Inducing the Entry of Third Stage Dispersal Juveniles of Bursaphelenchus xylophilus into Cryptobiosis Through Osmotic Regulation

Summary

The present protocol describes a method for inducing the dispersal of third-stage juveniles (pre-dauer juveniles, J_Ⅲ) of Bursaphelenchus xylophilus into cryptobiosis through osmotic regulation of potassium chloride (KCl) ions. This method can provide technical support for research on the stress resistance mechanisms of B. xylophilus.

Abstract

Cryptobiosis is a state where organisms lose nearly all their internal water and enter anhydrobiosis under extreme environmental stress. The dispersal third-stage juveniles (pre-dauer juveniles, ) of Bursaphelenchus xylophilus can enter cryptobiosis through dehydration and revive upon rehydration when environmental conditions improve. Osmotic regulation is crucial for their survival in this process. In this study, specimens of B. xylophilus were collected from dead Pinus massoniana due to pine wilt disease in Ningbo, Zhejiang Province, China. Following immersion in 8% potassium chloride (KCl) solution, B. xylophilus entered a cryptobiotic state after gradual dehydration due to increased external osmotic pressure by natural water evaporation. B. xylophilus could resist low-temperature stress at -20 °C. B. xylophilus could revive upon rehydration, with a survival rate of 92.1%. This process can regulate the entry of B. xylophilus into cryptobiosis, enabling them to resist extreme environments. This method described in our study is simple and reliable, providing technical support for studying the stress resistance mechanisms of B. xylophilus.

Introduction

Pine wilt disease is one of the most severe forest diseases in China¹. The pinewood nematode Bursaphelenchus xylophilus (B. xylophilus) poses a significant threat to pine species such as Pinus massoniana (P. massoniana) by reproducing within the host, leading to rapid wilting and death. Under unfavorable environmental conditions after the death of the host, the reproductive juveniles of B. xylophilus may enter pre-dauer juveniles (). These juveniles seek insect vectors to continue their transfer and spread, causing extensive pine forest die-offs².

Cryptobiosis is a state where organisms lose nearly all their internal water and enter anhydrobiosis under extreme environmental stress³. In this process, organisms gradually dehydrate, resulting in halted movement, reduced metabolism, and a suspended lifecycle⁴. Under suitable conditions, they can recover upon rehydration. Cryptobiosis enables organisms to resist extreme environments, such as low temperatures, and extend their lifespans⁵. Nematodes that survived in cryptobiosis for 30,000 years were found in Antarctic permafrost⁶.

Previous studies have shown that nematodes can adapt to various abiotic stressors -- such as low temperature, drought, and high osmotic pressure -- during dehydration by adjusting their surface permeability⁷. Additionally, nematodes can curl their bodies to decrease surface area and minimize water loss rate. For example, Caenorhabditis elegans regulates osmotic balance, allowing ions and small molecules to freely pass through its cuticle, while larger molecules cannot⁸. B. xylophilus also exhibits cryptobiosis. During the dehydration and rehydration stages of cryptobiosis, osmotic regulation is essential for controlling substance exchange across the cuticle, making it pivotal for entering or recovering from cryptobiosis⁹.

The present study introduces a method for inducing cryptobiosis in B. xylophilus through osmotic regulation. This method is simple, efficient, and highly reliable, providing technical support for researching the stress resistance mechanisms of B. xylophilus.

Protocol

1. Collection of J_III B. xylophilus

1. Collection of infected disks of P. massoniana
   NOTE: All procedures complied with the Administrative Measures for Pine Wilt Disease Epidemic Area and Infected Wood and other applicable regulations. A P. massoniana forest infected by B. xylophilus in Xiangshan County, Ningbo City, Zhejiang Province, was selected as the sample site. P. massoniana, which died from pine wilt disease, was felled within the sample site.
   1. Collect round logs (5 cm thick) from the upper, middle, and lower sections of the main trunk of the felled trees.
   2. Cut the collected round logs of P. massoniana into 0.5-1 cm thick slices, then trim them into small strips of 2-3 cm length.
   3. Place the small wood strips into sealed bags and store them in a laboratory for further use.
2. Extraction of B. xylophilus
   1. Attach a rubber tube to the narrow end of a long-neck glass funnel with an approximate diameter of 15 cm. Secure the rubber tube with a pinchcock. Place the assembled funnel on a funnel stand.
   2. Wrap the small wood strips completely in a single layer of tissue paper and then place them in the funnel. Add distilled water to the funnel until all wood strips are submerged.
      ​NOTE: After standing for 12-24 h, The nematodes migrated out of the wood strips and settled at the bottom of the funnel¹⁰. Tissue paper prevented wood debris from settling while allowing nematodes to pass through.
   3. Release the pinchcock at the bottom of the funnel slowly, and let 2000-5000 µL of the mixture leak into a Petri dish (5 cm in diameter) for microscopic examination and morphological identification.
   4. Identify B. xylophilus by observing features such as stylets, median esophageal bulbs, lipid droplets, and "finger-like" tail shapes¹¹.
   5. Under a microscope, transfer individual B. xylophilus onto a slide by aspirating 10 µL of liquid along with one nematode using a 10 µL pipette each time. Repeat this process 30 times to collect approximately 300 µL of the mixture containing nematodes.
   6. After extraction of B. xylophilus, sterilize the remaining wood strips and infected wood materials to prevent further contamination.

2. Osmotic regulation process control

1. Preparation of osmotic regulation mixture
   1. Prepare an 8% KCl aqueous solution.
   2. Using a pipette, add 10 µL of the 8% KCl aqueous solution to the slide containing the mixture of B. xylophilus, yielding approximately 310 µL of a mixture of the nematodes and KCl solution.
2. Regulation of increased osmotic pressure and dehydration of B. xylophilus​
   1. Place the slide on the microscope stage, allowing the water to evaporate naturally. It would gradually increase the osmotic pressure of the surrounding environment, causing nematodes to dehydrate over approximately 30-60 min.
      NOTE: The operator only needs to wait for the water to evaporate naturally until the KCL crystals precipitate and observe the state of the nematodes under a microscope. It takes about 30-60 min for 310 µL of the mixture to evaporate naturally until the KCL crystals precipitate.
   2. Observe the dehydration process (e.g., body curling, aggregation, etc.) of B. xylophilus under the microscope. When the water fully evaporates and KCl crystals precipitate, B. xylophilus stops moving and enters cryptobiosis.
3. Low-temperature stress treatment of cryptobiotic B. xylophilus
   1. Place the cryptobiotic B. xylophilus in a constant temperature environment of -20 °C for 24 h.
4. Regulation of decreased osmotic pressure and rehydration of B. xylophilus
   1. Add approximately 300 µL of distilled water around the juveniles that had undergone low-temperature treatment by pipette. The rehydration process, lasting for about 30-60 min, gradually decreases the osmotic pressure of their surrounding environment.
   2. Observe the rehydration process of B. xylophilus (e.g., body stretching, resumption of movement, etc.) under the microscope. Calculate the survival rate after 24 h.

Results

In the 8% KCl solution, as the water evaporated, B. xylophilus initially exhibited a noticeable "curling" while remaining motile (Figure 1A). Once the water evaporated completely, the nematodes ceased movement and curled, with depressed surfaces and accumulated lipid droplets (Figure 1B), indicating their entry into the cryptobiotic state. Upon the addition of distilled water to their surrounding environment, the nematodes rehydrated and revived (Figure 1C). After the water evaporates, KCL crystals will appear (Figure 1D). Figure 1E shows the morphological characteristics of B. xylophilus.

A dehydration experiment demonstrated that in an initial 8% KCl solution, water evaporation caused B. xylophilus to lose water. After dehydration, B. xylophilus were subjected to low-temperature stress at -20 °C for 24 h; the average survival rate was 92.1%¹¹. Compared with a survival rate of 0% in the control group that had not entered a cryptobiotic state, the above data suggested dehydration could significantly enhance the survival rate of B. xylophilus under low-temperature conditions.

During the rehydration phase following the cryptobiosis of B. xylophilus, we observed that 100% of the nematodes exhibited in vivo "bubbles" when being transferred from 40% KCl solution to distilled water (Figure 2).

Figure 1: The dehydration-cryptobiosis-rehydration process of B. xylophilus. This image illustrates the cryptobiosis process in B. xylophilus, including the (A) dehydration stage, (B) cryptobiosis stage, (C) rehydration stage, (D) KCL crystallization stage, and (E) B. xylophilus. Please click here to view a larger version of this figure.

Figure 2: Accumulation of fat granules in unrevived B. xylophilus. This image shows the state of B. xylophilus after rehydration, characterized by the presence of in vivo bubble-shaped fat granules. Please click here to view a larger version of this figure.

Discussion

In this study, B. xylophilus was collected from dead P. massoniana in Ningbo, Zhejiang Province. It should be noted that B. xylophilus was selected from a mixed population with different instars. Specifically, round logs with a high density of B. xylophilus were chosen, followed by the screening of B. xylophilus. During winter, when the daily minimum temperature in Liaoning Province is around -20 °C, a large number of single-instar B. xylophilus can be obtained from dead Pinus tabuliformis, which simplified the selection process in this experiment¹².

During the cryptobiotic process, the slow dehydration rate of the nematodes benefits their survival and prevents structural damage to their proteins¹³. The dehydration time is critical in determining the survival rate of B. xylophilus. In this study, a KCl solution at an initial concentration of 8% was used, and its concentration was increased gradually with the natural evaporation of water, allowing B. xylophilus to slowly dehydrate and enter cryptobiosis. If water evaporates too quickly or the nematodes are placed directly into a high-concentration KCl solution, their survival rate decreases significantly.

The method described in this study has several advantages. The experimental materials are easily accessible, requiring only KCl solution and distilled water to regulate the dehydration and rehydration of B. xylophilus. The procedures are straightforward: dehydration occurred through natural evaporation, and rehydration was achieved by adding distilled water. The entire process can be completed within 2 h. This method boasts high success and survival rates, with most of B. xylophilus entering cryptobiosis after dehydration and the majority reviving after rehydration, respectively¹¹.

The proposed method is applicable for regulating the cryptobiotic state of both B. xylophilus and the fourth-stage dispersal juveniles ()¹⁴. However, it may not be effective for the propagative B. xylophilus.

In recent years, pine wilt disease caused by B. xylophilus has expanded from southern China to northern areas. B. xylophilus, which inhabits the pine trees in these colder northern regions, tends to exhibit greater cold resistance¹⁵. The proposed method can induce cryptobiosis in B. xylophilus, enhancing their resistance to cold and drought. Collectively, this method can provide technical support for studying the stress resistance mechanisms of B. xylophilus and the transmission mechanism of pine wilt disease.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Glass slide	Bioland	GS7101-7525	75 mm × 25 mm × 1 mm
Long necked glass funnel	Nanjing Kangluoda Experimental Technology	N/A	150 mm
Microscope	Phenix	XSP-35-1600X	PH100-2B41L-IPL
Petri dish	Nanjing Kangluoda Experimental Technology	TB-624935792745	50 mm × 50 mm
Pinchcock	Nanjing Kangluoda Experimental Technology	TB-624973311743	36 mm × 11 mm  × 55 mm
Pipette	Eppendorf	3123000012	10–100 µL
Potassium chloride (KCL)	Solarbio Life Science	P9921	500 g
Rubber tube	Nanjing Kangluoda Experimental Technology	TB-624923457094	7 mm × 5 mm
Sterilizer	BinJiang	https://detail.1688.com/offer/644766597053.html?_t=1730892390541&spm= a2615.7691456.co_0_0_wangpu _score_0_0_0_0_1_0_0000_0.0	75L
Tissue paper	Heart to heart imprint	M3	22.5 cm × 21.5 cm