Our research primarily focus on the cold-receptive mechanisms of the dispersal third stage juveniles of pine wood nematode, specifically addressing how the pine wood nematode withstands low-temperature stress at approximately minus 20. The current experimental challenge is dehydration time, which is critical in determining the survival rate of third stage dispersal juveniles of Bursaphelenchus xylophilus. We advise that the longer the time, the better the emphasis.
We have established that the entry of third stage dispersal juveniles of Bursaphelenchus xylophilus into cryptobiosis enable them to withstand extreme low-temperature environments, highlighting a key survival mechanism in harsh conditions. Our protocol addresses the research gap by inducing cryptobiosis in pine wood nematodes and successfully reviving them architecturally. It enables controlled studies of their survival mechanisms and additional applications.
Our protocol is simple and reliable for very robust technical support for standing the stress resistance mechanisms of pine wood nematode, making it more accessible and efficient compared to other techniques. Begin by cutting the collected round logs of Pinus massoniana into slices measuring 0.5 to one centimeter in thickness using a saw. Trim the slices into small strips of two to three centimeters in length.
Place the small wood strips into sealed bags and store the bags in a laboratory for further use. Attach a rubber tube to the narrow end of a long neck glass funnel with an approximate diameter of 15 centimeters. Secure the rubber tube with a pinchcock and place the assembled funnel on a funnel stand.
Completely wrap the small wood strips in a single layer of tissue paper. Place the wrapped wood strips into the funnel and add distilled water until all the wood strips are submerged, then slowly release the pinchcock at the bottom of the funnel and allow 2000 to 5, 000 microliters of the mixture to leak into a five-centimeter diameter Petri dish for microscopic examination and morphological identification. Observe the mixture to identify J3 Bursaphelenchus xylophilus by noting features such as stylets, median esophageal bulbs, lipid droplets, and finger-like tail shapes.
Using a 10 microliter pipette, aspirate 10 microliters of liquid along with one nematode each time to transfer individual J3 Bursaphelenchus xylophilus onto a slide. Finally, sterilize the remaining wood strips and infected wood materials after extracting J3 Bursaphelenchus xylophilus to prevent further contamination. To begin, extract specimens of J3 Bursaphelenchus xylophilus from Pinus massoniana, then prepare an 8%potassium chloride aqueous solution for osmotic regulation.
Using a pipette, add 10 microliters of the 8%potassium chloride aqueous solution to the slide containing the mixture of J3 Bursaphelenchus xylophilus. Place the slide on the microscope stage and allow the water to evaporate naturally. Observe the dehydration process of J3 Bursaphelenchus xylophilus under the microscope.
Monitor signs such as body curling and aggregation. When the water fully evaporates and potassium chloride crystals precipitate, observe the nematodes stopping movement and entering cryptobiosis. Place the cryptobiotic J3 Bursaphelenchus xylophilus in a constant temperature environment of minus 20 degrees Celsius for 24 hours.
After 24 hours, use a pipette to add approximately 300 microliters of distilled water around the juveniles that underwent low temperature treatment. Observe the rehydration process of J3 Bursaphelenchus xylophilus under the microscope. Monitor signs such as body stretching and the resumption of movement.
