Microfluidic device integrated with a porous membrane for quantitative chemotaxis assay of plant-parasitic nematodes

Plant-parasitic root-knot nematodes (RKNs) cause significant damage to plant crops by inhibiting nutrient absorption in host plants through infection. Chemotaxis is an important factor in controlling RKNs behavior as well as in understanding the mechanisms of parasitic behavior of RKNs on plants. Thus, studies on RKN chemotaxis are important for developing more environmentally friendly strategies to manage RKN infestations instead of current control methods using environmentally harmful pesticides. To better understand the chemotactic behavior of RKNs, we developed an easy-to-use microfluidic device consisting of two-layer polydimethylsiloxane (PDMS) microchannel chips and a porous hydrophilic polycarbonate membrane. The porous membrane acts both as a filter in introducing agarose gel containing nematodes to the observation chamber and as a diffuser to generate chemical concentration gradients in chemotaxis assays. We demonstrated the chemical concentration gradient was formed within 5 min in the gel-filled chamber using fluorescence substance. Using this device, we analyzed the correlation between nematode activity (chemotactic behavior and mobility) and the concentration gradients of several chemicals including KNO₃, cadaverine, and putrescine (1, 10 and 100 mM). Finally, we confirmed the repellent effect of KNO₃ and the attractive effect of cadaverine and putrescine on the RKN, Meloidogyne incognita, which was cultured on tomatoes, within 10 min after injecting the chemicals and quantitatively identified the correlation between nematode activity and chemical environmental conditions.