ON-CHIP CHEMOTAXIS ASSAY OF PLANT-PARASITIC NEMATODE TOWARDS INCREASING GLOBAL CROP PRODUCTIVITY

Hirotaka Hida1,4, Hidetaka Nishiyama2, Shinichiro Sawa2, Tetsuya Higashiyama1,3, and Hideyuki Arata1

1JST, ERATO, Live-Holonics Project/Nagoya University, JAPAN 2Kumamoto University, JAPAN

3 Institute of Transformative Bio-Molecules (ITbM), Nagoya University, JAPAN 4Kobe University, JAPAN (Present affiliation)

ABSTRACT

This paper reports a new method for simple and efficient on-chip chemotaxis assay of plant-parasitic nematode, Meloidogyne incognita. To precisely characterize chemotaxis of the nematodes, we have developed a poly-dimethylsiloxane (PDMS) T-shaped microchannel-device which has a pair of micro-slits connected by narrow microchannel arrays with chemical inlets. This microchannel-device can efficiently create controllable chemical conditions for analyzing nematodes behavior. We have established a protocol to allow nematodes to swim freely in the microchannel by loading an agarose gel. Then, KNO3 was firstly identified as attractant/repellent and the nematode behavior depending on chemical conditions was quantitatively investigated. This method might contribute to global food and energy problems by improving crop productivity. KEYWORDS: Plant-parasitic nematode, Chemotaxis, Microchannel, Polydimethylsiloxane INTRODUCTION

Meloidogyne incognita, one of the representative plant-parasitic nematodes, causes significant damage to agricultural products all over the world. Nevertheless, using toxic pesticides is only way to efficiently exterminate the nematodes so far. Studying their chemotaxis properties is an inevitable issue to realize a new extermination method, which is harmless to the environments. In conventional in vitro chemotaxis assays, the nematodes and chemical compounds are placed on agar plate and attractive/repellent properties are defined by tracking the nematodes for one or a few days [1,2]. However, this on-agar-plate assay has several issues to be overcome considering chemotaxis analysis in quantitative manner. One of the issues is that the nematodes can swim both on the surface and inside the agar gel, thus it is difficult to track and analyze the nematodes behavior in detail. The other is the difficulty in keeping a chemical gradient with high spatial resolutions on agar plate [3]. To overcome these issues, we developed a microchannel device for chemotaxis assay of the plant-parasitic nematodes. MICROCHANNEL DEVICE

Figure 1 shows the developed microchannel device for chemotaxis assay of the plant-parasitic nematodes. The microchannel device consists of T-shaped microchannel, a couple of micro-slit, chemical inlets and narrow channel arrays between the inlets and the slits. The array of microchannels, which are narrower than the nematode’s width, have two roles; to confine the nematodes within the micro-slits and to generate concentration gradients by diffusion without an external power source. In the chemotaxis assay, a chemical substance is applied into one of the chemical inlets and diffuses through the narrow microchannel array into the micro-slit where the nematodes exist. Then, we can define the attractant and repellent behaviors by counting the nematodes in both micro-slits as shown in figure 1(b).

Figure 1: Schematic views of the on-chip chemotaxis assay of the nematodes. Microscopic view and schematics of the developed microchannel-device. (b) Illustration of the on-chip chemotaxis assay.

978-0-9798064-6-9/µTAS 2013/$20©13CBMS-0001 1752 17th International Conference on MiniaturizedSystems for Chemistry and Life Sciences27-31 October 2013, Freiburg, Germany

EXPERIMENTAL M. incognita juveniles were cultured on hydroponics tomato roots and isolated from the roots by Baermann funnel

method. The isolated nematodes were managed in DI water at 5 oC to be maintained in juvenile long time. The protocol for allowing the nematodes to swim in the microchannel is as follows. First, 20 L of 1 wt% ultra low-

melting agarose gel (A2576 Agarose Type IX-A, SIGMA-ALDRICH) was melted and injected into the chemical and nematode inlets and the gel was automatically loaded into the channel by power-free pumping method[4] at RT (room temperature). After the microchannel filled with the gel, the agar gel in the nematode inlet was drained with micropipette to allow the nematodes to move into the microchannel quickly. Then, 20 L of 1 wt% gel including the nematodes is applied into the nematode inlet. The agar gel was coagulated at 5 oC and the device was finally stored in dark place at RT.

For evaluating the concentration gradient in the micro-slits, we applied a water solution of fluorescent substance, Rhodamine B (Wako Pure Chemical Industries, Ltd., Japan) into the device. We loaded 1 wt% agarose gel into the microchannel by the same protocol as mentioned above and applied 3 L of 2.3 mM Rhodamine B into the chemical inlet. The time-dependent fluorescent intensity was measured at RT.

The several candidate chemicals were applied with various concentrations for identifying attractant/repellent conditions. We defined the attractant/repellent properties by comparing the number of the nematodes in both micro-slits after injecting 3 l of chemical solution into one chemical inlet. RESULTS AND DISCUSSION

We experimentally clarified that the nematodes can swim by themselves in the gel-filled microchannel. In contrast, no nematodes can swim in the water-filled microchannel. First, we counted the nematodes in both micro-slits to confirm the nematode’s behavior without applying any chemical substances (figure 2). After 10 hours, number of nematodes in the both micro-slits was almost the same and the population balance was maintained until 24 hours. From this result, we confirmed that the attractant/repellent conditions can be evaluated from the variation of number of the nematodes in the both micro-slits.

Figure 3 shows the time-dependent concentration of Rhodamin B in the micro-slit. We observed that the concentra-tion gradient was created in 1 hour and the rate of the concentration change gradually decreased after 4 hours. Thus, we estimated that the concentration gradient is kept in a steady-state around 3 hours.

Consequently, we have identified KNO3 as an effective chemical for the nematode Meloidogyne incognita as shown in figure 4. KNO3 acted as a repellent when applying 990 mM KNO3 while it showed attractant for around two hours with lower KNO3 concentrations, 49 mM and 9.9 mM. The relationship between the concentration gradient and the nematode behavior is discussed as below.

The concentration gradient of KNO3 (101.01 g mol-1) might be developed in a short time by comparing the experi-mental results using Rhodamin B (479.02 g mol-1) because the diffusion rate increases by decreasing molecular mass. Additionally, the value of concentration gradient is proportional to the amount of applied substance. When applying 990 mM KNO3, high concentration gradient might be rapidly generated in 1 hour even before reaching steady-state and act as a repellent. On the other hand, when applying 49 mM and 9.9 mM, the concentration gradient gradually increased and low/high concentration gradients might act as attractant/repellent respectively.

Figure 3. Time-dependent concentration gradient of 2.7mM Rhodamin B in the micro-slit detected from fluo-rescent signals.

Figure 2. Time course of the ratio between the number of plant-parasitic nematodes in L- and R-slit (N > 10).

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CONCLUSION

We presented a novel microchannel device for chemotaxis assay of the plant-parasitic nematodes, Meloidogyne in-cognita. We experimentally evaluated the concentration gradient in the microchannel and identified high- and low- con-centration KNO3 act as repellent and attractant for the nematodes.

From these results, we might develop a novel extermination method by controlling the chemical concentration-gradient in the soil where the plant-parasitic nematodes exist. This proposed method has a potential to replace conven-tional method using environmentally harmful pesticides and might contribute to improve the global crop productivity in the future. ACKNOWLEDGEMENTS

This work was supported by JSPS KAKENHI Grant-in-Aid for Young Scientists (B), Grant Number 60402509. REFERENCES [1] N. Wuyts, R. Swennenand D. D. Waele, “Effects of plant phenylpropanoid pathway products and selected terpe-

noids and alkaloids on the behaviour of the plant-parasitic nematodes Radopholus similis, Pratylenchus penetrans and Meloidogyne incognita”, Nematology, 8(1), pp. 89-101, (2006).

[2] T. E. Hewlett, E. M. Hewlett, and D. W. Dickson, “Response of Meloidogyne spp., Heterodera glycines, and Radopholus similis to Tannic Acid”, Journal of Nematology, 29, pp. 737-741, (1997).

[3] H. Zhang, and B.G. Forde ,“An Arabidopsis MADS box gene that controls nutrient-induced changes in root archi-tecture”, Science, 279, pp. 407–409, (1998).

[4] H. Arata, H. Komatsu, K. Hosokawa, and M. Maeda, "Sub-Attomole MicroRNA Detection with Laminar Flow-Assisted Dendritic Amplification on Power-Free Microfluidic Chip", PLoS One, Vol. 7, e48329, (2012).

CONTACT *H. Hida, tel: +81-78-803-6148; hida@mech.kobe-u.ac.jp

Figure 4. Time-dependent nematode behaviors with various KNO3 concentrations. This data suggests that KNO3

solutions with high- and low- concentration act as repellent and attractant, respectively, for the Meloidogyne in-cognita.

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