Effects of the wet catching method on the detection of Bursaphelenchus xylophilus from trapped longhorn beetle vectors

in Nematology
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The pine wood nematode (PWN), Bursaphelenchus xylophilus, causes pine wilt disease and is transmitted by Monochamus beetles. An efficient trapping system for these organisms is essential for their early detection in threatened regions. A wet catching method, using monoethylene glycol (MEG) in a collecting cup to preserve captured insects, was suitable for catching longhorn beetles. As a second step in developing this technique, we studied the influence of MEG on nematode detection. When M. galloprovincialis carrying PWN were submerged in MEG, nematodes died within 24 h. The preservative altered certain morphological features of nematodes immersed in solutions of 10, 30 or 60% MEG, impeding their morphological identification. However, molecular identification was possible as long as the MEG concentration was below 70%. At higher concentrations, a rinsing step with water before DNA extraction was enough to allow molecular detection. Wet trapping requires less frequent monitoring than dry trapping, thus reducing maintenance and related costs.

Effects of the wet catching method on the detection of Bursaphelenchus xylophilus from trapped longhorn beetle vectors

in Nematology

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References

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Figures

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    Changes in visibility (1 = not visible, 5 = clearly visible) over time of three morphological features of the genus Bursaphelenchus when submerged in rainwater (0% monoethylene glycol (MEG)) or different solutions of MEG (10, 30, 60, 100%). A: Cephalic region high and offset by a constriction with six lips; B: Lateral field with four lines; C: Excretory pore at/or behind median bulb.

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    Visibility scoring (1 = not visible, 5 = clearly visible) over time of four morphological features for females (A, B) or males (C, D) of Bursaphelenchus xylophilus, when submerged in 0, 10, 30, 60 or 100% monoethylene glycol (MEG) solution (mixed with rainwater). A: Female tail broadly sub-cylindrical with or without mucron; B: Female vulval flap straight, not ending in a deep depression; C: Male spicule with long and pointed rostrum; limbs of spicule with an angular curvature; D: Male spicule length < 30 μm.

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    Electrophoresis of the 18S rRNA PCR product of Bursaphelenchus xylophilus adults submerged in pure rainwater (lanes 1-9), 30% monoethylene glycol (MEG) (lanes 10-18) or 100% MEG (lanes 19-27) for 28 days. In the lanes marked with ∗, nematodes were rinsed in 25 μl pure water before DNA extraction; DNA in lanes 3, 6, 9, 12, 15, 18, 21, 24 and 27 was extracted from five nematodes; in the other lanes DNA was extracted from a single nematode; (–): negative control; L: 100 bp DNA ladder (Thermo Scientific).

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    Electrophoresis of the 18S rRNA PCR product of Bursaphelenchus xylophilus adults submerged in 20, 30, 40, 50, 60, 70, 80, 90 and 100% monoethylene glycol (lanes 1-9) for 28 days, DNA was extracted from one individual; (–): negative control; L: 100 bp DNA ladder (Thermo Scientific).

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