the experiment 5 more times with 800 and 400 eggs only (Fig. 2 B and S2). In all experiments, we again observed a statistically significant increase in signal intensity in wells containing 400 eggs compared to the positive control (between 161 and 450 RFU increase in water and 124 and 245 RFU increase in ZnCl 2 -incubated eggs). Again, the difference was more pronounced when 800 eggs were used (between 351 and 835 RFU increase in water and 246 and 544 RFU increase in ZnCl 2 -incubated eggs). Although the variance among the technical replicates within one plate was relatively low, we observed notable differences in the detected signal strength among the biological replicates. This can likely be attributed to factors such as differences in the age of the maintenance plates from which H. schachtii cysts were collected, inaccuracies in determining the egg concentration resulting in slight inconsistencies in egg population size, and other similar sources of biological variability. Therefore, we recommend presenting the data from each repetition as an individual graph. All the biological replicates followed the same significant trend, validating the reliability of the assay. Interestingly, we observed that the detected chitinase activity was slightly lower in the wells with ZnCl 2 than in the wells where the eggs were incubated in water. A possible explanation could be the direct interference of zinc with the enzyme. Heavy metals, including zinc, were previously reported to inhibit the activity of some chitinases ( 29 ). Nevertheless, this effect is clearly not significant enough to affect the ability of the juveniles to hatch, and the differences in chitinase activity in ZnCl 2 -incubated eggs and positive controls are reproducibly statistically significant. Therefore, researchers may use both depending on their specific needs. Although this assay could be easily adapted for many other nematode species, an obvious drawback could be its incompatibility with those species that can produce chitinase in life stages other than during hatching. For these cases, further optimization of the egg cleaning process would likely be necessary to ensure that the suspension used for the experiment contained only eggs. Another limiting factor is the need for a relatively large number of eggs. While the assay is undoubtedly more efficient than visual counting of hatched juveniles, one experiment typically requires several thousand eggs. This might make collecting a sufficient number of cysts for a large experiment quite tedious and unsuitable for high-throughput experiments. The assay might also not be useful in cases where only a limited amount of material is available, such as for monitoring the reproductive capacity of Heterodera spp. collected from the field. This is why we decided to evaluate the hatching of H. schachtii by detecting an increase in motility caused by the presence of newly hatched active juveniles via the WMicrotracker ONE. 3.2.2. Measuring the motility and hatching of Heterodera schachtii using the WMicrotracker ONE We tested two different setups—measuring the increase in motility in wells with intact cysts and in wells containing the egg mixtures prepared in the same way as for the chitinase assay. In both cases, we again compared the data for populations incubated in water and in ZnCl 2 . Here, the reported stimulatory effect of zinc salt on the hatching of Heterodera spp. was clearly apparent (Fig. 3 ). In wells containing eggs incubated in ZnCl 2, we detected a 60% increase in motility compared to water (195.9 mean activity counts compared to 78.8). For intact cysts, the difference was 44% (229.9 mean activity counts compared to 128.4). Therefore, we used ZnCl 2 in all subsequent experiments. To validate the robustness of the two assay setups, various concentrations of ethanol were used as a positive control, demonstrating the dose-dependent effect of the substance at various time points (Fig. 4 ). For both setups, we observed greater variability among the technical replicates, while the effect remained consistent among the biological replicates. We assume that this is due to inherent variability among the cysts and eggs and recommend using at least 8 technical replicates per condition to mitigate this issue. Furthermore, we noticed that the age of the plates from which the cysts were collected can significantly influence how soon hatching starts. Therefore, we recommend using maintenance plates of approximately the same age across all biological replicates. In the setup with intact cysts, we recommend using 3 cysts per well. Using fewer than 3 further increases the variability among technical replicates, while more cysts in the well seem to interfere with the instrument´s ability to measure the activity properly. For the egg suspension, we recommend using 50 eggs per well. The suspension obtained from crushed cysts, while enriched in eggs, also contained some J2 and mid-size debris. The presence of J2 at the beginning of the test results in some activity being detected in the wells during the initial measurement prior to adding the substances. These J2 tend to become inactive relatively quickly, likely due to stress caused during the preparation process. Accurately assessing the actual increase in the activity over time as the J2 hatch, while nematodes that were already present became inactive, might become difficult when a more concentrated mixture (with a stronger initial signal) is used. Another factor that might hinder the acquisition of reliable data is the presence of debris, which might interfere with beams passing through the wells. Using 50 eggs per well limits the number of unwanted juveniles and debris sufficiently, allowing the detection of a clear increase in activity counts over time due to hatching events. The difference between the positive and negative controls was reproducible and statistically significant in all biological replicates. For intact cysts, we observed a gradual increase in activity counts from approximately 10–16 during the initial measurement in all conditions to 220.1 mean activity counts in control wells after 1 week. For ethanol-treated cysts, the values obtained at the same time point were 209.2, 159.2, 26.7 and 2.9 for 0.88, 1.75, 3.5 and 7% ethanol, respectively. The effect was dose-dependent and ranged from none or marginal to severe. For isolated eggs, the initial mean activity counts ranged from approximately 33 to 50 under all conditions for the reasons outlined above. The movement rates in the wells gradually increased to approximately 150 mean activity counts at days 5 and 7 in both the control wells and the wells treated with 0.88% ethanol. A less prominent increase, to 111.6 and 73.8 mean activity counts after 1 week, was apparent in populations treated with 1.75 and 3.5% ethanol, respectively. The addition of 7% ethanol led to a decrease in the mean activity counts after 1 week compared to the initial value, to only 20.1. The chitinase assay has two obvious advantages over determining hatching activity via the WMicrotracker ONE. First, the presence of either J2 or debris does not interfere with the readout. Second, the population size of 50 eggs is relatively small and more prone to random error, contributing to greater deviation among technical replicates here than in the chitinase assay. Further cleaning of the suspension might be helpful, although we opted to not focus on this step further, as the assay provided reproducible and statistically significant data. Moreover, unlike in the chitinase assay, the WMicrotracker ONE experiment performed significantly better when ZnCl 2 was used. Researchers should be mindful of possible unwanted interference of the solution with substances or biological agents they plan to test on cyst nematodes and assess their possible interactions beforehand. This problem will not apply to many other species of PPN that do not require zinc salts to stimulate hatching. We believe that both the chitinase test and the WMicrotracker ONE measurements are compatible with other nematode species. The egg isolation process will need to be modified to account for differences in egg size and other species-specific parameters. Many laboratories already have well-optimized protocols for the isolation of eggs of the species on which they are focused. Overall, the established protocols are valuable, as both the chitinase assay and the WMicrotracker ONE assay are significantly less labor intensive than commonly used procedures and allow the testing of dozens of substances/conditions. The protocols are a good basis for further optimizations and adjustments that might even make them suitable for high-throughput screening. For example, while we worked with a 96-well plate format only, WMicrotracker instruments, depending on the model, also allow switching to a 384-well plate format. The same is true for the plate readers used for the chitinase assay. The advantages of the presented methods over other very promising modern approaches, such as automated image acquisition and image analysis ( 30 ), include the ease of data analysis and the significantly lower cost of the instrumentation needed. As outlined above, the current bottlenecks are the relative laboriousness of preparing the egg suspensions and/or collecting the cysts, the biological variability, and, in the case of setups that work better with ZnCl 2 , the necessity to perform prior tests. 4. CONCLUSIONS In this study, we optimized several assays to evaluate the motility and hatching of PPN. We primarily focused on H. schachtii and included the migratory species D. destructor for comparison when determining motility. From our experiments, we conclude that the developed methods can also be used with other economically important PPN, either in the presented form or with minor species-specific modifications. The methods are based on the detection of nematode movement in multiwell plates and the quantification of enzyme activity using a fluorogenic substrate. Inspired by protocols previously optimized for the model C. elegans and other nematodes, they also proved to be compatible with PPN species. These methods offer a more efficient alternative to commonly used approaches. They can find use in various areas of basic and applied research as well as in laboratories focused on PPN monitoring. Abbreviations PPN Plant–parasitic nematode ddH 2 O Demineralized water J2 Second–stage juveniles RFU Relative fluorescence units Declarations Ethics approval and consent to participate: NA Consent for publication: NA Availability of data and materials: All data used in this study are presented in the manuscript or supplementary information. The raw data are available from the corresponding author upon reasonable request. Competing interests: The authors declare that they have no competing interests. Funding: The work on this project was made possible by Deutscher Akademischer Austauschdienst (DAAD) and Podpora mobility na UP II. (CZ.02.2.69/0.0/0.0/18_053/0016919), which funded AK´s stay at ASSS´s home institution. Furthermore, the project was supported by project TA ČR TQ03000647. Authors' contributions: AK – conceptualization, experimental design, performing part of the experiments, data analysis and preparation of the manuscript. TJ – performed part of the experiments and other technical assistance. RH – assisted with the experimental design and establishment of the methods. VC – assisted with the experimental design and establishment of the methods. MH – assisted with the experimental design and methods. FMWG – assisted with the experimental design, method establishment, and manuscript preparation. ASSS – mentoring, assistance with conceptualization, experimental design, and manuscript preparation. Acknowledgements: The authors gratefully acknowledge the excellent technical assistance provided by Ute Schlee and Stefan Neumann, Molecular Phytomedicine Group, INRES, University Bonn, Germany. Furthermore, we would like to thank Kateřina Mikušková from the Central Institute for Supervising and Testing in Agriculture, the Ministry of Agriculture of the Czech Republic. References Jones JT, Haegeman A, Danchin EGJ, Gaur HS, Helder J, Jones MGK, et al. Top 10 plant-parasitic nematodes in molecular plant pathology. Mol Plant Pathol. 2013;14(9):946–61. Atolani O, Fabiyi OA. Plant parasitic nematodes management through natural products: current progress and challenges. Management of Phytonematodes: Recent Advances and Future Challenges. Singapore: Springer Singapore; 2020. pp. 297–315. Van Bezooijen J. Methods and techniques for nematology. Wageningen, The Netherlands: Wageningen University; 2006. O’Reilly LP, Luke CJ, Perlmutter DH, Silverman GA, Pak SC. C. elegans in high-throughput drug discovery. Adv Drug Deliv Rev. 2014;69–70:247–53. Carr JA, Parashar A, Gibson R, Robertson AP, Martin RJ, Pandey S. A microfluidic platform for high-sensitivity, real-time drug screening on C. elegans and parasitic nematodes. Lab Chip. 2011;11(14):2385–96. Buckingham SD, Partridge FA, Sattelle DB. Automated, high-throughput, motility analysis in Caenorhabditis elegans and parasitic nematodes: Applications in the search for new anthelmintics. Int J Parasitol Drugs Drug Resist. 2014;4(3):226–32. Liu M, Landuyt B, Klaassen H, Geldhof P, Luyten W. Screening of a drug repurposing library with a nematode motility assay identifies promising anthelmintic hits against Cooperia oncophora and other ruminant parasites. Vet Parasitol. 2019;265:15–8. Wählby C, Kamentsky L, Liu ZH, Riklin-Raviv T, Conery AL, O’Rourke EJ, et al. An image analysis toolbox for high-throughput C. elegans assays. Nat Methods. 2012;9(7):714–6. Stroustrup N, Ulmschneider BE, Nash ZM, López-Moyado IF, Apfeld J, Fontana W. The C. elegans Lifespan Machine. Nat Methods. 2013;10(7):665–70. Pulak R. Techniques for analysis, sorting, and dispensing of C. elegans on the COPAS flow-sorting system. Methods Mol Biol. 2006;351:275–86. Sijmons PC, Grundler FMW, von Mende N, Burrows PR, Wyss U. Arabidopsis thaliana as a new model host for plant-parasitic nematodes. Plant J. 1991;1(2):245–54. O’bannon JH, Taylor AL. Migratory endoparasite nematodes reared on carrot discs. Phytopathology. 1968;58(3):385. Steele AE, Toxopeus H, Heijbroek W. A comparison of the hatching of juveniles from cysts of Heterodera schachtii and H. trifolii. J Nematol. 1982;14(4):588. Zhang JH, Chung TD, Oldenburg KR. A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J Biomol Screen. 1999;4(2):67–73. Hunt PR, Keltner Z, Gao X, Oldenburg SJ, Bushana P, Olejnik N, et al. Bioactivity of nanosilver in Caenorhabditis elegans : Effects of size, coat, and shape. Toxicol Rep. 2014;1:923–44. Camicia F, Herz M, Prada LC, Kamenetzky L, Simonetta SH, Cucher MA, et al. The nervous and prenervous roles of serotonin in Echinococcus spp. Int J Parasitol. 2013;43(8):647–59. Bichara D, Calcaterra NB, Arranz S, Armas P, Simonetta SH. Set-up of an infrared fast behavioral assay using zebrafish ( Danio rerio ) larvae, and its application in compound biotoxicity screening. J Appl Toxicol. 2014;34(2):214–9. Davies KG, Curtis RHC. Cuticle surface coat of plant-parasitic nematodes. Annu Rev Phytopathol. 2011;49:135–56. Zheng SQ, Ding AJ, Li GP, Wu GS, Luo HR. Drug absorption efficiency in Caenorhbditis elegans delivered by different methods. PLoS ONE. 2013;8(2):e56877. Subbotin SA, Mundo-Ocampo M, Baldwin JG, Hunt DJ, Perry RN. Systematics of cyst nematodes (Nematodes: Heteroderinae). Nematology monographs and perspectives. Leiden-Boston: Brill; 2010. pp. 1–135. Stiernagle T. Maintenance of C. elegans . WormBook. 2006;(1999):1–11. Mes THM, Eysker M, Ploeger HW. A simple, robust and semi-automated parasite egg isolation protocol. Nat Protoc. 2007;2(3):486–9. Ellerbrock BR, Coscarelli EM, Gurney ME, Geary TG. Screening for presenilin inhibitors using the free-living nematode, Caenorhabditis elegans . J Biomol Screen. 2004;9(2):147–52. Milišiūnaitė V, Kadlecová A, Žukauskaitė A, Doležal K, Strnad M, Voller J, et al. Synthesis and anthelmintic activity of benzopyrano[2,3-c]pyrazol-4(2H)-one derivatives. Mol Divers. 2020;24(4):1025–42. Nisler J, Pěkná Z, Končitíková R, Klimeš P, Kadlecová A, Murvanidze N, et al. Cytokinin oxidase/dehydrogenase inhibitors: outlook for selectivity and high efficiency. J Exp Bot. 2022;73(14):4806–17. Addabbo TD’, Sasanelli N, Greco N, Stea V, Brandonisio A. Effect of water, soil temperatures, and exposure times on the survival of the sugar beet cyst nematode, Heterodera schachtii. Phytopathology. 2005;95(4):339–334. Sirimontree P, Fukamizo T, Suginta W. Azide anions inhibit GH-18 endochitinase and GH-20 Exo b-N-acetylglucosaminidase from the marine bacterium Vibrio harveyi. J Biochem. 2016;159(2):191–200. Tefft PM, Bone LW. Zinc-mediated hatching of eggs of soybean cyst nematode, Heterodera glycines. J Chem Ecol. 1984;10(2):361–72. Donderski W, Swiontek Brzezinska M. The influence of heavy metals on the activity of chitinases produced by planktonic, benthic and epiphytic bacteria. Pol J Environ Stud. 2005;14(6):851–9. Akintayo A, Tylka GL, Singh AK, Ganapathysubramanian B, Singh A, Sarkar S. A deep learning framework to discern and count microscopic nematode eggs. Scienific Rep. 2018;8:9145. Additional Declarations No competing interests reported. Supplementary Files 20240804SIfinal.docx Cite Share Download PDF Status: Published Journal Publication published 20 Jul, 2024 Read the published version in Plant Methods → Version 1 posted Editorial decision: Revision requested 29 May, 2024 Reviews received at journal 28 May, 2024 Reviewers agreed at journal 20 May, 2024 Reviews received at journal 28 Apr, 2024 Reviewers agreed at journal 19 Apr, 2024 Reviewers agreed at journal 19 Apr, 2024 Reviewers invited by journal 14 Apr, 2024 Editor assigned by journal 12 Apr, 2024 Submission checks completed at journal 12 Apr, 2024 First submitted to journal 10 Apr, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4235543","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Method Article","associatedPublications":[],"authors":[{"id":292385589,"identity":"ca933683-3780-4293-a841-f4a4e169191f","order_by":0,"name":"Alena Kadlecová","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/UlEQVRIiWNgGAWjYDACdjCZwMBwHMKXA2LGA3i1MMO0HIbwjUEEaVoSGwhp4W9mPvbgB0OaPN9h5oMfPrbZpW+43XzgAENNHU4tEofZ0g17GHIMZx5mS5ac2Zacu+HOsYQDDMfYcFtzmMdMgoehgnEDkMHMc4Y5d8ONHIMDjA08OHXIH+b/JvmHocJ+A5AB1FKfbgDRIoFTi8FhHjZpHoacRKAtbMw8FYcToFoMcGoxPMxmJi1jkJYM9Iux5IyK44YzQX5JOJaAU4vc8eZnkm8qkm37jjc//PDBoFqe73bzwQcf8IQY1HnIHJAvcNuBFeD2+CgYBaNgFIxQAADSXVQx9LsoTgAAAABJRU5ErkJggg==","orcid":"","institution":"Palacký University","correspondingAuthor":true,"prefix":"","firstName":"Alena","middleName":"","lastName":"Kadlecová","suffix":""},{"id":292385591,"identity":"bfe09c6e-a608-4558-ac15-29b8cfdb7a39","order_by":1,"name":"Romana Hendrychová","email":"","orcid":"","institution":"Palacký University","correspondingAuthor":false,"prefix":"","firstName":"Romana","middleName":"","lastName":"Hendrychová","suffix":""},{"id":292385596,"identity":"6e1a1226-cf18-42a7-8082-2e50b52dd2af","order_by":2,"name":"Tomáš Jirsa","email":"","orcid":"","institution":"Palacký University","correspondingAuthor":false,"prefix":"","firstName":"Tomáš","middleName":"","lastName":"Jirsa","suffix":""},{"id":292385597,"identity":"cafde56e-bebf-4f8d-9296-a2a86f3c1856","order_by":3,"name":"Václav Čermák","email":"","orcid":"","institution":"Central Institute for Supervising and Testing in Agriculture","correspondingAuthor":false,"prefix":"","firstName":"Václav","middleName":"","lastName":"Čermák","suffix":""},{"id":292385598,"identity":"293e3e70-41a6-499e-8fee-696e42c52a7b","order_by":4,"name":"Mengmeng Huang","email":"","orcid":"","institution":"University of Bonn","correspondingAuthor":false,"prefix":"","firstName":"Mengmeng","middleName":"","lastName":"Huang","suffix":""},{"id":292385600,"identity":"dac451e8-d1af-4b63-8e50-f12bcfcf3de5","order_by":5,"name":"Florian M.W. Grundler","email":"","orcid":"","institution":"University of Bonn","correspondingAuthor":false,"prefix":"","firstName":"Florian","middleName":"M.W.","lastName":"Grundler","suffix":""},{"id":292385601,"identity":"e3b22001-789e-4380-97cd-86ccc9676ee6","order_by":6,"name":"A. Sylvia S. Schleker","email":"","orcid":"","institution":"University of Bonn","correspondingAuthor":false,"prefix":"","firstName":"A.","middleName":"Sylvia S.","lastName":"Schleker","suffix":""}],"badges":[],"createdAt":"2024-04-08 10:08:45","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4235543/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4235543/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13007-024-01233-z","type":"published","date":"2024-07-20T16:05:01+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":55060445,"identity":"a415baad-4dbf-4eb0-89fe-ab761a2bddc0","added_by":"auto","created_at":"2024-04-22 02:24:48","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":56800,"visible":true,"origin":"","legend":"\u003cp\u003eMeasuring the motility of plant-parasitic nematodes using the WMicrotracker ONE. A) Effect of short (2-hour) exposure to 2 toxic chemicals (NaN\u003csub\u003e3\u003c/sub\u003e and NaClO) on the movement of \u003cem\u003eDitylenchus destructor\u003c/em\u003e (mixed age). The graph shows the means + SDs from 6 biological (at least 29 technical) replicates. B) The effect of longer (3 days) exposure to 2 toxic chemicals on the movement of \u003cem\u003eD. destructor\u003c/em\u003e (mixed age). The graph shows the means + SDs from 8 biological (at least 55 technical) replicates. C) The effect of short (2-hour) exposure to 2 toxic chemicals on the movement of \u003cem\u003eHeterodera schachtii\u003c/em\u003e J2. The graph shows the means + SDs from 7 biological (at least 44 technical) replicates. D) The effect of longer (3 days) exposure to 2 toxic chemicals on the movement of \u003cem\u003eH. schachtii\u003c/em\u003e J2. The graph shows the means + SDs from 4 biological (at least 29 technical) replicates. In all graphs, asterisks indicate statistically significant differences compared to the negative control at the indicated time points (**** p \u0026lt; 0.0001; two-way RM ANOVA, Dunnett´s multiple comparison test).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4235543/v1/27da9dcb456782d82902775c.png"},{"id":55060447,"identity":"6a87e8c5-18f5-42d2-bf12-103f8cafec82","added_by":"auto","created_at":"2024-04-22 02:24:48","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":26830,"visible":true,"origin":"","legend":"\u003cp\u003eEvaluation of egg hatching of \u003cem\u003eHeterodera schachtii\u003c/em\u003e using the chitinase assay. Panels A and B depict 2 representative experiments with different egg population sizes (X-axis; 1600–50 range) and heat-killed eggs (HK; 55 °C for 2 hours) as a positive control. The data are displayed as the means + SDs of 2 – 4 technical replicates. Refer to supplementary information S1 and S2 for results from repeated experiments. In all graphs, asterisks indicate statistically significant differences compared to nonviable, heat-killed eggs (**** p \u0026lt; 0.0001; ** 0.05 \u0026gt; p \u0026gt; 0.001; two-way ANOVA, Dunnett´s multiple comparison test). RFU – relative fluorescent units.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4235543/v1/fa689bc69d6bc1703dd1f888.png"},{"id":55060718,"identity":"debcd5a7-0e29-4c70-a65b-68c98b83b8ef","added_by":"auto","created_at":"2024-04-22 02:32:48","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":31034,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eHeterodera schachtii\u003c/em\u003e eggs and cysts incubated in ZnCl\u003csub\u003e2\u003c/sub\u003e for 7 days showed significantly greater activity than those incubated in water (measured by WMicrotracker ONE). A) Results from isolated eggs, 50 per well. The data are shown as the means + SDs from 5 biological (at least 16 technical) replicates. B) Results from intact cysts, 3 per well. The data are shown as the means + SDs from 3 biological (28 technical) replicates. In both graphs, asterisks indicate statistically significant differences compared to the water control group (**** p \u0026lt; 0.0001; two-way RM ANOVA, Dunnett multiple comparison test).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4235543/v1/b80e467e0dcb3e2076898e2f.png"},{"id":55060448,"identity":"0cdb5f42-c747-4340-9c54-f65f670fc3db","added_by":"auto","created_at":"2024-04-22 02:24:48","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":36152,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of different concentrations of ethanol (7% - 0.88%) on \u003cem\u003eHeterodera schachtii\u003c/em\u003e activity measured with WMicrotracker ONE. A) Results from isolated eggs, 50 per well, incubated in ZnCl\u003csub\u003e2\u003c/sub\u003e. The data are shown as the means + SDs from 8 biological (64 technical) replicates. B) Results from intact cysts, 3 per well, incubated in ZnCl\u003csub\u003e2\u003c/sub\u003e. The data are shown as the means + SDs from 4 biological (32 technical) replicates. In both graphs, asterisks indicate statistically significant differences compared to the water-treated control at the indicated time points (**** p \u0026lt; 0.0001; *** 0.001 \u0026gt; p \u0026gt; 0.0001; ** 0.05 \u0026gt; p \u0026gt; 0.001; two-way RM ANOVA, Dunnett´s multiple comparison test).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4235543/v1/fd752d1ebbd41ddc3bc28625.png"},{"id":61594686,"identity":"fb618703-fce2-44bf-bf5e-f2e1cccc3228","added_by":"auto","created_at":"2024-08-01 17:15:59","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":756893,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4235543/v1/b98cbd31-31c2-4e3c-b559-4175a85129f9.pdf"},{"id":55060449,"identity":"6439c327-e533-41fb-b821-164d52554564","added_by":"auto","created_at":"2024-04-22 02:24:48","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":161289,"visible":true,"origin":"","legend":"","description":"","filename":"20240804SIfinal.docx","url":"https://assets-eu.researchsquare.com/files/rs-4235543/v1/a9fdec66a04b372ea3c15740.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Advanced Screening Methods for Assessing Motility and Hatching in Plant- Parasitic Nematodes","fulltext":[{"header":"1. BACKGROUND","content":"\u003cp\u003ePlant-parasitic nematodes (PPN) are significant pathogens affecting nearly all major agricultural crops (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Given their economic importance, many nematologists have focused on monitoring PPN in the field or studying their biology and plant-parasite interactions to identify new avenues for plant breeders or potential molecular targets for nematicide development. Various substances or biological agents that can interfere with nematode survival or behaviour have also been studied (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThis research requires the performance of assays to evaluate basic characteristics such as survival, motility, and hatching of PPNs. Traditionally, these assays involve visually observing and counting nematodes and eggs under a dissecting microscope, making them time-consuming and laborious (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Several high-throughput methods for performing similar assays have been optimized for the model nematode \u003cem\u003eCaenorhabditis elegans\u003c/em\u003e (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e) and, to some extent, for some species of mammalian parasitic nematodes (\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). These methods are based on various principles, including automatic image acquisition and analysis (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e), large object flow cytometry systems (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e), and many others. While some of these methods require expensive machines, many can be performed using relatively affordable or common laboratory equipment. Surprisingly, attempts to adapt such methods for PPN appear to be relatively rare.\u003c/p\u003e \u003cp\u003eIn this work, we present a simple method for evaluating the motility of nematodes using the WMicrotracker ONE. This method provided reliable results for both motile infective juveniles (J2) of the sedentary cyst nematode \u003cem\u003eHeterodera schachtii\u003c/em\u003e and the migratory endoparasitic nematode \u003cem\u003eDitylenchus destructor\u003c/em\u003e, suggesting that the platform is compatible with various PPN species. Furthermore, we describe two robust and easy methods for determining the hatching of \u003cem\u003eH. schachtii\u003c/em\u003e \u0026ndash; one utilizing the WMicrotracker ONE and the other based on measuring the activity of the enzyme chitinase.\u003c/p\u003e"},{"header":"2. METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Nematode cultivation\u003c/h2\u003e \u003cdiv id=\"Sec4\" class=\"Section3\"\u003e \u003ch2\u003e2.1.1. Maintenance of \u003cem\u003eHeterodera schachtii\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eThe stock culture of nematodes was maintained on mustard (\u003cem\u003eSinapsis alba\u003c/em\u003e cv. Albatros) roots grown \u003cem\u003ein vitro\u003c/em\u003e on modified Knop media according to previously published protocols (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Mustard seeds were sterilized by successive treatment with 70% ethanol (1 min), 1.3% NaClO (5 min) and 96% ethanol (1 min) and washed 3 times with sterile double distilled water (ddH\u003csub\u003e2\u003c/sub\u003eO). Seeds were allowed to germinate on 0.8% H\u003csub\u003e2\u003c/sub\u003eO agar for 2 days in the dark at 25\u0026deg;C, and healthy seedlings (3 per plate) were then transferred onto 15 cm Petri dishes containing modified Knop medium supplemented with 3% sucrose (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). The plants were subsequently grown at 25\u0026deg;C under a 16-hour light regime. After 2\u0026ndash;3 weeks, each plate was inoculated with approximately 300 \u003cem\u003eH. schachtii\u003c/em\u003e J2. The infected plates were kept at 25\u0026deg;C in the dark. Mature cysts of the nematodes could be collected from the infected plates after approximately 2 months, when the worms had completed their life cycle, and females had transformed into mature cysts that were apparent on the roots.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003e2.1.2. Maintenance of \u003cem\u003eDitylenchus destructor\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eThe initial population of \u003cem\u003eD. destructor\u003c/em\u003e was extracted from a hop plant (Central Bohemia/CZ) and maintained on carrot discs according to previously published protocols (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Briefly, to prepare the discs, the carrots were surface-sterilized with 1% NaClO for 30 minutes, washed in sterile water in the hood, peeled and cut into ca. 1.5 cm pieces with sterilized equipment. The discs were placed on 10 cm Petri dishes and kept at 25\u0026deg;C in the dark until white callus specks were apparent (approximately 4 weeks). Plates that were not used immediately were stored in the refrigerator. The plates were inoculated with 50\u0026ndash;60 nematodes per disc. In our experience, the best time to collect healthy populations for experiments is after 2\u0026ndash;4 weeks at 25\u0026deg;C, when the discs start to develop brown colouration.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.1.3. Collecting nematodes for experiments\u003c/h2\u003e \u003cp\u003eThe motile \u003cem\u003eH. schachtii\u003c/em\u003e J2 required for the tests were collected from funnels filled with 3 mM ZnCl\u003csub\u003e2\u003c/sub\u003e to increase the hatching rate of the nematodes (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). Approximately 300 cysts were placed in a sieve (60 \u0026micro;m mesh size) in the funnel so that it was approximately half covered with the liquid. The hatched J2 passed through the sieve and settled at the exit of the funnel, which led to a silicone tube closed with a clip. By opening the clip, the hatched J2 could be easily collected. The best time to collect a healthy population of J2 for the experiment was between 3 and 10 days after dissection of the cysts. Juveniles can be collected repeatedly from a funnel.\u003c/p\u003e \u003cp\u003eFor experiments with \u003cem\u003eD. destructor\u003c/em\u003e, 5 ml of sterile ddH\u003csub\u003e2\u003c/sub\u003eO was added to plates containing infected carrot discs. Nematodes naturally migrate from the discs into the water. To increase the yield, the discs were partially submerged in water for approximately 30 min. The liquid containing nematodes was transferred from the plates to Eppendorf tubes. In case any debris from the carrot discs was collected, the nematodes were washed several times with water prior to the experiment (by allowing the nematodes to settle on the bottom of the tubes and exchanging the liquid). Populations containing a mixture of different developmental stages were used for the experiments.\u003c/p\u003e \u003cp\u003eThe concentration of nematodes was determined by counting the number of living nematodes in 3 10 \u0026micro;L drops. The suspension was further diluted with sterile ddH\u003csub\u003e2\u003c/sub\u003eO to achieve the desired final concentration.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Evaluation of nematode motility using WMicroTracker ONE\u003c/h2\u003e \u003cp\u003eThe WMicrotracker ONE device (Phylumtech S.A.) emits an infrared beam that passes through the wells of a microtiter plate. Moving animals scatter light, and interference is subsequently detected. The instrument evaluates the activity in all wells continuously and then displays the number of these interferences (\u0026ldquo;activity counts\u0026rdquo;) per user-defined time interval (\u0026ldquo;bin\u0026rdquo;). All the data presented in this study show the number of activity counts detected in wells in 30-minute bins.\u003c/p\u003e \u003cp\u003eThe suspension was distributed into U-bottom 96-well plates (54 \u0026micro;L per well). The plates were kept in an incubator set to 20\u0026deg;C for 20\u0026ndash;30 min prior to the measurement to allow the nematodes to settle on the bottom of the wells. Afterwards, the plates were placed into WMicrotracker ONE device, and the initial motility of the worms was recorded for 30 minutes. The device was operated according to the manufacturer\u0026rsquo;s instructions. Six microliters of the tested control chemicals (sodium hypochlorite and sodium azide at a concentration 10 times greater than the desired final concentration) or sterile ddH\u003csub\u003e2\u003c/sub\u003eO water was added to each well (at least 4 wells per condition), and the motility of the populations was remeasured using WMicrotracker ONE at different time points. Between the measurements, the experimental plates were sealed with parafilm or PCR seal, kept at 20\u0026deg;C, and gently shaken on an orbital shaker (150 rpm) to ensure aeration of the suspension.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Evaluation of \u003cem\u003eHeterodera schachtii hatching\u003c/em\u003e\u003c/h2\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.3.1. Measuring the movement of J2 emerging from cysts using WMicrotracker ONE\u003c/h2\u003e \u003cp\u003eThe wells of a U-bottom 96-well plate were filled with 54 \u0026micro;L of sterile ddH\u003csub\u003e2\u003c/sub\u003eO or 3 mM ZnCl\u003csub\u003e2\u003c/sub\u003e. Three cysts were collected from the maintenance plate and placed into each well, while trying to ensure that cysts of similar size and colour were evenly distributed across the wells. After measuring the initial motility on WMicrotracker ONE (which should be close to 0 because no juveniles have yet emerged), 6 \u0026micro;L of the control chemical (ethanol at a concentration 10 times higher than the desired final concentration) or sterile ddH\u003csub\u003e2\u003c/sub\u003eO was added to each well. Due to the greater inherent variability of this assay, at least 8 wells per condition were used. The experimental plates were kept under the same conditions and remeasured at different time points, as described in section 2.2.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.3.2. Measuring hatching using WMicrotracker ONE\u003c/h2\u003e \u003cp\u003eApproximately 300 cysts were collected from maintenance plates or retrieved from funnels previously used for other experiments and placed into a 100 ml glass bottle filled with 3\u0026ndash;5 ml of sterile ddH\u003csub\u003e2\u003c/sub\u003eO or 3 mM ZnCl\u003csub\u003e2\u003c/sub\u003e. A medium-sized stirring bar was added to the bottle, and the cysts were crushed on a magnetic stirrer (1000 rpm, 5 min). The suspension was passed through a sieve (30 \u0026micro;m pore size) to remove smaller debris and some J2 that had already hatched inside of the cysts. The sieve was placed bottom up on a piece of mesh (116 \u0026micro;m pore size) and washed with 3\u0026ndash;5 ml of ddH\u003csub\u003e2\u003c/sub\u003eO. The liquid passing through the mesh was collected. This step removes larger debris. The final suspension was enriched in eggs but was not completely clean, as some J2 and mid-sized debris were also collected. The concentration of eggs was determined by counting the number of intact eggs in three 10 \u0026micro;L drops under a microscope. Approximately 50 eggs per well were used. The experimental plates were prepared, stored, and measured as described in section 2.3.1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e2.3.3. Evaluation of hatching by the chitinase assay\u003c/h2\u003e \u003cp\u003eThis assay is based on measuring the enzymatic activity of chitinase (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). The enzyme is produced by hatching juveniles to dissolve the chitin-containing eggshells. Its activity, which correlates to the number of viable juveniles, can be measured by the addition of a fluorogenic substrate.\u003c/p\u003e \u003cp\u003eThe egg suspension was prepared as described in section 2.3.2. A total of 400\u0026ndash;800 eggs per well were used, and heat-killed eggs (55\u0026deg;C, 2 hours, these conditions were adapted from (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e)) served as a positive control. The experimental plates were stored as indicated in the previous sections for 7 days. Afterwards, 20 \u0026micro;M of the chitinase substrate (4-methylumbelliferyl β-D-N,N\u0026prime;,N\u0026Prime;-triacetylchitotrioside, stock diluted in DMSO) was added, and the plates were incubated at 37\u0026deg;C for 1 hour. Alkaline buffer (1 M glycine, 1 N NaOH, pH\u0026thinsp;=\u0026thinsp;10.6) was added at a 1:2 ratio (i.e., 30 \u0026micro;L to 60 \u0026micro;L of liquid in our case). The fluorescence was measured on a TECAN Infinite PRO plate reader (λex 365 nm, λem 460 nm, gain manually set to 65).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Data analysis\u003c/h2\u003e \u003cp\u003eThe data were analysed in GraphPad Prism. All the data are displayed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). The data were analysed by repeated measures two-way analysis of variance (RM ANOVA) followed by Dunnet\u0026rsquo;s post hoc test. To assess the quality of the assay, the Z factor (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e) was calculated from the mean values and standard deviations using the following formula:\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$Z=1-\\frac{\\left(3\\times SD of sample\\right)+\\left(3\\times SD of control\\right)}{mean of sample-mean ofcontrol}$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003c/div\u003e"},{"header":"3. RESULTS AND DISCUSSION","content":"\u003cp\u003eSearching for new strategies for PPN control (i.e., screening of compounds, biological substances, etc.) is an important matter of research but is laborious and time-consuming. Here, we present several easy, robust, and straightforward assays with the potential to be developed into semi- or high-throughput screening tools. The assays described below are based on two different principles described in parts 2.2. and 2.3.3.\u003c/p\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003e3.1. WMicrotracker ONE is a suitable tool for measuring the movement of plant-parasitic nematodes\u003c/h2\u003e\n \u003cp\u003eThe WMicrotracker ONE was previously successfully used to analyse the locomotion and behaviour of not only the free-living model nematode \u003cem\u003eC. elegans\u003c/em\u003e (\u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e) but also multiple species of mammalian parasitic helminths (\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e16\u003c/span\u003e), as well as insect and zebrafish larvae (\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e). Our experiments (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e) show that the platform can be used in a similar manner for PPN, including both migratory species and motile developmental stages of sedentary species, which are generally considered the most economically important (\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eTo validate the method, we exposed the experimental populations of \u003cem\u003eD. destructor\u003c/em\u003e (mixed population) and \u003cem\u003eH. schachtii\u003c/em\u003e (infective J2) to 2 toxic chemicals, sodium azide (10 mM) and sodium hypochlorite (1.4%). We chose to use these compounds as a positive control due to their reliable effect. During the selection process, we also tested the effect of a variety of commonly used anthelmintic drugs, such as levamisole, ivermectin and mebendazole, with unsatisfactory results (data not shown). These drugs are likely unable to efficiently penetrate the cuticle of nematodes (\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e). Better results would presumably be obtained if the nematodes were actively feeding and ingesting the substance during the exposure (\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e). It is important to consider this when, for example, testing new promising substances for possible nematicidal activity.\u003c/p\u003e\n \u003cp\u003eWe measured the motility of the treated and control nematodes for 2 hours immediately after the addition of the compounds and again 3 days after treatment (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). As expected, both species showed a rapid decline in movement after exposure to both substances. After 30 minutes, the activity was reduced by 73.8% (from mean 258.9 activity counts to 67.7) in populations of \u003cem\u003eD. destructor\u003c/em\u003e treated with NaN\u003csub\u003e3\u003c/sub\u003e, 84.9% (from mean 243.9 activity counts to 36.9) in \u003cem\u003eD. destructor\u003c/em\u003e treated with NaClO, 98.9% (from mean 227.8 activity counts to only 2.4) in \u003cem\u003eH. schachtii\u003c/em\u003e exposed to NaN\u003csub\u003e3\u003c/sub\u003e, and 79.7% (from mean 241.6 activity counts to 49) in \u003cem\u003eH. schachtii\u003c/em\u003e in NaClO. Less than 1 mean activity count could be detected in populations of both nematode species treated with both substances after 3 days. At the same time, nematode motility in the controls remained consistent, with no decrease in activity counts detected in the short-term experiment and only a small decrease in motility (6\u0026ndash;8% on average) after 3 days.\u003c/p\u003e\n \u003cp\u003eAccording to our results, approximately 100\u0026ndash;150 worms per well should be used for smaller and less active nematodes such as \u003cem\u003eH. schachtii\u003c/em\u003e J2. For \u003cem\u003eD. destructor\u003c/em\u003e and other more active PPN species, 30\u0026ndash;50 nematodes per well are sufficient. Using plates with round bottoms allows nematodes to accumulate more closely and further stimulate each other\u0026rsquo;s movement by touch, resulting in higher detected activity counts than in plates with flat bottoms. This finding is in agreement with the information published on the website of Phylumtech S.A., the manufacturer of WMicrotracker ONE. The wells should contain approximately 40\u0026ndash;100 \u0026micro;L of liquid in total so that the bottom of the plate is fully covered. Too large volumes of liquid in wells could hinder aeration of the suspension, especially during longer experiments.\u003c/p\u003e\n \u003cp\u003eThe assay is very efficient and easy to evaluate, especially in comparison to the visual counting of moving nematodes under a microscope. Nematodes cultivated \u003cem\u003ein vitro\u003c/em\u003e can be harvested in relatively large quantities. This makes the assay suitable for high-throughput screening. To assess the quality of the assay, we calculated the Z factor (\u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e). Values exceeding 0.5 (a threshold for the assay to be considered excellent) were achieved both overall and in all individual replicates, in longer experiments as well as in shorter assays, at 90 min and longer for \u003cem\u003eH. schachtii\u003c/em\u003e and 120 min for \u003cem\u003eD. destructor\u003c/em\u003e (supplementary table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). This indicates that the assay is reliable and could presumably be used for screening larger libraries of compounds or other substances for nematicidal activity, similar to what was shown for nematodes parasitizing mammals (\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e\u003cspan\u003e\n \u003cp\u003e\u003cstrong\u003e3.2 Both the WMicrotracker ONE and the chitinase assay are suitable for evaluating the hatching of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eHeterodera schachtii\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/span\u003e\n \u003cp\u003eThe hatching rate of a PPN and the ability of a (putative) control agent to decrease it are important parameters. \u003cem\u003eH. schachtii\u003c/em\u003e cysts can contain hundreds of eggs that remain vital in the soil for many years and are awaiting optimal conditions to hatch and infect a host plant (\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e). Thus, we established two streamlined protocols allowing us to reliably evaluate reproductive capacity of PPN.\u003c/p\u003e\n \u003cp\u003eTo obtain the best possible results, an important step in preparing these assays is acquiring a clean egg suspension. Unwanted debris can hinder the experiment in several ways. Poor visibility of the eggs in the suspension can make accurate determination of their concentration difficult. Larger pieces of debris tend to block the pipette, resulting in uneven distribution of the egg suspension. Reducing the amount of debris is especially crucial when using the WMicrotracker ONE device, where extra material present in wells could interfere with proper signal detection.\u003c/p\u003e\n \u003cp\u003eThe isolation of clean eggs from \u003cem\u003eC. elegans\u003c/em\u003e can be performed simply by dissolving juveniles and adult nematodes in bleach (\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e). However, this process is not suitable for PPN, where eggs are usually extracted by passing the suspension through a series of sieves or using floating and centrifugation (\u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e). In the case of \u003cem\u003eH. schachtii\u003c/em\u003e, we mechanically crushed the cysts and then carefully passed the suspension through 2 sieves with different pore sizes (see section 2.3.2 for details). While the final suspension was not completely debris or juvenile free, it was sufficient to achieve reliable data of acceptable quality using both methods described.\u003c/p\u003e\n \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e\n \u003ch2\u003e3.2.1. The chitinase assay\u003c/h2\u003e\n \u003cp\u003eThe chitinase assay was originally described for evaluating hatching in \u003cem\u003eC. elegans\u003c/em\u003e (\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e), and a modified version of this assay was used to test the toxic effects of various compounds on \u003cem\u003eC. elegans\u003c/em\u003e (\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e). As mentioned above, the principle of the assay involves the addition of the fluorogenic substrate 4-methylumbelliferyl \u0026beta;-D-N,N\u0026prime;,N\u0026Prime;-triacetylchitotrioside. The reagent is cleaved by the nematode-produced enzyme chitinase, leading to the release of fluorescent 4-methylumbelliferone that can be detected.\u003c/p\u003e\n \u003cp\u003eOur results show that the assay can also be used with PPN eggs (Figs. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e, S1 and S2).\u003c/p\u003e\n \u003cp\u003eAs a positive control, we used heat-inactivated eggs We avoided using chemicals due to possible unwanted interactions. For instance, sodium azide, which was used as a control in previous assays, was reported to directly inhibit the activity of some chitinases (\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e). On the other hand, sodium hypochlorite, our other control substance, could negatively interact with ZnCl\u003csub\u003e2\u003c/sub\u003e, in which the eggs were incubated.\u003c/p\u003e\n \u003cp\u003eOur rationale for using both water and ZnCl\u003csub\u003e2\u003c/sub\u003e was based on the reported ability of zinc salts to stimulate the hatching of several members of the genus \u003cem\u003eHeterodera\u003c/em\u003e, including \u003cem\u003eH. schachtii\u003c/em\u003e (\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e). Eggs of some species of PPN, including \u003cem\u003eHeterodera\u003c/em\u003e spp., usually only start to hatch after they detect suitable environmental conditions and/or cues from nearby hosts. In our experiment, we observed a noticeable increase in the number of juveniles after 1 week of incubation of the eggs in both water and ZnCl\u003csub\u003e2\u003c/sub\u003e (Fig. S3), suggesting that hatching occurs even without additional stimuli. An increased number of juveniles correlated with increased detected activity of the enzyme chitinase.\u003c/p\u003e\n \u003cp\u003eIn the initial experiments (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA and S1), we determined that a concentration of 400 eggs per well was sufficient to achieve a reproducible, statistically significant difference between the positive and negative controls. The signal detected was approximately 1.7\u0026ndash;2 times greater (increase of 71\u0026ndash;125 RFU \u0026ndash; relative fluorescence units) for the eggs incubated in both water and ZnCl\u003csub\u003e2\u003c/sub\u003e than for the heat-killed control. The difference was more obvious when more eggs were used, with 1600 eggs per well providing a signal more than 4 times greater than that of the positive control. Nevertheless, as the process of egg preparation is relatively laborious, using as few eggs as possible is desirable. To ensure that 400 eggs were indeed sufficient, we repeated the experiment 5 more times with 800 and 400 eggs only (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB and S2). In all experiments, we again observed a statistically significant increase in signal intensity in wells containing 400 eggs compared to the positive control (between 161 and 450 RFU increase in water and 124 and 245 RFU increase in ZnCl\u003csub\u003e2\u003c/sub\u003e-incubated eggs). Again, the difference was more pronounced when 800 eggs were used (between 351 and 835 RFU increase in water and 246 and 544 RFU increase in ZnCl\u003csub\u003e2\u003c/sub\u003e-incubated eggs).\u003c/p\u003e\n \u003cp\u003eAlthough the variance among the technical replicates within one plate was relatively low, we observed notable differences in the detected signal strength among the biological replicates. This can likely be attributed to factors such as differences in the age of the maintenance plates from which \u003cem\u003eH. schachtii\u003c/em\u003e cysts were collected, inaccuracies in determining the egg concentration resulting in slight inconsistencies in egg population size, and other similar sources of biological variability. Therefore, we recommend presenting the data from each repetition as an individual graph. All the biological replicates followed the same significant trend, validating the reliability of the assay.\u003c/p\u003e\n \u003cp\u003eInterestingly, we observed that the detected chitinase activity was slightly lower in the wells with ZnCl\u003csub\u003e2\u003c/sub\u003e than in the wells where the eggs were incubated in water. A possible explanation could be the direct interference of zinc with the enzyme. Heavy metals, including zinc, were previously reported to inhibit the activity of some chitinases (\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e). Nevertheless, this effect is clearly not significant enough to affect the ability of the juveniles to hatch, and the differences in chitinase activity in ZnCl\u003csub\u003e2\u003c/sub\u003e-incubated eggs and positive controls are reproducibly statistically significant. Therefore, researchers may use both depending on their specific needs.\u003c/p\u003e\n \u003cp\u003eAlthough this assay could be easily adapted for many other nematode species, an obvious drawback could be its incompatibility with those species that can produce chitinase in life stages other than during hatching. For these cases, further optimization of the egg cleaning process would likely be necessary to ensure that the suspension used for the experiment contained only eggs.\u003c/p\u003e\n \u003cp\u003eAnother limiting factor is the need for a relatively large number of eggs. While the assay is undoubtedly more efficient than visual counting of hatched juveniles, one experiment typically requires several thousand eggs. This might make collecting a sufficient number of cysts for a large experiment quite tedious and unsuitable for high-throughput experiments. The assay might also not be useful in cases where only a limited amount of material is available, such as for monitoring the reproductive capacity of \u003cem\u003eHeterodera\u003c/em\u003e spp. collected from the field. This is why we decided to evaluate the hatching of \u003cem\u003eH. schachtii\u003c/em\u003e by detecting an increase in motility caused by the presence of newly hatched active juveniles via the WMicrotracker ONE.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec16\" class=\"Section3\"\u003e\n \u003ch2\u003e3.2.2. Measuring the motility and hatching of \u003cem\u003eHeterodera schachtii\u003c/em\u003e using the WMicrotracker ONE\u003c/h2\u003e\n \u003cp\u003eWe tested two different setups\u0026mdash;measuring the increase in motility in wells with intact cysts and in wells containing the egg mixtures prepared in the same way as for the chitinase assay. In both cases, we again compared the data for populations incubated in water and in ZnCl\u003csub\u003e2\u003c/sub\u003e. Here, the reported stimulatory effect of zinc salt on the hatching of \u003cem\u003eHeterodera\u003c/em\u003e spp. was clearly apparent (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). In wells containing eggs incubated in ZnCl\u003csub\u003e2,\u003c/sub\u003e we detected a 60% increase in motility compared to water (195.9 mean activity counts compared to 78.8). For intact cysts, the difference was 44% (229.9 mean activity counts compared to 128.4). Therefore, we used ZnCl\u003csub\u003e2\u003c/sub\u003e in all subsequent experiments.\u003c/p\u003e\n \u003cp\u003eTo validate the robustness of the two assay setups, various concentrations of ethanol were used as a positive control, demonstrating the dose-dependent effect of the substance at various time points (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). For both setups, we observed greater variability among the technical replicates, while the effect remained consistent among the biological replicates. We assume that this is due to inherent variability among the cysts and eggs and recommend using at least 8 technical replicates per condition to mitigate this issue. Furthermore, we noticed that the age of the plates from which the cysts were collected can significantly influence how soon hatching starts. Therefore, we recommend using maintenance plates of approximately the same age across all biological replicates.\u003c/p\u003e\n \u003cp\u003eIn the setup with intact cysts, we recommend using 3 cysts per well. Using fewer than 3 further increases the variability among technical replicates, while more cysts in the well seem to interfere with the instrument\u0026acute;s ability to measure the activity properly. For the egg suspension, we recommend using 50 eggs per well. The suspension obtained from crushed cysts, while enriched in eggs, also contained some J2 and mid-size debris. The presence of J2 at the beginning of the test results in some activity being detected in the wells during the initial measurement prior to adding the substances. These J2 tend to become inactive relatively quickly, likely due to stress caused during the preparation process. Accurately assessing the actual increase in the activity over time as the J2 hatch, while nematodes that were already present became inactive, might become difficult when a more concentrated mixture (with a stronger initial signal) is used. Another factor that might hinder the acquisition of reliable data is the presence of debris, which might interfere with beams passing through the wells. Using 50 eggs per well limits the number of unwanted juveniles and debris sufficiently, allowing the detection of a clear increase in activity counts over time due to hatching events.\u003c/p\u003e\n \u003cp\u003eThe difference between the positive and negative controls was reproducible and statistically significant in all biological replicates. For intact cysts, we observed a gradual increase in activity counts from approximately 10\u0026ndash;16 during the initial measurement in all conditions to 220.1 mean activity counts in control wells after 1 week. For ethanol-treated cysts, the values obtained at the same time point were 209.2, 159.2, 26.7 and 2.9 for 0.88, 1.75, 3.5 and 7% ethanol, respectively. The effect was dose-dependent and ranged from none or marginal to severe. For isolated eggs, the initial mean activity counts ranged from approximately 33 to 50 under all conditions for the reasons outlined above. The movement rates in the wells gradually increased to approximately 150 mean activity counts at days 5 and 7 in both the control wells and the wells treated with 0.88% ethanol. A less prominent increase, to 111.6 and 73.8 mean activity counts after 1 week, was apparent in populations treated with 1.75 and 3.5% ethanol, respectively. The addition of 7% ethanol led to a decrease in the mean activity counts after 1 week compared to the initial value, to only 20.1.\u003c/p\u003e\n \u003cp\u003eThe chitinase assay has two obvious advantages over determining hatching activity via the WMicrotracker ONE. First, the presence of either J2 or debris does not interfere with the readout. Second, the population size of 50 eggs is relatively small and more prone to random error, contributing to greater deviation among technical replicates here than in the chitinase assay. Further cleaning of the suspension might be helpful, although we opted to not focus on this step further, as the assay provided reproducible and statistically significant data. Moreover, unlike in the chitinase assay, the WMicrotracker ONE experiment performed significantly better when ZnCl\u003csub\u003e2\u003c/sub\u003e was used. Researchers should be mindful of possible unwanted interference of the solution with substances or biological agents they plan to test on cyst nematodes and assess their possible interactions beforehand. This problem will not apply to many other species of PPN that do not require zinc salts to stimulate hatching. We believe that both the chitinase test and the WMicrotracker ONE measurements are compatible with other nematode species. The egg isolation process will need to be modified to account for differences in egg size and other species-specific parameters. Many laboratories already have well-optimized protocols for the isolation of eggs of the species on which they are focused.\u003c/p\u003e\n \u003cp\u003eOverall, the established protocols are valuable, as both the chitinase assay and the WMicrotracker ONE assay are significantly less labor intensive than commonly used procedures and allow the testing of dozens of substances/conditions. The protocols are a good basis for further optimizations and adjustments that might even make them suitable for high-throughput screening. For example, while we worked with a 96-well plate format only, WMicrotracker instruments, depending on the model, also allow switching to a 384-well plate format. The same is true for the plate readers used for the chitinase assay. The advantages of the presented methods over other very promising modern approaches, such as automated image acquisition and image analysis (\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e), include the ease of data analysis and the significantly lower cost of the instrumentation needed. As outlined above, the current bottlenecks are the relative laboriousness of preparing the egg suspensions and/or collecting the cysts, the biological variability, and, in the case of setups that work better with ZnCl\u003csub\u003e2\u003c/sub\u003e, the necessity to perform prior tests.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e"},{"header":"4. CONCLUSIONS","content":"\u003cp\u003eIn this study, we optimized several assays to evaluate the motility and hatching of PPN. We primarily focused on \u003cem\u003eH. schachtii\u003c/em\u003e and included the migratory species \u003cem\u003eD. destructor\u003c/em\u003e for comparison when determining motility. From our experiments, we conclude that the developed methods can also be used with other economically important PPN, either in the presented form or with minor species-specific modifications. The methods are based on the detection of nematode movement in multiwell plates and the quantification of enzyme activity using a fluorogenic substrate. Inspired by protocols previously optimized for the model \u003cem\u003eC. elegans\u003c/em\u003e and other nematodes, they also proved to be compatible with PPN species. These methods offer a more efficient alternative to commonly used approaches. They can find use in various areas of basic and applied research as well as in laboratories focused on PPN monitoring.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePPN\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePlant\u0026ndash;parasitic nematode\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eddH\u003csub\u003e2\u003c/sub\u003eO\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDemineralized water\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eJ2\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSecond\u0026ndash;stage juveniles\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eRFU\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eRelative fluorescence units\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cul type=\"disc\"\u003e\n \u003cli\u003eEthics approval and consent to participate: NA\u003c/li\u003e\n\u003c/ul\u003e\n\u003cul type=\"disc\"\u003e\n \u003cli\u003eConsent for publication: NA\u003c/li\u003e\n\u003c/ul\u003e\n\u003cul type=\"disc\"\u003e\n \u003cli\u003eAvailability of data and materials: All data used in this study are presented in the manuscript or supplementary information. The raw data\u0026nbsp;are available from the corresponding author\u0026nbsp;upon\u0026nbsp;reasonable request.\u003c/li\u003e\n\u003c/ul\u003e\n\u003cul type=\"disc\"\u003e\n \u003cli\u003eCompeting interests:\u0026nbsp;The authors declare that they have no competing interests.\u003c/li\u003e\n\u003c/ul\u003e\n\u003cul type=\"disc\"\u003e\n \u003cli\u003eFunding: The work on this project was made possible by\u0026nbsp;Deutscher Akademischer Austauschdienst\u0026nbsp;(DAAD) and\u0026nbsp;Podpora mobility na UP II. (CZ.02.2.69/0.0/0.0/18_053/0016919), which\u0026nbsp;funded AK´s stay at ASSS´s home institution. Furthermore, the project was supported by project TA ČR\u0026nbsp;TQ03000647.\u003c/li\u003e\n\u003c/ul\u003e\n\u003cul type=\"disc\"\u003e\n \u003cli\u003eAuthors' contributions:\u0026nbsp;AK – conceptualization, experimental design, performing part of the experiments, data analysis and preparation of the manuscript. TJ –\u0026nbsp;performed\u0026nbsp;part of the experiments and other technical assistance. RH –\u0026nbsp;assisted\u0026nbsp;with\u0026nbsp;the\u0026nbsp;experimental design and\u0026nbsp;establishment of the\u0026nbsp;methods. VC –\u0026nbsp;assisted\u0026nbsp;with\u0026nbsp;the\u0026nbsp;experimental design and\u0026nbsp;establishment of the\u0026nbsp;methods. MH –\u0026nbsp;assisted\u0026nbsp;with\u0026nbsp;the\u0026nbsp;experimental design and methods. FMWG –\u0026nbsp;assisted\u0026nbsp;with\u0026nbsp;the\u0026nbsp;experimental design,\u0026nbsp;method establishment, and manuscript preparation. ASSS –\u0026nbsp;mentoring, assistance with conceptualization, experimental design, and manuscript preparation.\u003c/li\u003e\n\u003c/ul\u003e\n\u003cul type=\"disc\"\u003e\n \u003cli\u003eAcknowledgements: The authors gratefully acknowledge the excellent technical assistance provided by Ute Schlee and Stefan Neumann, Molecular Phytomedicine Group, INRES, University Bonn, Germany. Furthermore, we would like to thank Kateřina Mikušková from the Central Institute for Supervising and Testing in Agriculture, the Ministry of Agriculture of the Czech Republic.\u0026nbsp;\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eJones JT, Haegeman A, Danchin EGJ, Gaur HS, Helder J, Jones MGK, et al. Top 10 plant-parasitic nematodes in molecular plant pathology. Mol Plant Pathol. 2013;14(9):946\u0026ndash;61.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAtolani O, Fabiyi OA. Plant parasitic nematodes management through natural products: current progress and challenges. Management of Phytonematodes: Recent Advances and Future Challenges. Singapore: Springer Singapore; 2020. pp. 297\u0026ndash;315.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVan Bezooijen J. Methods and techniques for nematology. Wageningen, The Netherlands: Wageningen University; 2006.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eO\u0026rsquo;Reilly LP, Luke CJ, Perlmutter DH, Silverman GA, Pak SC. \u003cem\u003eC. elegans\u003c/em\u003e in high-throughput drug discovery. Adv Drug Deliv Rev. 2014;69\u0026ndash;70:247\u0026ndash;53.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCarr JA, Parashar A, Gibson R, Robertson AP, Martin RJ, Pandey S. A microfluidic platform for high-sensitivity, real-time drug screening on \u003cem\u003eC. elegans\u003c/em\u003e and parasitic nematodes. Lab Chip. 2011;11(14):2385\u0026ndash;96.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBuckingham SD, Partridge FA, Sattelle DB. Automated, high-throughput, motility analysis in \u003cem\u003eCaenorhabditis elegans\u003c/em\u003e and parasitic nematodes: Applications in the search for new anthelmintics. Int J Parasitol Drugs Drug Resist. 2014;4(3):226\u0026ndash;32.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu M, Landuyt B, Klaassen H, Geldhof P, Luyten W. Screening of a drug repurposing library with a nematode motility assay identifies promising anthelmintic hits against Cooperia oncophora and other ruminant parasites. Vet Parasitol. 2019;265:15\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eW\u0026auml;hlby C, Kamentsky L, Liu ZH, Riklin-Raviv T, Conery AL, O\u0026rsquo;Rourke EJ, et al. An image analysis toolbox for high-throughput \u003cem\u003eC. elegans\u003c/em\u003e assays. Nat Methods. 2012;9(7):714\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStroustrup N, Ulmschneider BE, Nash ZM, L\u0026oacute;pez-Moyado IF, Apfeld J, Fontana W. The \u003cem\u003eC. elegans\u003c/em\u003e Lifespan Machine. Nat Methods. 2013;10(7):665\u0026ndash;70.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePulak R. Techniques for analysis, sorting, and dispensing of \u003cem\u003eC. elegans\u003c/em\u003e on the COPAS flow-sorting system. Methods Mol Biol. 2006;351:275\u0026ndash;86.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSijmons PC, Grundler FMW, von Mende N, Burrows PR, Wyss U. \u003cem\u003eArabidopsis thaliana\u003c/em\u003e as a new model host for plant-parasitic nematodes. Plant J. 1991;1(2):245\u0026ndash;54.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eO\u0026rsquo;bannon JH, Taylor AL. Migratory endoparasite nematodes reared on carrot discs. Phytopathology. 1968;58(3):385.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSteele AE, Toxopeus H, Heijbroek W. A comparison of the hatching of juveniles from cysts of Heterodera schachtii and H. trifolii. J Nematol. 1982;14(4):588.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang JH, Chung TD, Oldenburg KR. A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J Biomol Screen. 1999;4(2):67\u0026ndash;73.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHunt PR, Keltner Z, Gao X, Oldenburg SJ, Bushana P, Olejnik N, et al. Bioactivity of nanosilver in \u003cem\u003eCaenorhabditis elegans\u003c/em\u003e: Effects of size, coat, and shape. Toxicol Rep. 2014;1:923\u0026ndash;44.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCamicia F, Herz M, Prada LC, Kamenetzky L, Simonetta SH, Cucher MA, et al. The nervous and prenervous roles of serotonin in Echinococcus spp. Int J Parasitol. 2013;43(8):647\u0026ndash;59.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBichara D, Calcaterra NB, Arranz S, Armas P, Simonetta SH. Set-up of an infrared fast behavioral assay using zebrafish (\u003cem\u003eDanio rerio\u003c/em\u003e) larvae, and its application in compound biotoxicity screening. J Appl Toxicol. 2014;34(2):214\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDavies KG, Curtis RHC. Cuticle surface coat of plant-parasitic nematodes. Annu Rev Phytopathol. 2011;49:135\u0026ndash;56.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZheng SQ, Ding AJ, Li GP, Wu GS, Luo HR. Drug absorption efficiency in Caenorhbditis elegans delivered by different methods. PLoS ONE. 2013;8(2):e56877.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSubbotin SA, Mundo-Ocampo M, Baldwin JG, Hunt DJ, Perry RN. Systematics of cyst nematodes (Nematodes: Heteroderinae). Nematology monographs and perspectives. Leiden-Boston: Brill; 2010. pp. 1\u0026ndash;135.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStiernagle T. Maintenance of \u003cem\u003eC. elegans\u003c/em\u003e. WormBook. 2006;(1999):1\u0026ndash;11.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMes THM, Eysker M, Ploeger HW. A simple, robust and semi-automated parasite egg isolation protocol. Nat Protoc. 2007;2(3):486\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEllerbrock BR, Coscarelli EM, Gurney ME, Geary TG. Screening for presenilin inhibitors using the free-living nematode, \u003cem\u003eCaenorhabditis elegans\u003c/em\u003e. J Biomol Screen. 2004;9(2):147\u0026ndash;52.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMilišiūnaitė V, Kadlecov\u0026aacute; A, Žukauskaitė A, Doležal K, Strnad M, Voller J, et al. Synthesis and anthelmintic activity of benzopyrano[2,3-c]pyrazol-4(2H)-one derivatives. Mol Divers. 2020;24(4):1025\u0026ndash;42.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNisler J, Pěkn\u0026aacute; Z, Končit\u0026iacute;kov\u0026aacute; R, Klimeš P, Kadlecov\u0026aacute; A, Murvanidze N, et al. Cytokinin oxidase/dehydrogenase inhibitors: outlook for selectivity and high efficiency. J Exp Bot. 2022;73(14):4806\u0026ndash;17.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAddabbo TD\u0026rsquo;, Sasanelli N, Greco N, Stea V, Brandonisio A. Effect of water, soil temperatures, and exposure times on the survival of the sugar beet cyst nematode, Heterodera schachtii. Phytopathology. 2005;95(4):339\u0026ndash;334.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSirimontree P, Fukamizo T, Suginta W. Azide anions inhibit GH-18 endochitinase and GH-20 Exo b-N-acetylglucosaminidase from the marine bacterium Vibrio harveyi. J Biochem. 2016;159(2):191\u0026ndash;200.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTefft PM, Bone LW. Zinc-mediated hatching of eggs of soybean cyst nematode, Heterodera glycines. J Chem Ecol. 1984;10(2):361\u0026ndash;72.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDonderski W, Swiontek Brzezinska M. The influence of heavy metals on the activity of chitinases produced by planktonic, benthic and epiphytic bacteria. Pol J Environ Stud. 2005;14(6):851\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAkintayo A, Tylka GL, Singh AK, Ganapathysubramanian B, Singh A, Sarkar S. A deep learning framework to discern and count microscopic nematode eggs. Scienific Rep. 2018;8:9145.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"plant-methods","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"plme","sideBox":"Learn more about [Plant Methods](http://plantmethods.biomedcentral.com/)","snPcode":"13007","submissionUrl":"https://submission.nature.com/new-submission/13007/3","title":"Plant Methods","twitterHandle":"@PlantMethods","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Plant-parasitic nematodes, Heterodera schachtii, cyst nematodes, Ditylenchus destructor, protocol, methods, screening, motility, viability, hatching, WMicrotracker ONE, chitinase.","lastPublishedDoi":"10.21203/rs.3.rs-4235543/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4235543/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cb\u003eBackground\u003c/b\u003e\u003c/p\u003e \u003cp\u003ePlant-parasitic nematodes are economically important pests responsible for substantial losses in agriculture. Researchers focusing on plant-parasitic nematodes often need to assess basic parameters such as their motility, viability, and reproduction. Traditionally, these assays involve visually counting juveniles and eggs under a dissecting microscope, making this investigation time-consuming and laborious.\u003c/p\u003e\u003cp\u003e\u003cb\u003eResults\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn this study, we established a procedure to efficiently determine the motility of two plant-parasitic nematode species, \u003cem\u003eHeterodera schachtii\u003c/em\u003e and \u003cem\u003eDitylenchus destructor\u003c/em\u003e, using the WMicrotracker ONE platform. Additionally, we demonstrated that hatching of the cyst nematode \u003cem\u003eH. schachtii\u003c/em\u003e can be evaluated using both the WMicrotracker ONE and by assessing the enzymatic activity of chitinase produced during hatching.\u003c/p\u003e\u003cp\u003e\u003cb\u003eConclusions\u003c/b\u003e\u003c/p\u003e \u003cp\u003eWe present easy and straightforward protocols for studying nematode motility and hatching that allow us to draw conclusions about viability and survival. Thus, these methods are useful tools for facilitating fast and efficient evaluation in various fields of research focused on plant-parasitic nematodes. The methods should also be compatible with other plant-parasitic nematode species.\u003c/p\u003e","manuscriptTitle":"Advanced Screening Methods for Assessing Motility and Hatching in Plant- Parasitic Nematodes","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-22 02:24:43","doi":"10.21203/rs.3.rs-4235543/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-05-29T21:48:30+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-05-29T00:55:54+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"156071943831062354009298152323383781024","date":"2024-05-20T13:13:12+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-04-28T16:30:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"b6b8f589-93f9-4905-a63c-3e8f76eb3397","date":"2024-04-19T13:37:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"15e61b05-80f0-4906-919a-96a752568e67","date":"2024-04-19T13:22:23+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-04-14T13:59:42+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-04-12T22:26:22+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-04-12T11:58:42+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant Methods","date":"2024-04-10T10:10:42+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"plant-methods","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"plme","sideBox":"Learn more about [Plant Methods](http://plantmethods.biomedcentral.com/)","snPcode":"13007","submissionUrl":"https://submission.nature.com/new-submission/13007/3","title":"Plant Methods","twitterHandle":"@PlantMethods","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1ace42fa-9ad6-4f37-a6ae-26556a9ee915","owner":[],"postedDate":"April 22nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-08-01T16:15:35+00:00","versionOfRecord":{"articleIdentity":"rs-4235543","link":"https://doi.org/10.1186/s13007-024-01233-z","journal":{"identity":"plant-methods","isVorOnly":false,"title":"Plant Methods"},"publishedOn":"2024-07-20 16:05:01","publishedOnDateReadable":"July 20th, 2024"},"versionCreatedAt":"2024-04-22 02:24:43","video":"","vorDoi":"10.1186/s13007-024-01233-z","vorDoiUrl":"https://doi.org/10.1186/s13007-024-01233-z","workflowStages":[]},"version":"v1","identity":"rs-4235543","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4235543","identity":"rs-4235543","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}